Polypeptides that bind to CD123 and their uses

Abstract

The present invention provides VHH-containing polypeptides that bind to CD123. Also provided are uses of the VHH-containing polypeptides.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority benefit of U.S. Provisional Application No. 62 / 843,407, filed on May 4, 2019, and the entire disclosure of the provisional application is incorporated herein by reference for any purpose. Technical field

[0003] The present invention relates to polypeptides that bind to CD123, and methods of using polypeptides that bind to CD123 to modulate the biological activity of CD123. Such methods include, but are not limited to, methods of treating cancer. Background art

[0004] CD123, or interleukin - 3 receptor alpha chain (IL - 3Rα), has been identified as a leukemia stem cell marker and is generally up - regulated on AML blasts, while its expression is low or absent on normal hematopoietic stem cells. CD123 may play a role in the proliferation, differentiation, and survival of hematopoietic cells. In blood cancers such as leukemia, poor prognosis, high blast counts, and resistance to apoptotic cell death have been associated with high expression of CD123. Thus, there is a therapeutic need for more effective treatments for cancers that express CD123. Summary of the invention

[0005] The present disclosure provides polypeptides that bind to CD123 and methods of using polypeptides that bind to CD123 to treat, for example, leukemia. In some embodiments, the polypeptide that binds to CD123 comprises at least one VHH domain. Some embodiments are provided below.

[0006] Embodiment 1. A polypeptide comprising at least one VHH domain that binds to CD123 and comprises a CDR1 having an amino acid sequence containing SEQ ID NO: 33, 42, 3, 7, 11, 15, 19, 23, 36, 39, 45, 48, 51, or 93; a CDR2 having an amino acid sequence containing SEQ ID NO: 34, 43, 4, 8, 12, 16, 20, 24, 37, 40, 46, 49, 52, or 94; and a CDR3 having an amino acid sequence containing SEQ ID NO: 35, 44, 5, 9, 13, 17, 21, 25, 38, 41, 47, 50, 53, or 95.

[0007] Embodiment 2. The polypeptide according to Embodiment 1, wherein the at least one VHH domain comprises a CDR1 having an amino acid sequence containing SEQ ID NO: 33 or 3; a CDR2 having an amino acid sequence containing SEQ ID NO: 34 or 4; and a CDR3 having an amino acid sequence containing SEQ ID NO: 35 or 5.

[0008] Embodiment 3. The polypeptide according to Embodiment 1 or Embodiment 2, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 42, 19 or 45; a CDR2 containing the amino acid sequence of SEQ ID NO: 43, 20 or 46; and a CDR3 containing the amino acid sequence of SEQ ID NO: 44, 21 or 47.

[0009] Embodiment 4. The polypeptide according to any one of Embodiments 1-3, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 7 or 36; a CDR2 containing the amino acid sequence of SEQ ID NO: 8 or 37; and a CDR3 containing the amino acid sequence of SEQ ID NO: 9 or 38.

[0010] Embodiment 5. The polypeptide according to any one of Embodiments 1-4, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 15 or 39; a CDR2 containing the amino acid sequence of SEQ ID NO: 16 or 40; and a CDR3 containing the amino acid sequence of SEQ ID NO: 17 or 41.

[0011] Embodiment 6. The polypeptide according to any one of Embodiments 1-5, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 23, 48, 51 or 93; a CDR2 containing the amino acid sequence of SEQ ID NO: 24, 49, 52 or 94; and a CDR3 containing the amino acid sequence of SEQ ID NO: 25, 50, 53 or 95.

[0012] Embodiment 7. The polypeptide according to any one of Embodiments 1-6, wherein at least one VHH domain comprises CDR1, CDR2 and CDR3 containing the amino acid sequences of SEQ ID NO: 33, 34 and 35; 42, 43 and 44; 3, 4 and 5; 7, 8 and 9; 11, 12 and 13; 15, 16 and 17; 19, 20 and 21; 23, 24 and 25; 36, 37 and 38; 39, 40 and 41; 45, 46 and 47; 48, 49 and 50; 51, 52 and 53; or 93, 94 and 95, respectively.

[0013] Embodiment 8. The polypeptide according to any one of Embodiments 1-7, wherein at least one VHH domain is humanized.

[0014] Embodiment 9. The polypeptide according to any one of Embodiments 1-8, 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, 26, 27, 28, 29, 30, 31 or 92.

[0015] Embodiment 10. The polypeptide according to any one of Embodiments 1-8, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 32, 26, 27, 28, 29, 30, 31 or 92.

[0016] Embodiment 11. The polypeptide according to any one of Embodiments 1-7, 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, 6, 10, 14, 18 or 22.

[0017] Embodiment 12. The polypeptide according to any one of Embodiments 1-7, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO: 2, 6, 10, 14, 18 or 22.

[0018] Embodiment 13. The polypeptide according to any one of Embodiments 1-12, wherein the polypeptide comprises two VHH domains.

[0019] Embodiment 14. The polypeptide according to any one of Embodiments 1-12, wherein the polypeptide comprises three VHH domains.

[0020] Embodiment 15. The polypeptide according to any one of Embodiments 1-14, wherein the polypeptide comprises at least one binding domain that binds an antigen other than CD123.

[0021] Embodiment 16. The polypeptide according to Embodiment 15, wherein the polypeptide comprises at least one binding domain that binds CD3, T cell receptor (TCR) α, TCRβ, CD28, CD16, CD32A, CD64, CD89, NKp46 or NKG2D.

[0022] Embodiment 17. The polypeptide according to Embodiment 13 or 14, wherein each VHH domain binds CD123.

[0023] Embodiment 18. The polypeptide according to Embodiment 17, wherein each VHH domain comprises the same CDR1, CDR2 and CDR3 amino acid sequences.

[0024] Embodiment 19. The polypeptide according to Embodiment 17, wherein each VHH domain contains the same VHH sequence.

[0025] Embodiment 20. The polypeptide according to any one of Embodiments 1-12, the polypeptide comprising one VHH domain.

[0026] Embodiment 21. The polypeptide according to any one of Embodiments 1-20, wherein the polypeptide comprises an Fc region.

[0027] Embodiment 22. The polypeptide according to Embodiment 21, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NOs: 54-89.

[0028] Embodiment 23. The polypeptide according to Embodiment 21 or Embodiment 22, the polypeptide forming a dimer under physiological conditions.

[0029] Embodiment 24. The polypeptide according to any one of Embodiments 1-23, wherein the CD123 is human CD123.

[0030] Embodiment 25. The polypeptide according to Embodiment 24, wherein the human CD123 comprises the sequence of SEQ ID NO: 1.

[0031] Embodiment 26. The polypeptide according to any one of the foregoing embodiments, wherein the polypeptide blocks the binding of CD123 to IL-3.

[0032] Embodiment 27. An immunoconjugate comprising the polypeptide according to any one of Embodiments 1-26 and a cytotoxic agent.

[0033] Embodiment 28. The immunoconjugate according to Embodiment 31, wherein the cytotoxic agent is selected from calicheamicin, auristatin, dolastatin, tubulicin, maytansinoid, cryptophycin, duocarmycin, esperamicin, pyrrolobenzodiazepine, and enediyne antibiotics.

[0034] Embodiment 29. A pharmaceutical composition comprising the polypeptide according to any one of Embodiments 1-26 or the immunoconjugate according to Embodiment 27 or Embodiment 28 and a pharmaceutically acceptable carrier.

[0035] Embodiment 30. An isolated nucleic acid encoding a polypeptide according to any one of Embodiments 1-26.

[0036] Embodiment 31. A vector comprising the nucleic acid according to Embodiment 30.

[0037] Embodiment 32. A host cell comprising the nucleic acid according to Embodiment 30 or the vector according to Embodiment 31.

[0038] Embodiment 33. A host cell expressing a polypeptide according to any one of Embodiments 1-26.

[0039] Embodiment 34. A method for producing a polypeptide according to any one of Embodiments 1-26, the method comprising incubating a host cell according to Embodiment 32 or Embodiment 33 under conditions suitable for expressing the polypeptide.

[0040] Embodiment 35. The method according to Embodiment 34, the method further comprising isolating the polypeptide.

[0041] Embodiment 36. A method for treating cancer, the method comprising administering to a subject suffering from cancer a pharmaceutically effective amount of a polypeptide according to any one of Embodiments 1-26, an immunoconjugate according to Embodiment 27 or Embodiment 28, or a pharmaceutical composition according to Embodiment 30.

[0042] Embodiment 37. The method according to Embodiment 36, wherein the cancer is selected from lymphoma; Hodgkin lymphoma; non-Hodgkin lymphoma; B-cell lymphoma; 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-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; and chronic myelogenous leukemia.

[0043] Embodiment 38. The method according to Embodiment 36 or 37, wherein the cancer is acute myeloid leukemia (AML).

[0044] Embodiment 39. The method according to any one of Embodiments 36-38, the method further comprising administering an additional therapeutic agent.

[0045] Embodiment 40. The method according to embodiment 39, wherein the additional therapeutic agent is an anti-cancer agent.

[0046] Embodiment 41. The method according to embodiment 40, wherein the anti-cancer agent is selected from chemotherapeutic agents, anti-cancer biologics, radiotherapy, CAR-T therapy, and oncolytic viruses.

[0047] Embodiment 42. The method according to any one of embodiments 36-41, wherein the cancer is a cancer expressing CD123. Description of the Drawings

[0048] Figures 1A-1C show biolayer interferometry data of polypeptides comprising a VHH domain that binds CD123. Figure 1A shows biolayer interferometry data of hzA5v2 compared to other CD123-binding sdAbs described herein. Figure 1B shows biolayer interferometry data of hzF3v22 compared to other CD123-binding sdAbs described herein. Figure 1C shows biolayer interferometry data of hz1B11v28 compared to hz4F2v3.

[0049] Figures 2A-2N show the binding of certain single-domain antibodies (sdAbs) to CD123 expressed on HEK 293 cells or Molm-13 cells. "CD123-FL" indicates HEK293 cells transfected with a plasmid encoding full-length CD123, as described in Example 2. "HEK 293" or "parental HEK 293" indicates untransfected HEK 293 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.

[0050] Figures 3A-3B show the inhibition of CD123 and IL-3 binding by sdAbs A5, C5, and F3 (3A) and sdAb hz1F5v1 (3B). DETAILED DESCRIPTION

[0051] The embodiments provided herein relate to polypeptides that bind CD123 and their use in various methods of treating cancer.

[0052] Definitions and various embodiments

[0053] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0054] All references cited herein (including patent applications, patent publications, and Genbank accession numbers) are incorporated herein by reference as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

[0055] Those skilled in the art are generally familiar with and typically use conventional methods to employ the techniques and procedures described or cited herein. Such conventional methods include, for example, widely utilized methods described in the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (edited by F.M. Ausubel et al., (2003)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (edited by M.J. MacPherson, B.D. Hames and G.R. Taylor (1995)), ANTIBODIES, A LABORATORY MANUAL edited by Harlow and Lane (1988), and ANIMAL CELL CULTURE (edited by R.I. Freshney (1987)); Oligonucleotide Synthesis (edited by M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by J.E. Cellis, 1998), Academic Press; Animal Cell Culture (edited by R.I. Freshney, 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998), Plenum Press; Cell and Tissue Culture Laboratory Procedures (edited by A. Doyle, J.B. Griffiths and D.G. Newell, 1993 - 8), J.Wiley and Sons; Handbook of Experimental Immunology (edited by D.M. Weir and C.C. Blackwell); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P.Calos, ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. 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 J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993); and their updated versions.

[0056] Unless otherwise defined, scientific and technical terms used in connection with this disclosure shall have the meaning commonly understood by one of ordinary skill in the art. In addition, unless the context otherwise requires or clearly indicates, the singular terms shall include the plural, and the plural terms shall include the singular. For any conflict in definitions between various sources or references, the definitions provided herein shall control.

[0057] Generally, the residue numbering in immunoglobulin heavy chains is as in the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0058] It is to be understood that the embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" the embodiments. Unless otherwise indicated, as used herein, the singular forms "a", "an", and "the" include plural referents. The use of the term "or" herein is not intended to imply that the alternatives are mutually exclusive.

[0059] In this application, unless explicitly stated or understood by those skilled in the art, the use of "or" means "and / or". In the context of multiple dependent claims, the use of "or" refers back to more than one of the preceding independent or dependent claims.

[0060] The phrases "reference sample", "reference cell", or "reference tissue" denote a sample having at least one known characteristic that can be used as a comparator to a sample having at least one unknown characteristic. In some embodiments, the reference sample can be used as a positive or negative indicator. The reference sample can be used to determine the level of a protein and / or mRNA present in, for example, healthy tissue as compared to the level of the protein and / or mRNA present in a sample having unknown characteristics. In some embodiments, the reference sample is from the same subject but from a different part of the subject than the part being tested. In some embodiments, the reference sample is from a tissue area around or adjacent to cancer. In some embodiments, the reference sample is not from the subject being tested but from a sample of a subject known to have or not have the disorder under discussion (e.g., a particular cancer or CD123-related disorder). In some embodiments, the reference sample is from the same subject but from a time point prior to the subject developing cancer. In some embodiments, the reference sample is from a benign cancer sample from the same or a different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule under discussion in the negative reference sample will indicate the level that those skilled in the art would consider to be absent and / or present at a low level given the present disclosure. When a positive reference sample is used for comparison, the expression level or amount of the molecule under discussion in the positive reference sample will indicate the level that those skilled in the art would consider to be present at a certain level given the present disclosure.

