CLEC12a-linked polypeptide and its uses

Abstract

A VHH-containing polypeptide that binds to CLEC12a is provided herein. Additionally, uses of the VHH-containing polypeptide are also provided.

Description

Technology Field

[0001] Cross-reference of related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 843,411 filed on May 4, 2019 and U.S. Provisional Application No. 62 / 844,426 filed on May 7, 2019, the full text of each of which is incorporated herein by reference for any purpose.

[0003] Technology field

[0004] The present invention relates to a CLEC12a-binding polypeptide and a method of using the CLEC12a-binding polypeptide to regulate the biological activity of CLEC12a. Such methods include, but are not limited to, methods for treating cancer. Background Technology

[0005] CLEC12a, also known as CLL-1, DCAL-2, MICL, CD371, or C-lectin domain family 12 member A, is a member of the C-lectin / C-lectin-like domain (CTL / CTLD) superfamily that regulates myeloid cell activation. Furthermore, CLEC12a is a marker for blast cells and leukemia stem cells in patients with acute myeloid leukemia (AML). Additionally, CLEC12a has been used to detect minimal residual disease in patients following AML treatment. Therefore, there is a therapeutic need for more potent agents for cancers expressing CLEC12a.

[0006] outline

[0007] For example, a CLEC12a binding polypeptide and a method of using the CLEC12a binding polypeptide for treating leukemia are provided herein. In some embodiments, the CLEC12a binding polypeptide comprises at least one VHH domain. Some embodiments below are provided.

[0008] Embodiment 1. A polypeptide comprising at least one VHH domain that binds to CLEC12a, comprising: CDR1 having the amino acid sequence of SEQ ID NO. 47, 32, 3, 7, 11, 15, 19, 23, 35, 38, 41, or 44; CDR2 having the amino acid sequence of SEQ ID NO. 48, 33, 4, 8, 12, 16, 20, 24, 36, 39, 42, or 45; and CDR3 having the amino acid sequence of SEQ ID NO. 49, 34, 5, 9, 13, 17, 21, 25, 37, 40, 43, or 46.

[0009] Embodiment 2. A polypeptide according to Embodiment 1, wherein at least one VHH domain comprises a CDR1 having the amino acid sequence of SEQ ID NO. 47 or 23; a CDR2 having the amino acid sequence of SEQ ID NO. 48 or 24; and a CDR3 having the amino acid sequence of SEQ ID NO. 49 or 25.

[0010] Embodiment 3. A polypeptide according to Embodiment 1 or 2, wherein at least one VHH domain comprises a CDR1 having the amino acid sequence of SEQ ID NO. 32 or 3; a CDR2 having the amino acid sequence of SEQ ID NO. 33 or 4; and a CDR3 having the amino acid sequence of SEQ ID NO. 34 or 5.

[0011] Embodiment 4. A polypeptide in any one of embodiments 1 to 3, wherein at least one VHH domain comprises CDR1 having the amino acid sequence of SEQ ID NO. 7 or 35; CDR2 having the amino acid sequence of SEQ ID NO. 8 or 36; and CDR3 having the amino acid sequence of SEQ ID NO. 9 or 37.

[0012] Embodiment 5. A polypeptide in any one of embodiments 1 to 4, wherein at least one VHH domain comprises a CDR1 having an amino acid sequence of SEQ ID NO. 11 or 38; a CDR2 having an amino acid sequence of SEQ ID NO. 12 or 39; and a CDR3 having an amino acid sequence of SEQ ID NO. 13 or 40.

[0013] Embodiment 6. A polypeptide in any one of embodiments 1 to 5, wherein at least one VHH domain comprises a CDR1 having an amino acid sequence of SEQ ID NO. 15 or 41; a CDR2 having an amino acid sequence of SEQ ID NO. 16 or 42; and a CDR3 having an amino acid sequence of SEQ ID NO. 17 or 43.

[0014] Embodiment 7. A polypeptide in any one of embodiments 1 to 6, wherein at least one VHH domain comprises a CDR1 having the amino acid sequence of SEQ ID NO. 19 or 44; a CDR2 having the amino acid sequence of SEQ ID NO. 20 or 45; and a CDR3 having the amino acid sequence of SEQ ID NO. 21 or 46.

[0015] Embodiment 8. A polypeptide in any one of embodiments 1 to 7, wherein at least one VHH domain comprises CDR1, CDR2, and CDR3, each comprising the amino acid sequences of SEQ ID NOs 32, 33, and 34; SEQ ID NOs 47, 48, and 49; 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 35, 36, and 37; SEQ ID NOs 38, 39, and 40; SEQ ID NOs 41, 42, and 43; and SEQ ID NOs 44, 45, and 46.

[0016] Embodiment 9. A polypeptide in which, in any one of embodiments 1 to 8, at least one VHH domain is humanized.

[0017] Embodiment 10. A polypeptide in any one of embodiments 1 to 9, 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. 26, 94, 31, 99, 27, 28, 29, 30, 95, 96, 97, or 98.

[0018] Embodiment 11. A polypeptide in any one of embodiments 1 to 9, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO. 26, 94, 31, 99