[0061] As used herein, the terms "benefit", "clinical benefit", "reactivity", and "treatment reactivity" in the context of benefiting from or responding to the administration of a therapeutic agent can be measured by assessing various endpoints, such as inhibition of disease progression to some extent, including slowing and complete arrest; reduction in the frequency of disease onset and / or symptoms; reduction in lesion size; inhibition (i.e., reduction, slowing, or complete cessation) of infiltration of diseased cells into adjacent peripheral organs and / or tissues; inhibition (i.e., reduction, slowing, or complete cessation) of disease spread; alleviation of one or more symptoms associated with the disorder to some extent; increase in the duration of disease-free manifestation (e.g., progression-free survival) after treatment; increase in overall survival; higher response rate; and / or reduced mortality at a given time point after treatment. A subject or cancer that is "non-responsive" or "fails to respond" is a subject or cancer that fails to meet the eligibility criteria for "responsive" as defined above.

[0062] The terms "nucleic acid molecule", "nucleic acid", and "polynucleotide" may be used interchangeably and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides and include, but are 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.

[0063] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. The definition encompasses both full-length proteins and fragments thereof. The terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In addition, for the purposes of the present disclosure, a "polypeptide" refers to a protein that includes modifications to the native sequence (such as deletions, additions, and substitutions, which are generally conservative in nature), provided that the protein retains the desired activity. These modifications may be intentional (such as by site-directed mutagenesis) or may be accidental (such as by mutations in the host producing the protein or due to errors in PCR amplification).

[0064] As used herein, "CD123" refers to any naturally occurring mature CD123 produced by the processing of the CD123 precursor in a cell. Unless otherwise indicated, 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). The term also includes naturally occurring CD123 variants such as splice variants or allelic variants. A non-limiting exemplary mature human CD123 amino acid sequence is shown, for example, in UniProt accession number P26951-1. See SEQ ID NO.1.

[0065] The term "specifically binds" to an antigen or epitope is a term well known 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, for a longer duration, and / or with greater affinity with a particular cell or substance compared to its reaction or association with alternative cells or substances. An antibody or polypeptide "specifically binds" or "preferentially binds" to a target if it binds to the target with greater affinity, avidity, more readily, and / or for a longer duration compared to the binding of a single domain antibody (sdAb) or a polypeptide containing a VHH to other substances. For example, an sdAb or a polypeptide containing a VHH that specifically or preferentially binds to a CD123 epitope is an sdAb or a polypeptide containing a VHH that binds to this epitope with greater affinity, avidity, more readily, and / or for a longer duration compared to its binding to other CD123 epitopes or non-CD123 epitopes. It should also be understood from reading this definition that, for example, an sdAb or a polypeptide containing a VHH 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 can include) exclusive binding. Generally, but not necessarily, a reference to binding means preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind an antigen.

[0066] The terms "inhibition" or "inhibit" refer to a decrease or cessation of any phenotypic trait, or a decrease or cessation in the incidence, degree, or likelihood of that trait. "Reduce" or "inhibit" refers to a decrease, lowering, or blocking of activity, function, and / or amount compared to a reference. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 10% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 50% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 75%, 85%, 90%, 95% or more. In some embodiments, the above amount is inhibited or reduced over a period of time relative to a control over the same period of time. As used herein, the term "inhibition" with respect to the activity of CD123 refers to a decrease in the activity of CD123 (such as binding to IL-3). In some embodiments, "inhibition" refers to a decrease in CD123 activity compared to CD123 activity in the absence of a modulator. In some embodiments, the polypeptides that bind CD123 described herein inhibit the binding of CD123 to IL-3.

[0067] 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) that binds to an antigen-binding molecule (e.g., an sdAb or a polypeptide containing a VHH). Epitopes generally comprise chemically reactive surface groupings of molecules such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural features as well as specific charge characteristics. Epitopes can be formed by contiguous and / or juxtaposed non-contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of the target molecule. Epitopes formed by contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) generally remain upon exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost upon 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, the length of the epitope is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues. If two antibodies exhibit competitive binding to a particular antigen, they can bind to the same epitope within the antigen. In some embodiments, an epitope can be identified by a certain minimum distance from CDR residues on the antigen-binding molecule. In some embodiments, an epitope can be identified by the aforementioned distance and is further limited to those residues involved in a bond (e.g., a hydrogen bond) between the residues of the antigen-binding molecule and the antigen residues. Epitopes can also be identified by various scans, e.g., alanine or arginine scans can indicate one or more residues with which the antigen-binding molecule can interact. Unless explicitly stated, a group of residues identified as an epitope does not exclude other residues from being part of the epitope for a particular antigen-binding molecule. Instead, the presence of such a group represents a minimum series (or species group) of the epitope. Thus, in some embodiments, a group of residues identified as an epitope represents the minimal epitope associated with the antigen, rather than an exclusive list of the residues of the epitope on the antigen.

[0068] A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides, amino acids, and / or sugars within an antigen protein, to which an antigen-binding molecule specific for the epitope binds. In some embodiments, at least one of the residues will not be adjacent to the other indicated residues of the epitope; however, one or more of the residues can also be adjacent to other residues.

[0069] "Linear epitope" includes contiguous polypeptides, amino acids, and / or sugars within an antigenic protein to which an antigen-binding molecule specific for the epitope binds. It should be noted that in some embodiments, not every residue within the linear epitope needs to be directly bound (or participate in bonding) by the antigen-binding molecule. In some embodiments, a linear epitope can be derived from immunization with a peptide effectively consisting of the sequence of the linear epitope, or from a structural portion of a protein that is relatively separated from the rest of the protein (such that the antigen-binding molecule can interact with at least primarily only that sequence portion).

[0070] The term "antibody" is used in the broadest sense and encompasses various polypeptides containing antibody-like antigen-binding domains, including but not limited to conventional antibodies (usually containing at least one heavy chain and at least one light chain), single-domain antibodies (sdAbs, containing at least one VHH domain and an Fc region), polypeptides containing VHHs (polypeptides containing at least one VHH domain), and fragments of any of the foregoing, so long as they exhibit the desired antigen-binding activity. In some embodiments, an antibody contains a dimerization domain. Such dimerization domains include but are not limited to heavy chain constant domains (containing CH1, hinge, CH2, and CH3, where CH1 typically pairs with the light chain constant domain CL and the hinge mediates dimerization) and Fc regions (containing hinge, CH2, and CH3, where the hinge mediates dimerization).

[0071] The term antibody also includes but is not limited to chimeric antibodies, humanized antibodies, and antibodies from various species such as camelids (including llamas), sharks, mice, humans, cynomolgus monkeys, etc.

[0072] The term "antigen-binding domain" as used herein refers to the portion of an antibody that is sufficient to bind an antigen. In some embodiments, the antigen-binding domain of a conventional antibody contains three heavy chain CDRs and three light chain CDRs. Thus, in some embodiments, the antigen-binding domain contains a heavy chain variable region and a light chain variable region, the heavy chain variable region containing CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen, and the light chain variable region containing 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 a polypeptide containing a VHH contains the three CDRs of the VHH domain. Thus, in some embodiments, the antigen-binding domain of an sdAb or a polypeptide containing a VHH contains a VHH domain, the VHH domain containing CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen.

[0073] As used herein, the terms "VHH" or "VHH domain" or "VHH antigen-binding domain" refer to the antigen-binding portion of a single-domain antibody (such as a camelid antibody or a shark antibody). In some embodiments, a VHH contains three CDRs and four framework regions, designated as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH can be truncated at the N-terminus or C-terminus such that it contains only a portion of FR1 and / or FR4, or lacks one or both of those framework regions, provided that the VHH substantially maintains antigen binding and specificity.

[0074] The terms "single-domain antibody" and "sdAb" are used interchangeably herein to refer to an antibody that contains at least one monomeric domain (such as a VHH domain) and does not contain a light chain and an Fc region. In some embodiments, an sdAb is a dimer of two polypeptides, wherein each polypeptide contains at least one VHH domain and an Fc region. As used herein, the terms "single-domain antibody" and "sdAb" encompass polypeptides that contain multiple VHH domains, such as polypeptides having the structure VHH1-VHH2-Fc or VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 can be the same or different.

[0075] The term "VHH-containing polypeptide" refers to a polypeptide that contains at least one VHH domain. In some embodiments, a VHH polypeptide contains two, three, or four or more VHH domains, wherein each VHH domain can be the same or different. In some embodiments, a VHH-containing polypeptide contains an Fc region. In some such embodiments, a VHH-containing polypeptide can be referred to as an sdAb. Additionally, in some such embodiments, a VHH polypeptide can form a dimer. Non-limiting structures of VHH-containing polypeptides (which are also sdAbs) include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 can be the same or different. In some embodiments of such structures, one VHH can be linked to another VHH by a linker, or one VHH can be linked to an Fc by a linker. In some such embodiments, the linker contains 1-20 amino acids, preferably 1-20 amino acids consisting primarily of glycine and optionally serine. In some embodiments, when a VHH-containing polypeptide contains an Fc, it forms a dimer. Thus, if the structure VHH1-VHH2-Fc forms a dimer, it is considered tetravalent (i.e., the dimer has four VHH domains). Similarly, if the structure VHH1-VHH2-VHH3-Fc forms a dimer, it is considered hexavalent (i.e., the dimer has six VHH domains).

[0076] The term "monoclonal antibody" refers to an antibody (including sdAb or a polypeptide containing VHH) within a substantially homogeneous population of antibodies, i.e., the individual antibodies making up 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. In addition, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Thus, a monoclonal antibody sample can bind to the same epitope on the antigen. The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be made by any particular method. For example, monoclonal antibodies can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or can be prepared by recombinant DNA methods (such as those described in U.S. Patent No. 4,816,567). For example, monoclonal antibodies can also be isolated from phage libraries generated using the techniques described by McCafferty et al., 1990, Nature 348:552-554.

[0077] The term "CDR" refers to complementarity-determining regions as defined by at least one mode of identification by a person of ordinary skill in the art. In some embodiments, the CDRs can be defined according to any one of the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition. VHH contains three CDRs, designated CDR1, CDR2, and CDR3.

[0078] As used herein, the term "heavy chain constant region" refers to a region that includes at least three heavy chain constant domains, CH1, hinge, CH2, and CH3. Of course, unless otherwise specified, non-functional altering deletions and alterations within the domains are encompassed within the term "heavy chain constant region". Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy constant region corresponds to an antibody isotype. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, and an antibody containing an α constant region is an IgA antibody. In addition, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε 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 the γ1 constant region), IgG2 (containing the γ2 constant region), IgG3 (containing the γ3 constant region), and IgG4 (containing the γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing the α1 constant region) and IgA2 (containing the α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0079] As used herein, "Fc region" refers to the portion of the heavy chain constant region that includes CH2 and CH3. In some embodiments, the Fc region includes the hinge, CH2, and CH3. In various embodiments, when the Fc region includes the hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any heavy chain constant region isotype of an antibody discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0080] As used herein, "receptor human framework" is a framework that contains the amino acid sequence of a heavy chain variable domain (V H ) framework derived from a human immunoglobulin framework or a human consensus framework, as discussed herein. A receptor human framework derived from a human immunoglobulin framework or a human consensus framework can contain the same amino acid sequence as it, or it can contain amino acid sequence variations. In some embodiments, across all human frameworks in a single antigen-binding domain (such as a VHH), the number of amino acid variations 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.

[0081] "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 polypeptide containing a VHH) and its binding partner (e.g., an antigen). The affinity or apparent affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K d ) or K d-表观 respectively. Affinity can be measured by common methods known in the art (such as ELISA K d , KinExA, flow cytometry, and / or surface plasmon resonance devices) (including those described herein). Such methods include, but are not limited to, methods involving or flow cytometry.

[0082] As used herein, the term "K d " refers to the equilibrium dissociation constant of an antigen-binding molecule / antigen interaction. When the term "K d " is used herein, it includes K d and K d-表观 .

[0083] In some embodiments, the K d of an antigen-binding molecule is measured by flow cytometry using a cell line expressing the antigen and fitting the mean fluorescence measured at each antibody concentration to a non-linear one-site binding equation (Prism Softwaregraphpad). In some such embodiments, K d is K d-表观 .

[0084] The term "bioactive" refers to any one or more biological properties of a molecule (whether naturally occurring as found in vivo or provided or achieved recombinantly).

[0085] An "agonist" or "activating" antibody is an antibody that increases and / or activates the biological activity of a target antigen. In some embodiments, the agonist antibody binds to the antigen and increases the biological activity of the antigen by at least about 20%, 40%, 60%, 80%, 85% or more.

[0086] 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, the neutralizing antibody binds to the antigen and reduces the biological activity of the antigen by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 99% or more.

[0087] An "affinity matured" sdAb or VHH-containing polypeptide is an sdAb or VHH-containing polypeptide that has one or more alterations in one or more CDRs compared to a parental sdAb or VHH-containing polypeptide that does not have such alterations, such alterations resulting in an increase in the affinity of the sdAb or VHH-containing polypeptide for the antigen.

[0088] As used herein, a "humanized VHH" is 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 with the corresponding non-human residues. In addition, a humanized VHH can contain residues that are not found in either the original VHH or the human framework sequence but are included to further improve and optimize the properties of the sdAb or VHH-containing polypeptide. In some embodiments, the humanized sdAb or VHH-containing polypeptide contains a human Fc region. As should be understood, a humanized sequence can be identified by its primary sequence and does not necessarily represent the process by which the antibody is produced.

[0089] An "effector-positive Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation, among others. Such effector functions generally require the combination of an Fc region with a binding domain (e.g., an antibody variable domain) and can be evaluated using a variety of assays.

[0090] "Native sequence Fc region" encompasses an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. The native sequence human Fc region includes the native sequence human IgG1 Fc region (non-A allotype and A allotype); the native sequence human IgG2 Fc region; the native sequence human IgG3 Fc region; and the native sequence human IgG4 Fc region, as well as its naturally occurring variants.

[0091] "Variant Fc region" refers to an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region due to at least one amino acid modification. In some embodiments, the "variant Fc region" refers to an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region due to at least one amino acid modification but still retains at least one effector function of the native sequence Fc region. In some embodiments, compared to the native sequence Fc region or the Fc region of the parental polypeptide, the variant Fc region has at least one amino acid substitution in the native sequence Fc region or in the Fc region of the parental polypeptide, such as from about one to about ten amino acid substitutions, preferably from about one to about five amino acid substitutions. In some embodiments, the variant Fc regions herein will have at least about 80% sequence identity with the native sequence Fc region and / or with the Fc region of the parental polypeptide, at least about 90% sequence identity therewith, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith.

[0092] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, FcγR is a native human FcR. In some embodiments, an FcR is a receptor that binds an IgG antibody (γ receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of those receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, e.g., in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" herein encompasses other FcRs, including those to be identified in the future. For example, the term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for 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 regulates immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.)).

[0093] As used herein, "chimeric antigen receptor" refers to an engineered polypeptide that comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular antigen recognition domain comprises a VHH domain.

[0094] As used herein, the terms "substantially similar" or "substantially identical" indicate a high enough degree of similarity between two or more values such that one of ordinary skill in the art would consider the difference between the two or more values to be of little or no biological and / or statistical significance in the context of the biological characteristic being measured by the values. In some embodiments, the difference between two or more substantially similar values does not exceed any one of about 5%, 10%, 15%, 20%, 25%, or 50%.

[0095] A polypeptide "variant" means a bioactive polypeptide that has at least about 80% amino acid sequence identity to a native sequence polypeptide after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity and not considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides having one or more amino acid residues added or deleted at the N-terminus or C-terminus of the polypeptide. In some embodiments, the variant will have at least about 80% amino acid sequence identity. In some embodiments, the variant will have at least about 90% amino acid sequence identity. In some embodiments, the variant will have at least about 95% amino acid sequence identity to the native sequence polypeptide.

[0096] 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 specific peptide or polypeptide sequence after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity and not considering any conservative substitutions as part of the sequence identity. The alignment for the purpose of determining the percent amino acid sequence identity can be achieved in various ways within the skill in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGNTM (DNASTAR) software. One of ordinary skill in the art can determine the appropriate parameters for measuring the alignment, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared.

[0097] 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 an antibody of interest, and the resulting product can be screened for the desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0098] Table 1

[0099] Original residue Exemplary substitutions Ala(A) Val; Leu; Ile Arg(R) Lys; Gln; Asn Asn(N) Gln; His; Asp; Lys; Arg Asp(D) Glu; Asn Cys(C) Ser; Ala Gln(Q) Asn; Glu Glu(E) Asp; Gln Gly(G) Ala His(H) Asn; Gln; Lys; Arg Ile(I) Leu; Val; Met; Ala; Phe; norleucine Leu(L) norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg; Gln; Asn Met(M) Leu; Phe; Ile Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Pro(P) Ala Ser(S) Thr Thr(T) Val; Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe; Thr; Ser Val(V) Ile; Leu; Met; Phe; Ala; norleucine

[0100] Amino acids can be grouped according to common side-chain characteristics:

[0101] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0102] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0103] (3) Acidic: Asp, Glu;

[0104] (4) Basic: His, Lys, Arg;

[0105] (5) Residues affecting chain orientation: Gly, Pro;

[0106] (6) Aromatic: Trp, Tyr, Phe.

[0107] Non-conservative substitutions would require the exchange of a member of one of these categories for another.

[0108] The term "vector" is used to describe a polynucleotide that can be engineered to contain one or more cloned polynucleotides that can replicate in a host cell. Vectors 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 a promoter and / or enhancer), and / or one or more selectable marker genes (such as antibiotic resistance genes and genes that can be used for colorimetric assays (e.g., β-galactosidase)). The term "expression vector" refers to a vector used for expressing a polypeptide of interest in a host cell.

[0109] "Host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic cells. 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, Crucell, and 293 and CHO cells and their derivatives (such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells). Host cells include the progeny of a single host cell, and due to natural, accidental, or deliberate mutations, the progeny may not necessarily be identical to the original parental cell (in terms of morphology or genomic DNA complementary sequence). Host cells include cells transfected in vivo with one or more of the polynucleotides provided herein.

[0110] As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the components with which it is normally found or produced in nature. For example, a polypeptide is referred to as "isolated" when it has been separated from at least some of the components of the cell that produced it. In the case where a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" when it is not part of a larger polynucleotide that is normally found in nature (such as genomic DNA or mitochondrial DNA in the case of DNA polynucleotides), or when, for example in the case of an RNA polynucleotide, it has been separated from at least some of the components of the cell that produced it. Thus, a DNA polynucleotide contained within a vector in a host cell can be referred to as "isolated".

[0111] The terms "individual" and "subject" are used interchangeably herein to refer to an animal; for example a mammal. In some embodiments, methods of treating a mammal are provided, the mammal including but not limited to humans, rodents, primates, felines, canines, equines, bovines, porcines, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sport animals, and mammalian pets. In some instances, an "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, a subject being treated can be a patient who has been identified as having a disorder relevant to the treatment, or at sufficient risk of developing the disorder.

[0112] As used herein, a "disease" or "disorder" refers to a condition that requires and / or is amenable to treatment.

[0113] Unless otherwise specified, the terms "tumor cell", "cancer cell", "cancer", "tumor", and / or "neoplasm" are used interchangeably herein and refer to a cell (or cells) that exhibits uncontrolled growth and / or abnormal increase in cell survival and / or inhibition of apoptosis that interferes with the normal functioning of body organs and systems. This definition includes benign and malignant cancers, blood cancers (such as leukemia, lymphoma, and multiple myeloma), polyps, hyperplasia, and dormant tumors or micrometastases.

[0114] The terms “cancer” and “tumor” encompass solid cancers and blood / lymph cancers, and also encompass malignant, pre-malignant, and benign growths, 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 cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancers; endometrial cancer; esophageal cancer; eye cancer; head and neck cancers; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; liver tumors; intraepithelial neoplasia; kidney cancer or renal carcinoma; 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 (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancers; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine cancer or endometrial cancer; urinary system cancers; vulvar cancer; lymphomas, including Hodgkin lymphoma and non-Hodgkin lymphoma, and B cell lymphomas (including low grade / follicular non-Hodgkin lymphoma (NHL); small lymphocyte (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myelogenous leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with phakomatosis, edema (such as edema associated with brain tumors), and Meigs syndrome.

[0115] As used herein, the term “non-tumor cell” or “non-cancer cell” refers to normal cells or tissues. Exemplary non-tumor cells include, but are not limited to: T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, macrophages, epithelial cells, fibroblasts, hepatocytes, renal interstitial cells, fibroblast-like synoviocytes, osteoblasts, and cells located in the breast, skeletal muscle, pancreas, stomach, ovary, small intestine, placenta, uterus, testis, kidney, lung, heart, brain, liver, prostate, colon, lymphoid organs, bone, and mesenchymal stem cells of bone origin. As used herein, the term “cells or tissues located in the periphery” refers to non-tumor cells that are not located near tumor cells and / or within the tumor microenvironment.

[0116] As used herein, the term "cells or tissues within the tumor microenvironment" refers to the 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 blood vessels; 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; myeloid-derived suppressor cells (MDSCs); and other immune cells located near the tumor. Methods for identifying tumor cells and / or cells / tissues within the tumor microenvironment are well known in the art and are described below.

[0117] In some embodiments, "increase" or "decrease" refers to a statistically significant increase or decrease, respectively. As will be apparent to those skilled in the art, "modulation" can also involve achieving a change (which can be an increase or decrease) in the affinity, avidity, specificity, and / or selectivity of a target or antigen for one or more of its ligands, binding partners, partners for association into homomeric or heteromeric forms, or substrates, compared to the same conditions but in the absence of the test agent; achieving a change (which can be an increase or decrease) in the sensitivity of a target or antigen to one or more conditions in the medium or environment in which the target or antigen is present, such as pH, ionic strength, presence of cofactors, etc.; and / or cell proliferation or cytokine production. 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.

[0118] As used herein, "immune response" is intended to encompass a cellular immune response and / or a humoral immune response that is sufficient to inhibit or prevent the onset of a disease (e.g., cancer or cancer metastasis) or to ameliorate the symptoms of said disease. "Immune response" can encompass aspects of both the innate and adaptive immune systems.

[0119] As used herein, "treatment" is a means for obtaining a beneficial or desired clinical outcome. As used herein, "treatment" includes any administration or application of a therapeutic agent to a disease in a mammal, including a human. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction of the degree of the disease, prevention or delay of the spread of the disease (e.g., metastasis, e.g., to the lung or lymph nodes), prevention or delay of the recurrence of the disease, delay or slowing of the progression of the disease, improvement of the disease state, inhibition of the disease or the progression of the disease, inhibition or slowing of the disease or the progression of the disease, arrest of the development of the disease, and remission (whether partial or complete). "Treatment" also encompasses reducing the pathological consequences of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. Consistent with the foregoing, the term treatment does not require the complete elimination of all aspects of the disorder one hundred percent.

[0120] "Improve" means a reduction or improvement of one or more symptoms as compared to not administering a therapeutic agent. "Improve" also includes shortening or reducing the duration of the symptoms.

[0121] The term "anticancer agent" is used herein in its broadest sense to refer to an agent for treating one or more cancers. Exemplary classes of such agents include, but are not limited to, chemotherapeutic agents, anticancer biologics (such as cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiotherapy, CAR-T therapy, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNA), and oncolytic viruses.

[0122] The term "biological sample" means an amount of material from a living or once-living thing. Such materials include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, white blood cells, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0123] In an experimental or comparative context, the terms "control" or "reference" refer to a composition known to be free of an analyte ("negative control") or containing an analyte ("positive control"). The positive control may contain a known concentration of the analyte. The control or reference may also refer to a control agent known to lack the activity of the agent being tested, such as an antibody.

[0124] As used herein, "delay the development of a disease" means to postpone, impede, slow, retard, stabilize, inhibit, and / or defer the development of a disease, such as cancer. This delay can have different lengths of time, depending on the medical history and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can actually encompass prevention, since the individual does not develop the disease. For example, it may delay the development of advanced cancer, such as metastasis.

[0125] As used herein, "prevention" includes providing prophylaxis against the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but has not been diagnosed with the disease. Unless otherwise stated, the terms "reduce", "inhibit", or "prevent" do not imply or require complete prevention at all times, but only during the period being measured.

[0126] A "therapeutically effective amount" of a substance / molecule, agonist, or antagonist can vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the substance / molecule, agonist, or antagonist to elicit the desired response in the individual. A therapeutically effective amount is also an amount where the therapeutic beneficial effects exceed any toxic or detrimental effects of the substance / molecule, agonist, or antagonist. A therapeutically effective amount can be delivered in one or more administrations. A therapeutically effective amount is an amount that is effective to achieve the desired therapeutic and / or prophylactic outcome at the necessary dosage and for the necessary period of time.

[0127] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a formulation that is in a form that permits the bioactivity of one or more active ingredients to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. Such formulations can be sterile.

[0128] A "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 that is used in conjunction with a therapeutic agent and that together constitutes a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosages and concentrations employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation employed.

[0129] Administering "in combination" with one or more other therapeutic agents includes administering simultaneously (concurrently) and sequentially in any order.

[0130] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents where at least a portion of the administrations overlap in time, or where the administration of one therapeutic agent occurs within a very short time period relative to the administration of another therapeutic agent, or where the therapeutic effects of the two agents overlap for at least a period of time.

[0131] 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 agents do not overlap.

[0132] As used herein, "in combination with" refers to administering one mode of treatment in addition to another mode of treatment. Thus, "in combination with" refers to administering one mode of treatment before, during, or after administering another mode of treatment to an individual.

[0133] The term "package insert" is used to refer to the instructions typically included in the commercial packaging of a therapeutic product, which contains information on indications, usage, dosage, administration, combination therapies, contraindications, and / or warnings regarding the use of such therapeutic product.

[0134] An "article" is any manufactured item (e.g., a package or container) or kit that contains at least one reagent (e.g., a drug for treating a disease or disorder such as cancer) or a probe for specifically detecting a biomarker described herein. In some embodiments, the manufactured item or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0135] The terms "label" and "detectable label" mean, for example, a moiety attached to an antibody or antigen such that a reaction (e.g., binding) between members of a specific binding pair is detectable. The labeled member of the specific binding pair is referred to as "detectably labeled". Thus, the term "labeled binding protein" refers to a protein that incorporates a label provided for identifying the binding protein. In some embodiments, the label is a detectable label that can generate a signal detectable by visual or instrumental means, such as a radioactively labeled amino acid incorporated or a polypeptide attached to a biotinyl moiety that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent label or enzyme activity that can be detected by an optical or colorimetric method). Examples of labeling of polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho or 153 Sm); chromogens; fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors); enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent labels; biotinyl groups; a predetermined polypeptide epitope recognized by a second reporter (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 produce light, such as acridine compounds; and moieties that produce fluorescence, such as fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with yet another moiety.

[0136] Exemplary polypeptides that bind to CD123

[0137] The present disclosure provides polypeptides that bind to CD123. In various embodiments, the CD123-binding polypeptide comprises at least one VHH domain that binds to CD123. In some embodiments, the CD123 is human CD123. In some embodiments, the CD123-binding polypeptide blocks 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 polypeptide 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.

[0138] In some embodiments, the CD123-binding polypeptide comprises at least one VHH domain that binds to CD123 and an Fc region. In some embodiments, the CD123-binding polypeptides provided herein comprise 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 that 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.

[0139] In some embodiments, the CD123-binding polypeptide comprises at least two VHH domains, wherein a first VHH domain binds to a first epitope of CD123 and a second VHH domain binds to a second epitope of CD123. When the 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 "bispecific" or "dual-epitope". In some embodiments, the CD123-binding polypeptide comprises at least two VHH domains, wherein a first VHH domain binds to CD123 and a second VHH domain binds to an antigen other than CD123. Such polypeptides may be referred to as "bispecific" or "multispecific".

[0140] Non-limiting exemplary CD123-binding polypeptides are shown in Table 2. The sequences of the indicated single-domain antibodies are shown in the list of certain sequences herein. Polypeptide names starting with "hz" indicate that it is a humanized version of the corresponding parental polypeptide.

[0141] Table 2: Polypeptides Comprising at Least One VHH That Binds CD123

[0142]

[0143]

[0144] CD123-binding polypeptide

[0145] In various embodiments, the VHH domain that binds CD123 comprises a CDR1 sequence selected from SEQ ID NOs: 3, 7, 11, 15, 19, 23, 33, 36, 39, 42, 45, 48, 51, and 93; a CDR2 sequence selected from SEQ ID NOs: 4, 8, 12, 16, 20, 24, 34, 37, 40, 43, 46, 49, 52, and 94; and a CDR3 sequence selected from SEQ ID NOs: 5, 9, 13, 17, 21, 25, 35, 38, 41, 44, 47, 50, 53, and 95. In various embodiments, the VHH domain that binds CD123 comprises CDR1, CDR2, and CDR3 sequences selected from: SEQ ID NOs: 3, 4, and 5; SEQ ID NOs: 7, 8, and 9; SEQ ID NOs: 11, 12, and 13; SEQ ID NOs: 15, 16, and 17; SEQ ID NOs: 19, 20, and 21; SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 36, 37, and 38; SEQ ID NOs: 39, 40, and 41; SEQ ID NOs: 42, 43, and 44; SEQ ID NOs: 45, 46, and 47; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 51, 52, and 53; and SEQ ID NOs: 93, 94, and 95. In various embodiments, the VHH domain is humanized.

[0146] 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%, or at least 99% identical to an amino acid sequence selected from SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 27, 28, 29, 30, 31, 32, and 92. In some embodiments, the VHH domain that binds CD123 comprises an amino acid sequence selected from SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 27, 28, 29, 30, 31, 32, and 92.

[0147] In various embodiments, the polypeptide that binds CD123 comprises one, two, three, or four VHH domains that bind CD123.

[0148] In various embodiments, the polypeptide that binds CD123 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, the polypeptide that binds CD123 may be referred to as a bispecific antibody.

[0149] In some embodiments, the polypeptide that binds CD123 comprises at least one VHH domain as described herein fused to an Fc region. In some embodiments, the Fc region has a sequence selected from: SEQ ID NO: 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 85, 86, 87, 88, and 89.

[0150] In some embodiments, the VHH domain that binds CD123 is humanized. Humanized antibodies (such as sdAbs or polypeptides containing VHHs) can be used as therapeutic molecules because humanized antibodies reduce or eliminate the human immune response to non-human antibodies, which can lead to an immune response to antibody therapeutics and reduced efficacy of the therapeutic agent. 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 a human antibody sequence. A humanized antibody optionally also will comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), for example to restore or improve antibody specificity or affinity.

[0151] Reviews of humanized antibodies and methods of making them are found, for example, in Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633, and are further described in, for example, Riechmann et al., (1988) Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86:10029-10033; U.S. Patent 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 "surface reshaping"); Dall'Acqua et al., (2005) Methods 36:43-60 (describing "FR shuffling"); and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing "directed selection" methods of FR shuffling).

[0152] Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best fit" method (see, for example, Sims et al. (1993) J. Immunol. 151:2296); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of heavy chain variable regions (see, for example, Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol, 151:2623); human mature (somatic hypermutated) framework regions or human germline framework regions (see, for example, Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633); and framework regions derived from screening FR libraries (see, for example, Baca et al., (1997) J. Biol. Chem. 272:10678-10684 and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Generally, the FR region of a VHH is replaced with a human FR region to produce a humanized VHH. In some embodiments, certain FR residues of the human FR are replaced to improve one or more properties of the humanized VHH. A VHH domain having such replacement residues is still referred to herein as "humanized".

[0153] In various embodiments, the Fc region included in a polypeptide that binds CD123 is a human Fc region, or is derived from a human Fc region.

[0154] In some embodiments, the Fc region included in a polypeptide that binds CD123 is derived from a human Fc region and contains a deletion of three amino acids corresponding to IgG1 E233, L234, and L235 in the lower hinge, referred to herein as "Fc xELL". The FcxELL polypeptide does not engage FcγR and is thus referred to as "effector silent" or "effector null"; however, in some embodiments, the xELL Fc region binds FcRn and thus has an extended half-life and endocytic trafficking associated with FcRn-mediated recycling.

[0155] In some embodiments, the Fc region included in a polypeptide that binds CD123 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 (near 6.5) of the endosome while losing detectable binding at neutral pH (about 7.2), thereby allowing enhanced FcRn-mediated recycling and an extended half-life.

[0156] In some embodiments, the Fc region included in a polypeptide that binds CD123 is derived from a human Fc region and contains mutations designed for heterodimerization, referred to herein as "knob" and "hole". In some embodiments, the "knob" Fc region contains the mutation T366W. In some embodiments, the "hole" Fc region contains the mutations T366S, L368A, and Y407V. In some embodiments, the Fc region for heterodimerization contains additional mutations, such as the mutation S354C on the first member of the heterodimeric Fc pair, which forms an asymmetric disulfide bond 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 unmodified at H435. In various embodiments, the "hole" Fc region contains the modification H435R or H435K (referred to as "hole R" in some cases when the modification is H435R), while the "knob" Fc region does not. In some cases, the "hole R" mutation improves the purification of the heterodimer compared to the homodimeric "hole" Fc region that may be present.

[0157] Non-limiting exemplary Fc regions that can be used for a polypeptide that binds CD123 include Fc regions comprising the amino acid sequences of SEQ ID NOs: 54 to 89.

[0158] Chimeric receptors and engineered cells

[0159] The present disclosure provides chimeric antigen receptors (CARs) having extracellular domains that include one or more VHH domains that bind CD123 as provided herein. The CAR constructs provided herein include an extracellular domain that contains the one or more VHH domains that bind CD123, a transmembrane domain, and an intracellular signaling region. The one or more VHH domains that form the antigen-binding unit of the CAR bind or are capable of binding (i.e., targeting) CD123 with sufficient affinity such that the CAR can be used in therapies that target cells or tissues that express CD123.

[0160] A CAR is a synthetic receptor that typically contains an extracellular targeting / binding portion associated with one or more signaling domains in a single fusion molecule that is expressed on the surface of a cell such as a T cell. Thus, a CAR combines antigen specificity and T cell activation properties in a single fusion molecule. First-generation CARs typically include the cytoplasmic region of CD3ζ or the Fc receptor γ chain as their signaling domain. First-generation CARs have been tested in phase I clinical studies in patients with ovarian cancer, renal cancer, lymphoma, and neuroblastoma, in which they have induced modest responses (reviewed in Sadelain et al., Curr Opin Immunol, 21(2):215-223, 2009). Second-generation CARs that contain signaling domains of costimulatory molecules such as CD28 and CD3ζ provide dual signaling to direct combined activation and costimulatory signals. Third-generation CARs are more complex, having 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).

[0161] In some embodiments, the provided CAR comprises a VHH domain that binds CD123. In some embodiments, the CAR contains 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 VHH domains that bind CD123, thereby providing a bivalent molecule. In one embodiment, the antigen-binding domain comprises two or at least two VHH domains that bind CD123, but bind to different epitopes on CD123. In such cases, the antigen-binding domain comprises a first VHH domain that binds CD123 and 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 bispecific, and the CAR is a bispecific CAR. In yet another embodiment, the antigen-binding domain comprises two VHH domains that bind to the same epitope on CD123.

[0162] The transmembrane domain of the CAR provided herein is a domain that generally traverses or is capable of traversing or spanning the plasma membrane and is directly or indirectly (e.g., via a spacer such as an immunoglobulin hinge sequence) linked to the extracellular antigen-binding domain and the endoplasmic portion containing the intracellular signaling domain. In one embodiment, the transmembrane domain of the CAR is the 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 the CD3ζ domain or the CD28 transmembrane domain. Other transmembrane domains will be apparent to those skilled in the art and can be used in combination with the embodiments of the CAR provided herein.

[0163] The intracellular signaling region of the CAR provided herein contains one or more intracellular signaling domains that transmit signals to a T cell upon engagement of the antigen-binding domain of the CAR (such as upon binding of an antigen). In some embodiments, the intracellular region contains an intracellular signaling domain that is or contains an ITAM signaling domain. Exemplary intracellular signaling domains include, for example, signaling domains derived from the zeta chain of the T cell receptor complex or any analog thereof (e.g., eta chain, FcsRIy and beta chain, MB1 (Iga) chain, B29 (Ig) chain, etc.), the human CD3 zeta chain, CD3 polypeptides (delta, delta, and epsilon), syk family tyrosine kinases (Syk, ZAP70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction (such as CD2, CD5, OX40, and CD28). In certain embodiments, the intracellular signaling region contains an intracellular signaling domain derived from the human CD3 zeta chain.

[0164] In some embodiments, the intracellular signaling region of the CAR can further contain an intracellular signaling domain derived from a co-stimulatory molecule. In such instances, for example, compared to a CAR that contains only a signaling domain containing an ITAM (such as CD3 zeta), such a signaling domain can enhance CAR-T cell activity, such as by enhancing the proliferation, survival, and / or development of memory cells, upon antigen-specific engagement. In some embodiments, the co-stimulatory domain is a functional signaling domain obtained from a protein selected from: CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, or a combination thereof. In certain embodiments, the co-stimulatory signaling domain is derived from or obtained from a human protein. In some aspects, the co-stimulatory signaling domain is derived from or obtained from human CD28 or human CD137 (4-1BB).

[0165] In some embodiments, the co-stimulatory signaling domain is derived from CD28 or 41BB.

[0166] In certain embodiments, the CAR further comprises a hinge region or spacer region that connects the extracellular antigen-binding domain and the transmembrane domain. This hinge region or spacer region can be used to achieve different lengths and flexibilities of the resulting CAR. Examples of hinge regions or spacer regions that can be used include, but are not limited to, the Fc fragment of an antibody or its fragment or derivative, the hinge region of an antibody or its fragment or derivative, the CH2 region of an antibody, the CH3 region of an antibody, an artificial spacer sequence (such as a peptide sequence), or a combination thereof. Other hinge regions 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.

[0167] In some embodiments, the spacer and transmembrane domain are the hinge and transmembrane domains derived from CD8.

[0168] Also provided herein is an isolated nucleic acid construct comprising at least one nucleic acid encoding a CAR as provided herein. In some aspects, the construct is an expression vector for expressing the 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). A variety of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviral (such as adenoviral) vectors are used. In one embodiment, a lentiviral vector is used.

[0169] In a further aspect, also provided is an isolated cell or cell population comprising one or more of the nucleic acid constructs described above. Also provided is an isolated cell or cell population that has been genetically modified to express a CAR as provided herein. Thus, provided herein are genetically engineered cells comprising (such as stably expressing) a CAR as provided herein. In one embodiment, the cell is selected from T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, hematopoietic stem cells, and / or pluripotent embryonic / induced stem cells. In some cases, the cell is a T cell, such as a CD4 and / or 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, such as transfection or transduction with a CAR nucleic acid construct.

[0170] In an exemplary example, primary T cells (CD4 cells or CD8 cells or both) can be purified ex vivo and the cells can be 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 the CAR can be stably introduced into the primary T cells by standard lentiviral or retroviral transduction protocols or plasmid electroporation strategies. CAR expression in the cells can be monitored, for example, by flow cytometry using an anti-epitope tag or antibody that cross-reacts with the native parental molecule. T cells expressing the CAR can be enriched by sorting with an anti-epitope tag antibody or enriched for high or low expression depending on the application.

[0171] The proper function of CAR-engineered T cells can be determined by a variety of means. In some cases, in vitro cytotoxicity, proliferation, or cytokine assays (e.g., IFN-γ expression) can be used to evaluate the function of the engineered T cells. Exemplary standard endpoints are the percentage of lysis of a tumor line in the culture supernatant, proliferation of the engineered T cells, or IFN-γ protein expression. In some cases, the ability to stimulate T cell activation after CAR stimulation (e.g., via antigen) can be evaluated, such as by monitoring the expression of activation markers (such as CD69, CD44, or CD62L), proliferation, and / or cytokine production.

[0172] Polypeptide expression and production

[0173] There is provided a nucleic acid molecule comprising a polynucleotide encoding a polypeptide that binds CD123. In some embodiments, the nucleic acid molecule may further encode a leader sequence that directs secretion of the polypeptide that binds CD123, the leader sequence typically being cleaved such that it is not present in the secreted polypeptide. The leader sequence can be a native heavy chain (or VHH) leader sequence or can be another heterologous leader sequence.

[0174] The nucleic acid molecule can be constructed using conventional recombinant DNA techniques in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0175] There is provided a vector comprising a nucleic acid encoding a polypeptide that binds CD123 as described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector optimized for expression of the polypeptide in a desired cell type (such as CHO or CHO-derived cells) or NSO cells is selected. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0176] In some embodiments, a CD123-binding polypeptide can be expressed in a prokaryotic cell (such as a bacterial cell); or in a eukaryotic cell (such as a fungal cell (such as yeast), a plant cell, an insect cell, and a mammalian cell). Such expression can be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used for expressing the polypeptide include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44.Lec13 CHO cells, and FUT8 CHO cells; Crucell; and NSO cells. In some embodiments, the CD123-binding polypeptide can be expressed in yeast. See, for example, U.S. Publication No. US 2006 / 0270045A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform the desired post-translational modifications on the polypeptide. For example, in some embodiments, the level of sialylation of a polypeptide produced by CHO cells is higher than the level of sialylation of the same polypeptide produced in 293 cells.

[0177] One or more nucleic acids (such as a vector) can be introduced into a desired host cell 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, 3rd ed. Cold Spring Harbor Laboratory Press (2001). According to any suitable method, nucleic acids can be transiently or stably transfected into the desired host cell.

[0178] Also provided are host cells comprising any of the nucleic acids or vectors described herein. In some embodiments, host cells are provided that express a CD123-binding polypeptide described herein. The CD123-binding polypeptide expressed in the host cell can be purified by any suitable method. Such methods include, but are not limited to, using an affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include ROR1 ECD and agents that bind to the Fc region. For example, protein A, protein G, protein A / G, or an antibody affinity column can be used to bind to the Fc region and purify a CD123-binding polypeptide that contains an 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., reverse phase / anion exchange, reverse phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) can also be suitable for purifying some polypeptides, such as antibodies. Many methods for purifying polypeptides are known in the art.

[0179] 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); Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0180] In some embodiments, a CD123-binding polypeptide prepared by the above methods is provided. In some embodiments, the CD123-binding polypeptide is prepared in a host cell. In some embodiments, the CD123-binding polypeptide is prepared in a cell-free system. In some embodiments, the CD123-binding polypeptide is purified. In some embodiments, a cell culture medium comprising a CD123-binding polypeptide is provided.

[0181] In some embodiments, a composition comprising an antibody prepared by the above methods is provided. In some embodiments, the composition comprises a CD123-binding polypeptide prepared in a host cell. In some embodiments, the composition comprises a CD123-binding polypeptide prepared in a cell-free system. In some embodiments, the composition comprises a purified CD123-binding polypeptide.

[0182] Exemplary methods of treating diseases using polypeptides that bind to CD123

[0183] In some embodiments, methods for treating a disease in an individual are provided, the methods comprising administering a polypeptide that binds CD123 or a cell that expresses a polypeptide that binds CD123. In some embodiments, methods for treating cancer in an individual are provided. In some embodiments, methods for treating CD123-expressing or CD123-positive cancer in an individual are provided. The methods comprise administering to the individual an effective amount of a polypeptide that binds CD123 provided herein or a cell that expresses a polypeptide that binds CD123. In some embodiments, the polypeptide that binds CD123 blocks the binding of CD123 to IL-3. In some embodiments, the polypeptide that binds CD123 is used to deliver a cytotoxic agent to cells that express CD123. In some such embodiments, the polypeptide that binds CD123 comprises a binding domain that binds to a cytotoxic T cell or NK cell. In some such embodiments, the binding domain binds CD3, T cell receptor (TCR) α, TCRβ, CD28, CD16, CD32A, CD64, CD89, NKp46 or NKG2D. In some embodiments, the binding domain can be a VHH domain or an antibody binding domain comprising a heavy chain variable region and a light chain variable region (such as VH / VL, scFv, Fab fragment, etc.).

[0184] In some embodiments, the polypeptide that binds CD123 is linked to a cytotoxic agent to form an immunoconjugate. A variety of cytotoxic agents for immunoconjugates are known in the art and include, but are not limited to, calicheamicin, auristatin, dolastatin, duocarmycin, maytansine, nostocarphin, enediyne antibiotics, esperamicin, pyrrolobenzodiazepine and enediyne antibiotics.

[0185] In some embodiments, the polypeptide that binds CD123 is a chimeric antigen receptor expressed on a cytotoxic cell (such as a T cell (CAR-T) or NK cell (CAR-NK)). Such treatment methods can be used in humans or animals. In some embodiments, methods for treating humans are provided.

[0186] Non-limiting exemplary cancers that can be treated with the CD123-binding polypeptides provided herein or cells expressing CD123-binding polypeptides include, but are not limited to, lymphoma; Hodgkin lymphoma; non-Hodgkin lymphoma; B-cell lymphoma; 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-cleaved cell NHL; bulky lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenström macroglobulinemia; acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myelogenous leukemia. In some embodiments, the cancer is a CD123-expressing (i.e., CD123-positive) cancer.

[0187] The CD123-binding polypeptides or cells expressing CD123-binding polypeptides can be administered to a subject as needed. The determination of the frequency of administration can be made by a person skilled in the art, such as an attending physician, taking into account the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, etc. In some embodiments, an effective dose of the CD123-binding polypeptide or cells expressing CD123-binding polypeptides is administered to the subject one or more times. In some embodiments, an effective dose of the CD123-binding polypeptide or cells expressing CD123-binding polypeptides is administered to the subject daily, semi-weekly, weekly, bi-weekly, monthly, etc. An effective dose of the CD123-binding polypeptide or cells expressing CD123-binding polypeptides is administered to the subject at least once. In some embodiments, an effective dose of the CD123-binding polypeptide or cells expressing CD123-binding polypeptides can be administered multiple times, including multiple administrations over a period of at least one month, at least six months, or at least one year.

[0188] 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 body weight of the subject being treated, his or her physical or health condition, the extent of the condition to be treated, or the age of the subject being treated. Generally, the antibody can be administered in an amount in the range of about 0.05 mg / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, the antibody can be administered in an amount in the range of about 10 μg / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, the antibody can be administered in an amount in the range of about 50 μg / kg body weight to about 5 mg / kg body weight per dose. In some embodiments, the antibody can be administered in an amount in the range of about 100 μg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody can be administered in an amount in the range of about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody can be administered in an amount in the range of about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody can be administered in an amount in the range of about 0.5 mg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody can be administered in an amount in the range of about 0.05 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody can be administered in an amount in the range of about 0.05 mg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody can be administered in an amount in the range of about 5 mg / kg body weight or less (such as less than 4, less than 3, less than 2, or less than 1 mg / kg).

[0189] In some embodiments, the CD123-binding polypeptide or the cell expressing the CD123-binding polypeptide can be administered in vivo by various routes, including but not limited to intravenous, intraarterial, parenteral, intraperitoneal, or subcutaneous. The appropriate formulation and route of administration can be selected according to the intended application.

[0190] In some embodiments, the therapeutic treatment using the CD123-binding polypeptide is achieved by targeting cytotoxic agents to cells expressing CD123, such as cancer cells expressing CD123. In some such embodiments, the CD123-binding polypeptide is a chimeric antigen receptor expressed on cytotoxic cells, such as T cells or NK cells.

[0191] Pharmaceutical compositions

[0192] In some embodiments, a composition comprising a CD123-binding polypeptide is provided in a formulation having a variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th Edition (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd Edition, Pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers can be used, including vehicles, adjuvants, and diluents. In addition, a variety of pharmaceutically acceptable auxiliary substances can also be used, such as pH regulators and buffers, tonicity regulators, stabilizers, wetting agents, etc. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0193] In some embodiments, the pharmaceutical composition comprises a CD123-binding polypeptide at the following concentrations: 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.

[0194] Combination therapies

[0195] The CD123-binding polypeptide or engineered cells can be administered alone or in combination with other treatment modalities (such as other anti-cancer agents). They can be provided before, substantially simultaneously with, or after (i.e., in parallel or sequentially) other treatment modalities. In some embodiments, the treatment methods described herein can further comprise administering: radiotherapy, chemotherapy, vaccination, targeted tumor therapy, CAR-T therapy, oncolytic virus therapy, cancer immunotherapy, cytokine therapy, surgical resection, chromatin modification, ablation, cryotherapy, antisense agents against tumor targets, siRNA agents against tumor targets, microRNA agents against tumor targets, or anti-cancer / anti-tumor agents, or biological agents (such as antibodies, cytokines, or receptor extracellular domain-Fc fusions).

[0196] In some embodiments, the CD123-binding polypeptides provided herein are administered in parallel with one or more chemotherapeutic agents, CAR-T (chimeric antigen receptor T cell) therapy, oncolytic virus therapy, cytokine therapy, and / or agents targeting other checkpoint molecules (such as VISTA, gpNMB, B7H4, HHLA2, CD73, CTLA4, TIGIT, etc.).

[0197] In some embodiments, the CD123-binding polypeptides or engineered cells of the present disclosure are combined with other anti-tumor agents that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, BCMA, PD-1, PDL1, PDL2, CTLA4, or one or more VEGF receptors, TRAIL / Apo2 (such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib , platelet-derived growth factor inhibitors (e.g., (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, CTLA4 inhibitors (e.g., anti-CTLA antibody ipilimumab ), 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, antagonists (e.g., neutralizing antibodies)), and other bioactive agents and organic chemical agents, etc.

[0198] In some embodiments, the CD123-binding polypeptides or engineered cells provided herein are administered in parallel with PD-1 / PD-L1 therapies. Examples of PD-1 / PD-L1 therapies include nivolumab (BMS); pidilizumab (CureTech, CT-011); pembrolizumab (Merck); durvalumab (Medimmune / AstraZeneca); atezolizumab (Genentech / Roche); avelumab (Pfizer); AMP-224 (Amplimmune); BMS-936559; AMP-514 (Amplimmune); MDX-1105 (Merck); TSR-042 (Tesaro / AnaptysBio, ANB-011); STI-A1010 (Sorrento Therapeutics); STI-A1110 (Sorrento Therapeutics); and other agents directed against programmed death-1 (PD-1) or programmed death ligand 1 (PD-L1).

[0199] In some embodiments, the CD123-binding polypeptides or engineered cells of the present disclosure can be used in combination with chemotherapeutic agents. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; polyacetyls (especially bullatacin and bullatacinone); camptothecin (including synthetic analogues such as topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1 (see, e.g., Agnew, Chem Intl.Ed.Engl., 33:183-186(1994)); dynemicin, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin D, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,. Doxorubicin (including morpholino-doxorubicin, cyano-morpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy-doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, streptozotocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine; androgens, such as carpronium, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folic acid; glucuronolactone; aldophosphamide glycoside; aminolevulinic acid; enuracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; colchicine amide; diaziquone; elfornithine; elisidepsin; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine alkaloids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-acetylhydrazine; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, OR); bis(2-chloroethyl)methylamine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichloroethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), the Cremophor-free albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, IL) and docetaxel ( -Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including oxaliplatin treatment regimens (FOLFOX); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib ) and VEGF-A; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0200] Other non-limiting exemplary chemotherapeutic agents include antihormonal agents that act to modulate or inhibit the hormonal effects on cancer, such as antiestrogens and selective estrogen receptor modulators (SERM), including for example tamoxifen (including tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal gland, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprorelin, and goserelin; and cytarabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit gene expression in signal transduction pathways involved in abnormal cell proliferation, such as PKC-α, Ralf, and H-Ras; ribozymes such as VEGF expression inhibitors (e.g., ribozymes) and HER2 expression inhibitors; vaccines such as gene therapy vaccines, e.g., vaccines, vaccines, and vaccines; (aldesleukin) rIL-2; topoisomerase 1 inhibitors; GnRH agonists; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0201] 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 cell and the additional agent are separate from each other, e.g., each formulated into a separate therapeutic composition, and the CD123-binding polypeptide or engineered cell and the additional agent are administered simultaneously, or the CD123-binding polypeptide or engineered cell and the additional agent are administered at different times during a treatment regimen. For example, the CD123-binding polypeptide or engineered cell is administered before the additional agent, the CD123-binding polypeptide or engineered cell is administered after the additional agent, or the CD123-binding polypeptide or engineered cell and the additional agent are administered in an alternating manner. The CD123-binding polypeptide and the additional agent may be administered as a single dose or as multiple doses.

[0202] In some embodiments, the CD123-binding polypeptide or engineered cell and the one or more additional agents are administered simultaneously. For example, the CD123-binding polypeptide and the one or more additional agents can be formulated as a single composition or administered as two or more separate compositions. In some embodiments, the CD123-binding polypeptide or engineered cell and the one or more additional agents are administered sequentially, or the CD123-binding polypeptide or engineered cell and the additional agent are administered at different times during a treatment regimen.

[0203] Non-limiting exemplary methods of diagnosis and treatment

[0204] In some embodiments, the methods described herein can be used to evaluate 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.

[0205] In some embodiments, the methods described herein include evaluating the presence, absence, or level of a protein. In some embodiments, the methods described herein include evaluating the presence, absence, or expression level 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 sample or cells cultured from a tumor with a therapeutic agent as described herein.

[0206] In some embodiments, the evaluation can guide treatment (including treatment with the antibodies described herein). In some embodiments, the evaluation can guide the use or discontinuation of adjuvant therapy after resection. Adjuvant therapy (also known as adjuvant care) is treatment given in addition to primary, main, or initial treatment. By way of non-limiting example, adjuvant therapy can be additional treatment typically given after surgery in which all detectable disease has been removed but there is still a statistical risk of recurrence due to occult disease. In some embodiments, the polypeptide is used as adjuvant therapy for treating cancer. In some embodiments, the polypeptide is used as the sole adjuvant therapy for treating cancer. In some embodiments, the polypeptides described herein are not used as adjuvant therapy for treating cancer. For example, if a patient is unlikely to respond or will have a minimal response to the antibodies described herein, treatment may not be administered in order to improve quality of life and avoid unnecessary toxicity from ineffective chemotherapy. In such cases, palliative care can be used.

[0207] In some embodiments, the polypeptide is administered as neoadjuvant therapy prior to resection. In some embodiments, neoadjuvant therapy refers to a therapy that shrinks and / or downstages a tumor prior to any surgery. In some embodiments, neoadjuvant therapy means chemotherapy administered to a cancer patient prior to surgery. In some embodiments, neoadjuvant therapy means an antibody administered to a cancer patient prior to surgery. Cancer types commonly considered for neoadjuvant chemotherapy include, for example, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, and lung cancer. In some embodiments, the polypeptide is used as neoadjuvant therapy for treating cancer. In some embodiments, it is used prior to resection.

[0208] In some embodiments, the tumor microenvironment contemplated in the methods described herein is one or more of the following: tumor blood vessels; 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 lymphocytes; neutrophils; and other immune cells located near the tumor.

[0209] Kits

[0210] Articles and kits are also provided, the articles and kits comprising any CD123-binding polypeptide as described herein and a suitable package. In some embodiments, the invention includes a kit having (i) a CD123-binding polypeptide and (ii) instructions for administering the CD123-binding polypeptide to an individual using the kit.

[0211] Suitable packages for the compositions described herein are known in the art and include, for example, vials (e.g., sealed vials), vessels, ampoules, bottles, jars, flexible packages (e.g., sealed Mylar or plastic bags), etc. These articles can be further sterilized and / or sealed. Unit dosage forms comprising the compositions described herein are also provided. These unit dosage forms can be stored in suitable packages in single or multiple unit doses and can also be further sterilized and sealed. The instructions provided in the kits of the invention are generally written instructions on a label or package insert (e.g., a sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. The instructions related to the use of the antibody generally include information on dosage, dosing schedule, and route of administration for the intended therapeutic or industrial use. The kit can further include a description of selecting a suitable individual or treatment.

[0212] The container can be a unit dose, a bulk package (e.g., a multi-dose package), or a sub-unit dose. For example, a kit containing a sufficient dose of the molecules disclosed herein can also be provided to provide effective treatment for an individual over an extended period of time, such as any one of about 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 longer. The kit can also include multiple unit doses of the molecule and instructions for use, and is packaged in an amount sufficient for storage and use in a pharmacy (e.g., a hospital pharmacy and a compounding pharmacy). In some embodiments, the kit includes a dry (e.g., lyophilized) composition that can be reconstituted, resuspended, or rehydrated to form an aqueous suspension of the antibody that is generally stable.

[0213] Example

[0214] The examples discussed below are intended solely as illustrations of the invention and should not be regarded as limiting the invention in any way. The examples are not intended to represent that the following experiments are all or the only experiments conducted. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are atmospheric or near atmospheric.

[0215] Example 1: CD123 single-domain antibody

[0216] Single-domain antibodies targeting human CD123 were generated by immunizing llamas and alpacas with a recombinant version of the extracellular domain of human CD123.

[0217] After generating specific anti-CD123 antibody titers, llama / alpaca peripheral blood mononuclear cells (PBMCs) were isolated from 500 mL of blood from the immunized animals, and total mRNA was isolated using the Qiagen RNeasy Maxi kit and subsequently converted to first-strand cDNA using the Thermo Superscript IV reverse transcriptase and oligo dT primers. The VHH sequences were specifically amplified via PCR using the cDNA as a template and cloned into a yeast surface display vector as a VHH-Fc-AGA2 fusion protein.

[0218] The yeast library displaying the VHH-Fc-AGA2 fusion protein was enriched via magnetic bead separation and then via fluorescence-activated cell sorting (FACS) using a recombinant form of CD123ECD. The sorted yeast were plated out, and the isolated colonies were picked into 96-well blocks and grown in a medium that converted the expression of surface-displayed VHH-Fc to secretion into the medium. The supernatant from the 96-well yeast secretion cultures was applied to 293F cells (CD123 positive) transiently transfected with CD123 or untransfected 293F cells (CD123 negative), washed, treated with a fluorophore-labeled anti-human IgG1Fc secondary antibody, and analyzed by 96-well flow cytometry.

[0219] The nucleic acid sequence encoding a VHH that binds to CD123-positive cells but not to CD123-negative cells was cloned in-frame with the human Fc-encoding region into a mammalian expression vector and expressed by transient transfection into HEK293Freestyle cells (293F cells) or CHO cells using polyethylenimine. The supernatant was collected 3 - 7 days later, the secreted recombinant protein was purified by protein A chromatography, and the concentration was calculated from the absorbance at 280 nm and the extinction coefficient.

[0220] The epitopes of single-domain antibodies (sdAbs) containing a VHH domain that binds CD123 were compared using biolayer interferometry. 5 μg / mL of histidine-tagged human CD123 was immobilized on a nickel-nitrilotriacetic acid (Ni-NTA)-coated capture sensor. Then 100 nM of one sdAb was loaded onto the CD123 antigen and allowed to reach equilibrium. The sensor was then transferred into 100 nM of a second sdAb. An increase in the measured signal indicates binding, showing that the second sdAb targets an epitope different from that of the first sdAb.

[0221] Camelid CD123 VHHs were humanized using the human VH3-23 germline as a scaffold. Camelid residues that contribute to solubility, specificity, stability, and / or affinity were left unmodified. Additionally, amino acid sequences with potential developability liabilities were modified where possible and as needed to mitigate this risk. Additionally, all humanized variants contain the Leu11Glu (L11E) modification as described in US20160207981.

[0222] The results showed that four distinct epitopes were found in the humanized versions of sdAb A5, F3, C5, 1B11, 1F5, and 4F2. As shown in Figure 1A, none of the tested sdAbs had the same epitope as the hzA5v2 sdAb. As shown in Figure 1B, sdAb C5 and hz1F5v1 had the same epitope as the hzF3v22 sdAb, which was different from the epitopes of sdAb hz1B11v28 and hz4F2v3. As shown in 1C, the hz4F2v3 sdAb had a different epitope from the hz1B11v28 sdAb. A summary of the results is shown in Table 3 below.

[0223] Table 3

[0224] sdAb Epitope bin hzA5v2 1 hzF3v22 2 C5 2 hz1F5v1 2 hz1B11v28 3 hz4F2v3 4

[0225] Example 2: Binding of Polypeptides to CD123

[0226] The binding of sdAbs to human CD123 was evaluated by flow cytometry. Each sdAb contained the VHH domain and the human IgG1 xELL Fc region as indicated in Table 4 below, in which amino acids Glu233, Leu234, and Leu235 (according to EU numbering) (SEQ ID NO:55) were deleted. HEK 293 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 to 2M, and untransfected HEK 293 cells were used as CD123-negative cells. In Figure 2N, Molm-13 cells expressing CD123 were used as the positive cell line. Each cell type was plated at 30,000 cells / well in FACS buffer (PBS 1% BSA, 0.1% NaN3 pH 7.4) in a 96-well round-bottom plate. The sdAbs were diluted in FACS buffer in a 3-fold 11-point serial dilution. The sdAb dilutions were added to the plated cells, and the assay plates were incubated at 4°C for 30 minutes. After washing twice in 150 μL of FACS buffer, the cells in each well were resuspended in 100 μL of a 1:2000 Alexa Fluor 647-conjugated anti-human IgG secondary dilution in FACS buffer and incubated at 4°C for 30 minutes. The cells were washed twice more, and then the bound antibodies were detected by flow cytometry.

[0227] Flow cytometry analysis was performed on an Intellicyte iQue Plus, and fluorescence was plotted as median fluorescence intensity. Apparent affinity (K d , nM) was determined using a one-site binding non-linear regression in PRISM graphing software.

[0228] As shown in FIGS. 2A to 2N, the tested sdAbs exhibited CD123 binding and did not bind to untransfected HEK 293 cells that do not express CD123. The apparent binding affinities are shown in Table 4 below.

[0229] Table 4

[0230] sdAb <![CDATA[Apparent K d (nM)]]> SEQ ID NO of the VHH domain A5-IgG1 0.12 2 hzA5v2-IgG1 0.11 26 F3-IgG1 0.53 18 hzF3v22-IgG1 0.59 29 hzF3v26-IgG1 0.93 30 1F5-IgG1 0.97 22 hz1F5v1-IgG1 0.58 31 hz1F5v2-IgG1 0.92 32 1B11-IgG1 0.52 6 hz1B11v28-IgG1 0.96 27 4F2-IgG1 1.37 14 hz4F2v3-IgG1 2.40 28 hz1F5v6-IgG1 1.36 92

[0231] Example 3: Inhibition of the binding of CD123 to IL-3

[0232] In the presence of a fixed amount of recombinant IL-3-mFc, the ability of certain CD123-binding sdAbs to block the binding of CD123 to IL-3 was tested by titrating the sdAbs onto HEK-293 cells expressing CD123. Bound IL-3 was detected by flow cytometry using a fluorophore-conjugated anti-mouse IgG specific secondary antibody.

[0233] The results of this experiment are shown in FIG. 3. sdAbs A5, C5, and F3 were all able to block the binding of CD123 to IL-3 (FIG. 3A). hz1F5v1 and the anti-CD123 antibody analogue CSL362 (see US Publication No. 2013 / 0137855) were also able to block the binding of CD123 to IL-3 (FIG. 3B).

[0234] Without departing from the spirit or essential characteristics of the present disclosure, the present disclosure may be implemented in other specific forms. Accordingly, the foregoing embodiments are considered illustrative in all respects and not restrictive of the present disclosure. Thus, the scope of the present disclosure is indicated by the appended claims rather than by the foregoing description, and all changes within the meaning and range of equivalents of the claims are intended to be included herein.

[0235] List of certain sequences

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242] Sequence Listing <110> Inhibio Co., Ltd. <120> Polypeptides Binding to CD123 and Their Uses <130> 01202-0021-00PCT <150> US 62 / 843,407 <151> May 4, 2019 <160> 95 <170> PatentIn version 3.5 <210> 1 <211> 360 <212> PRT <213> Homo sapiens <400> 1 Thr Lys Glu Asp Pro Asn Pro Pro Ile Thr Asn Leu Arg Met Lys Ala 1 5 10 15 Lys Ala Gln Gln Leu Thr Trp Asp Leu Asn Arg Asn Val Thr Asp Ile 20 25 30 Glu Cys Val Lys Asp Ala Asp Tyr Ser Met Pro Ala Val Asn Asn Ser 35 40 45 Tyr Cys Gln Phe Gly Ala Ile Ser Leu Cys Glu Val Thr Asn Tyr Thr 50 55 60 Val Arg Val Ala Asn Pro Pro Phe Ser Thr Trp Ile Leu Phe Pro Glu 65 70 75 80 Asn Ser Gly Lys Pro Trp Ala Gly Ala Glu Asn Leu Thr Cys Trp Ile 85 90 95 His Asp Val Asp Phe Leu Ser Cys Ser Trp Ala Val Gly Pro Gly Ala 100 105 110 Pro Ala Asp Val Gln Tyr Asp Leu Tyr Leu Asn Val Ala Asn Arg Arg 115 120 125 Gln Gln Tyr Glu Cys Leu His Tyr Lys Thr Asp Ala Gln Gly Thr Arg 130 135 140 Ile Gly Cys Arg Phe Asp Asp Ile Ser Arg Leu Ser Ser Gly Ser Gln 145 150 155 160 Ser Ser His Ile Leu Val Arg Gly Arg Ser Ala Ala Phe Gly Ile Pro 165 170 175 Cys Thr Asp Lys Phe Val Val Phe Ser Gln Ile Glu Ile Leu Thr Pro 180 185 190 Pro Asn Met Thr Ala Lys Cys Asn Lys Thr His Ser Phe Met His Trp 195 200 205 Lys Met Arg Ser His Phe Asn Arg Lys Phe Arg Tyr Glu Leu Gln Ile 210 215 220 Gln Lys Arg Met Gln Pro Val Ile Thr Glu Gln Val Arg Asp Arg Thr 225 230 235 240 Ser Phe Gln Leu Leu Asn Pro Gly Thr Tyr Thr Val Gln Ile Arg Ala 245 250 255 Arg Glu Arg Val Tyr Glu Phe Leu Ser Ala Trp Ser Thr Pro Gln Arg 260 265 270 Phe Glu Cys Asp Gln Glu Glu Gly Ala Asn Thr Arg Ala Trp Arg Thr 275 280 285 Ser Leu Leu Ile Ala Leu Gly Thr Leu Leu Ala Leu Val Cys Val Phe 290 295 300 Val Ile Cys Arg Arg Tyr Leu Val Met Gln Arg Leu Phe Pro Arg Ile 305 310 315 320 Pro His Met Lys Asp Pro Ile Gly Asp Ser Phe Gln Asn Asp Lys Leu 325 330 335 Val Val Trp Glu Ala Gly Lys Ala Gly Leu Glu Glu Cys Leu Val Thr 340 345 350 Glu Val Gln Val Val Gln Lys Thr 355 360 <210> 2 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> A5 <400> 2 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ala Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Ser Asn Ser Trp Ile Ala Gly Ser Thr Tyr Tyr Ala Gly Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Leu Leu Ala Thr Ala Asp Asp Glu Tyr Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val 115 <210> 3 <211> 10 <212> PRT <213> Artificial sequence <220> <223> A5 CDR1 <400> 3 Gly Gly Thr Phe Ser Ser Tyr Gly Met Ala 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial sequence <220> <223> A5 CDR2 <400> 4 Ser Asn Ser Trp Ile Ala Gly Ser Thr Tyr 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> A5 CDR3 <400> 5 Asp Leu Leu Ala Thr Ala Asp Asp Glu Tyr Asp Tyr 1 5 10 <210> 6 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 1B11 <400> 6 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ala Gly Arg Thr Gln Ser Ala Val 20 25 30 Ala Met Gly Trp Phe Arg Gln Asp Pro Gly Lys Asp Arg Asp Phe Val 35 40 45 Ala Ala Ile Arg Trp Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Ala 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Ser Cys 85 90 95 Ala Ile Ser Met Asn His Phe Gly Met Tyr Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val 115 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 1B11 CDR1 <400> 7 Gly Arg Thr Gln Ser Ala Val Ala Met Gly 1 5 10 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 1B11 CDR2 <400> 8 Ala Ile Arg Trp Ser Gly Gly Asn Thr Tyr 1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 1B11 CDR3 <400> 9 Ser Met Asn His Phe Gly Met Tyr Asp Tyr 1 5 10 <210> 10 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> C5 <400> 10 Gln Val Thr Leu Arg Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Gly Ser Gly Arg Ala Ile Asn Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Asn Gly Gly His Thr Arg Tyr Ala Asp Ser Val 50 55 60 Gln Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Asp Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Ala Tyr Ser Asp Tyr His Arg Ile Ala Thr Met Glu Ala Asp Ala 100 105 110 Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> C5 CDR1 <400> 11 Gly Arg Ala Ile Asn Thr Tyr Ala Met Gly 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial sequence <220> <223> C5 CDR2 <400> 12 Ala Ile Ser Trp Asn Gly Gly His Thr Arg 1 5 10 <210> 13 <211> 16 <212> PRT <213> Artificial sequence <220> <223> C5 CDR3 <400> 13 Tyr Ser Asp Tyr His Arg Ile Ala Thr Met Glu Ala Asp Ala Asp Ser 1 5 10 15 <210> 14 <211> 120 <212> PRT <213> Artificial sequence <220> <223> 4F2 <400> 14 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Val Ser Asn Tyr 20 25 30 Pro Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala His Ile Ser Trp Ser Gly Ile Thr Ser Ile Leu Asn Ser Val Asn 50 55 60 Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Ala Gln Arg Pro Thr Ala Gly Pro Lys Gly Pro Phe Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val 115 120 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 4F2 CDR1 <400> 15 Gly Arg Thr Val Ser Asn Tyr Pro Met Ala 1 5 10 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 4F2 CDR2 <400> 16 His Ile Ser Trp Ser Gly Ile Thr Ser 1 5 <210> 17 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> 4F2 CDR3 <400> 17 Ala Gln Arg Pro Thr Ala Gly Pro Lys Gly Pro Phe Gly Tyr 1 5 10 <210> 18 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> F3 <400> 18 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Arg Ala Ser Gly Arg Ala Ile Asn Ser Tyr 20 25 30 Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Asn Gly Ala Arg Thr Tyr Tyr Gln Asp Ala Leu 50 55 60 Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Arg Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Arg Trp Ser Ala Ala Val Pro Ser Gly Glu Asp Gln 100 105 110 Tyr Asn Phe Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 <210> 19 <211> 10 <212> PRT <213> Artificial sequence <220> <223> F3 CDR1 <400> 19 Gly Arg Ala Ile Asn Ser Tyr Asn Met Gly 1 5 10 <210> 20 <211> 10 <212> PRT <213> Artificial sequence <220> <223> F3 CDR2 <400> 20 Ala Ile Asn Trp Asn Gly Ala Arg Thr Tyr 1 5 10 <210> 21 <211> 17 <212> PRT <213> Artificial sequence <220> <223> F3 CDR3 <400> 21 Ala Gly Arg Trp Ser Ala Ala Val Pro Ser Gly Glu Asp Gln Tyr Asn 1 5 10 15 Phe <210> 22 <211> 123 <212> PRT <213> Artificial sequence <220> <223> 1F5 <400> 22 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Thr Val Ser Cys Thr Ala Ser Gly Arg Ala Ile Asn Met Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Asn Trp Asn Gly Ala Tyr Thr Gln Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Gln Tyr Tyr Cys 85 90 95 Ser Ala Asp Ala Asp Tyr Asn Thr Tyr Val Ser Pro Asn Lys Arg Val 100 105 110 Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr Val 115 120 <210> 23 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 1F5 CDR1 <400> 23 Gly Arg Ala Ile Asn Met Tyr Ala Met Gly 1 5 10 <210> 24 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> 1F5 CDR2 <400> 24 Ala Ile Asn Trp Asn Gly Ala Tyr Thr Gln 1 5 10 <210> 25 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> 1F5 CDR3 <400> 25 Asp Ala Asp Tyr Asn Thr Tyr Val Ser Pro Asn Lys Arg Val Ser Tyr 1 5 10 15 <210> 26 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> hzA5v2 <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Ser Asn Ser Trp Ile Ala Gly Ser Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Leu Leu Ala Thr Ala Asp Asp Glu Tyr Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val 115 <210> 27 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> hz1B11v28 <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Gln Ser Ala Val 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Asp Phe Val 35 40 45 Ala Ala Ile Arg Trp Ser Gly Gly Asn Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Ser Cys 85 90 95 Ala Ile Ser Leu Asn His Phe Gly Leu Tyr Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val 115 <210> 28 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> hz4F2v3 <400> 28 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Val Ser Asn Tyr 20 25 30 Pro Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala His Ile Ser Trp Ser Gly Ile Thr Ser Ile Leu Asn Ser Val Asn 50 55 60 Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Ala Gln Arg Pro Thr Ala Gly Pro Lys Gly Pro Phe Gly Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val 115 120 <210> 29 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> hzF3v22 <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Ile Asn Ala Tyr 20 25 30 Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Asn Ala Ala Arg Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Gly Arg Trp Ser Ala Ala Val Pro Ser Gly Glu Asp Gln 100 105 110 Tyr Asn Phe Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 <210> 30 <211> 124 <212> PRT <213> Artificial sequence <220> <223> hzF3v26 <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Arg Ala Ser Gly Arg Ala Ile Asn Ala Tyr 20 25 30 Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Asn Ala Ala Arg Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ser Gly Arg Trp Ser Ala Ala Val Pro Ser Gly Glu Asp Gln 100 105 110 Tyr Asn Phe Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 <210> 31 <211> 123 <212> PRT <213> Artificial sequence <220> <223> hz1F5v1 <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Ile Asn Met Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Asn Trp Asn Gly Ala Tyr Thr Gln Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Ala Asp Ala Asp Tyr Asn Thr Tyr Val Ser Pro Asn Lys Arg Val 100 105 110 Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 <210> 32 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> hz1F5v2 <400> 32 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Ala Ile Asn Met Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Asn Trp Asn Gly Ala Tyr Thr Gln Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Ala Asp Ala Asp Tyr Asn Thr Tyr Val Ser Pro Asn Lys Arg Val 100 105 110 Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 115 120 <210> 33 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of hzA5v2 <400> 33 Gly Gly Thr Phe Ser Ser Tyr Gly Met Ala 1 5 10 <210> 34 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR2 of HzA5v2 <400> 34 Ser Asn Ser Trp Ile Ala Gly Ser Thr Tyr 1 5 10 <210> 35 <211> 12 <212> PRT <213> Artificial sequence <220> <223> CDR3 of hzA5v2 <400> 35 Asp Leu Leu Ala Thr Ala Asp Asp Glu Tyr Asp Tyr 1 5 10 <210> 36 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR1 of hz1B11v28 <400> 36 Gly Arg Thr Gln Ser Ala Val Ala Met Gly 1 5 10 <210> 37 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR2 of hz1B11v28 <400> 37 Ala Ile Arg Trp Ser Gly Gly Asn Thr Tyr 1 5 10 <210> 38 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR3 of hz1B11v28 <400> 38 Ser Leu Asn His Phe Gly Leu Tyr Asp Tyr 1 5 10 <210> 39 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR1 of hz4F2v3 <400> 39 Gly Arg Thr Val Ser Asn Tyr Pro Met Ala 1 5 10 <210> 40 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR2 of hz4F2v3 <400> 40 His Ile Ser Trp Ser Gly Ile Thr Ser 1 5 <210> 41 <211> 14 <212> PRT <213> Artificial sequence <220> <223> CDR3 of hz4F2v3 <400> 41 Ala Gln Arg Pro Thr Ala Gly Pro Lys Gly Pro Phe Gly Tyr 1 5 10 <210> 42 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR1 of hzF3v22 <400> 42 Gly Arg Ala Ile Asn Ala Tyr Asn Met Gly 1 5 10 <210> 43 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of hzF3v22 <400> 43 Ala Ile Asn Trp Asn Ala Ala Arg Thr Tyr 1 5 10 <210> 44 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of hzF3v22 <400> 44 Ser Gly Arg Trp Ser Ala Ala Val Pro Ser Gly Glu Asp Gln Tyr Asn 1 5 10 15 Phe <210> 45 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of hzF3v26 <400> 45 Gly Arg Ala Ile Asn Ala Tyr Asn Met Gly 1 5 10 <210> 46 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of HzF3v26 <400> 46 Ala Ile Asn Trp Asn Ala Ala Arg Thr Tyr 1 5 10 <210> 47 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDR3 of hzF3v26 <400> 47 Ser Gly Arg Trp Ser Ala Ala Val Pro Ser Gly Glu Asp Gln Tyr Asn 1 5 10 15 Phe <210> 48 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR1 of hz1F5v1 <400> 48 Gly Arg Ala Ile Asn Met Tyr Ala Met Gly 1 5 10 <210> 49 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR2 of hz1F5v1 <400> 49 Ala Ile Asn Trp Asn Gly Ala Tyr Thr Gln 1 5 10 <210> 50 <211> 16 <212> PRT <213> Artificial sequence <220> <223> CDR3 of hz1F5v1 <400> 50 Asp Ala Asp Tyr Asn Thr Tyr Val Ser Pro Asn Lys Arg Val Ser Tyr 1 5 10 15 <210> 51 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR1 of hz1F5v2 <400> 51 Gly Arg Ala Ile Asn Met Tyr Ala Met Gly 1 5 10 <210> 52 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR2 of hz1F5v2 <400> 52 Ala Ile Asn Trp Asn Gly Ala Tyr Thr Gln 1 5 10 <210> 53 <211> 16 <212> PRT <213> Artificial sequence <220> <223> CDR3 of hz1F5v2 <400> 53 Asp Ala Asp Tyr Asn Thr Tyr Val Ser Pro Asn Lys Arg Val Ser Tyr 1 5 10 15 <210> 54 <211> 227 <212> PRT <213> Homo sapiens <400> 54 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 55 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> Human IgG1 xELL Fc Region <400> 55 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 56 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> M252Y and M428V (YV) S354C T366W club-shaped structure in the Fc region <400> 56 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 57 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Fc region M252Y, M428V, H435R (YVR) T366S, L368A, Y407V Mortar structure <400> 57 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val 195 200 205 His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 58 <211> 224 <212> PRT <213> Artificial sequence <220> <223> Fc region xELL H435R <400> 58 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200 205 Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 59 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> Fc region xELL M252Y and M428V (YV) <400> 59 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 60 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> Fc region xELL M252Y and M428L (YL) <400> 60 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 61 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> Fc region xELL M252Y, M428L, H435R (YLR) <400> 61 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala 195 200 205 Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 62 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> Fc region xELL M252Y, M428V, H435R (YVR) <400> 62 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val His Glu Ala 195 200 205 Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 63 <211> 224 <212> PRT <213> Artificial sequence <220> <223> Fc region xELL S354C T366W club-shaped structure <400> 63 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 64 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> Fc region xELL H435R S354C T366W club-shaped structure <400> 64 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200 205 Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 65 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> Fc region xELL M252Y and M428V (YV) S354C T366W club-shaped structure <400> 65 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 66 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> Fc region xELL M252Y and M428L (YL) S354C T366W club-shaped structure <400> 66 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 67 <211> 224 <212> PRT <213> Synthetic sequence <220> <223> Fc region xELL M252Y, M428L, H435R (YLR) S354C T366W club-shaped structure <400> 67 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala 195 200 205 Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 68 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> Fc region xELL M252Y, M428V, H435R (YVR) S354C T366W club-shaped structure <400> 68 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val His Glu Ala 195 200 205 Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 69 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> Fc region xELL T366S, L368A, Y407V socket-like structure <400> 69 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 130 135 140 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 70 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> Fc region xELL H435R, T366S, L368A, Y407V cagelike structure <400> 70 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 130 135 140 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200 205 Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 71 <211> 224 <212> PRT <213> 人工序列 <220> <223> Fc区xELL M252Y和M428V (YV) T366S, L368A, Y407V臼状结构 <400> 71 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 130 135 140 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 72 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> Fc region xELL M252Y and M428L (YL) T366S, L368A, Y407V socket-like structure <400> 72 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 130 135 140 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 73 <211> 224 <212> PRT <213> Artificial sequence <220> <223> Fc region xELL M252Y, M428L, H435R (YLR) T366S, L368A, Y407V Mortar structure <400> 73 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 130 135 140 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala 195 200 205 Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 74 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> Fc region xELL M252Y, M428V, H435R (YVR) T366S, L368A, Y407V Mortar structure <400> 74 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 35 40 45 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 115 120 125 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 130 135 140 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val His Glu Ala 195 200 205 Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 220 <210> 75 <211> 227 <212> PRT <213> Artificial sequence <220> <223> Fc region H435R <400> 75 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 76 <211> 227 <212> PRT <213> Artificial sequence <220> <223> Fc region M252Y and M428V (YV) <400> 76 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 77 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Fc region M252Y and M428L (YL) <400> 77 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 78 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Fc region M252Y, M428L, H435R (YLR) <400> 78 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 195 200 205 His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 79 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Fc region M252Y, M428V, H435R (YVR) <400> 79 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val 195 200 205 His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 80 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Fc region S354C T366W club-shaped structure <400> 80 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 81 <211> 227 <212> PRT <213> Artificial sequence <220> <223> Fc region H435R S354C T366W club-shaped structure <400> 81 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 82 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Fc region M252Y and M428L (YL) S354C T366W club-shaped structure <400> 82 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 83 <211> 227 <212> PRT <213> Artificial sequence <220> <223> Fc region M252Y, M428L, H435R (YLR) S354C T366W club-shaped structure <400> 83 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 195 200 205 His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 84 <211> 227 <212> PRT <213> Artificial sequence <220> <223> Fc region M252Y, M428V, H435R (YVR) S354C T366W club-shaped structure <400> 84 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val 195 200 205 His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 85 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Fc region T366S, L368A, Y407V socket-like structure <400> 85 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 86 <211> 227 <212> PRT <213> Artificial sequence <220> <223> Fc region H435R, T366S, L368A, Y407V socket-like structure <400> 86 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 87 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Fc区M252Y和M428V (YV) T366S, L368A, Y407V臼状结构 <400> 87 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Val 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 88 <211> 227 <212> PRT <213> Synthetic Sequence <220> <223> Fc region M252Y and M428L (YL) T366S, L368A, Y407V socket-like structure <400> 88 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 89 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Fc region M252Y, M428L, H435R (YLR) T366S, L368A, Y407V Mortar structure <400> 89 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 195 200 205 His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 90 <211> 287 <212> PRT <213> Artificial Sequence <220> <223> CD123 ECD <400> 90 Thr Lys Glu Asp Pro Asn Pro Pro Ile Thr Asn Leu Arg Met Lys Ala 1 5 10 15 Lys Ala Gln Gln Leu Thr Trp Asp Leu Asn Arg Asn Val Thr Asp Ile 20 25 30 Glu Cys Val Lys Asp Ala Asp Tyr Ser Met Pro Ala Val Asn Asn Ser 35 40 45 Tyr Cys Gln Phe Gly Ala Ile Ser Leu Cys Glu Val Thr Asn Tyr Thr 50 55 60 Val Arg Val Ala Asn Pro Pro Phe Ser Thr Trp Ile Leu Phe Pro Glu 65 70 75 80 Asn Ser Gly Lys Pro Trp Ala Gly Ala Glu Asn Leu Thr Cys Trp Ile 85 90 95 His Asp Val Asp Phe Leu Ser Cys Ser Trp Ala Val Gly Pro Gly Ala 100 105 110 Pro Ala Asp Val Gln Tyr Asp Leu Tyr Leu Asn Val Ala Asn Arg Arg 115 120 125 Gln Gln Tyr Glu Cys Leu His Tyr Lys Thr Asp Ala Gln Gly Thr Arg 130 135 140 Ile Gly Cys Arg Phe Asp Asp Ile Ser Arg Leu Ser Ser Gly Ser Gln 145 150 155 160 Ser Ser His Ile Leu Val Arg Gly Arg Ser Ala Ala Phe Gly Ile Pro 165 170 175 Cys Thr Asp Lys Phe Val Val Phe Ser Gln Ile Glu Ile Leu Thr Pro 180 185 190 Pro Asn Met Thr Ala Lys Cys Asn Lys Thr His Ser Phe Met His Trp 195 200 205 Lys Met Arg Ser His Phe Asn Arg Lys Phe Arg Tyr Glu Leu Gln Ile 210 215 220 Gln Lys Arg Met Gln Pro Val Ile Thr Glu Gln Val Arg Asp Arg Thr 225 230 235 240 Ser Phe Gln Leu Leu Asn Pro Gly Thr Tyr Thr Val Gln Ile Arg Ala 245 250 255 Arg Glu Arg Val Tyr Glu Phe Leu Ser Ala Trp Ser Thr Pro Gln Arg 260 265 270 Phe Glu Cys Asp Gln Glu Glu Gly Ala Asn Thr Arg Ala Trp Arg 275 280 285 <210> 91 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> His-tagged CD123 ECD <400> 91 Thr Lys Glu Asp Pro Asn Pro Pro Ile Thr Asn Leu Arg Met Lys Ala 1 5 10 15 Lys Ala Gln Gln Leu Thr Trp Asp Leu Asn Arg Asn Val Thr Asp Ile 20 25 30 Glu Cys Val Lys Asp Ala Asp Tyr Ser Met Pro Ala Val Asn Asn Ser 35 40 45 Tyr Cys Gln Phe Gly Ala Ile Ser Leu Cys Glu Val Thr Asn Tyr Thr 50 55 60 Val Arg Val Ala Asn Pro Pro Phe Ser Thr Trp Ile Leu Phe Pro Glu 65 70 75 80 Asn Ser Gly Lys Pro Trp Ala Gly Ala Glu Asn Leu Thr Cys Trp Ile 85 90 95 His Asp Val Asp Phe Leu Ser Cys Ser Trp Ala Val Gly Pro Gly Ala 100 105 110 Pro Ala Asp Val Gln Tyr Asp Leu Tyr Leu Asn Val Ala Asn Arg Arg 115 120 125 Gln Gln Tyr Glu Cys Leu His Tyr Lys Thr Asp Ala Gln Gly Thr Arg 130 135 140 Ile Gly Cys Arg Phe Asp Asp Ile Ser Arg Leu Ser Ser Gly Ser Gln 145 150 155 160 Ser Ser His Ile Leu Val Arg Gly Arg Ser Ala Ala Phe Gly Ile Pro 165 170 175 Cys Thr Asp Lys Phe Val Val Phe Ser Gln Ile Glu Ile Leu Thr Pro 180 185 190 Pro Asn Met Thr Ala Lys Cys Asn Lys Thr His Ser Phe Met His Trp 195 200 205 Lys Met Arg Ser His Phe Asn Arg Lys Phe Arg Tyr Glu Leu Gln Ile 210 215 220 Gln Lys Arg Met Gln Pro Val Ile Thr Glu Gln Val Arg Asp Arg Thr 225 230 235 240 Ser Phe Gln Leu Leu Asn Pro Gly Thr Tyr Thr Val Gln Ile Arg Ala 245 250 255 Arg Glu Arg Val Tyr Glu Phe Leu Ser Ala Trp Ser Thr Pro Gln Arg 260 265 270 Phe Glu Cys Asp Gln Glu Glu Gly Ala Asn Thr Arg Ala Trp Arg Gly 275 280 285 Gly Ser Gly Gly Ser His His His His His His His His His His 290 295 300 <210> 92 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> hz1F5v6 <400> 92 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Glu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Ile Asn Met Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Asn Trp Asn Ala Ala Tyr Thr Gln Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Ala Asp Ala Asp Tyr Asn Thr Tyr Val Ser Pro Asn Lys Arg Val 100 105 110 Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Lys Pro 115 120 125 <210> 93 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of hz1F5v6 <400> 93 Gly Arg Ala Ile Asn Met Tyr Ala Met Gly 1 5 10 <210> 94 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of hz1F5v6 <400> 94 Ala Ile Asn Trp Asn Ala Ala Tyr Thr Gln 1 5 10 <210> 95 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of hz1F5v6 <400> 95 Asp Ala Asp Tyr Asn Thr Tyr Val Ser Pro Asn Lys Arg Val Ser Tyr 1 5 10 15

Claims

1. A VHH domain that binds to CD123, wherein the VHH domain comprises CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are shown as SEQ ID NO: 23, 24, and 25, respectively; or 23, 94, and 25.

2. The VHH domain according to claim 1, wherein the VHH domain is humanized.

3. The VHH domain according to claim 1, wherein the VHH domain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 32, 31, or 92.

4. The VHH domain according to claim 1, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 32, 31, or 92.

5. The VHH domain according to claim 1, wherein the VHH domain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:

22.

6. The VHH domain according to claim 1, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO:

22.

7. The VHH domain according to claim 1, wherein the CD123 is human CD123, which has the sequence of SEQ ID NO:

1.

8. An isolated nucleic acid encoding the VHH domain according to any one of claims 1-7.

9. A vector comprising the nucleic acid according to claim 8.

10. A host cell comprising the nucleic acid according to claim 8 or the vector according to claim 9.

11. A host cell that expresses the VHH domain according to any one of claims 1-7.

12. The host cell according to claim 11, wherein the host cell is a natural killer (NK) cell or a cytotoxic T lymphocyte (CTL).

13. The host cell according to claim 12, wherein the host cell is a CD4 and / or CD8 T cell.

14. Use of the VHH domain according to any one of claims 1-7 in the preparation of a medicament for the treatment of blood cancer, wherein the blood cancer is a cancer that expresses CD123.

15. The use according to claim 14, wherein the blood cancer is selected from chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; and chronic myelogenous leukemia.

16. The use according to claim 14, wherein the cancer is acute myeloid leukemia (AML).

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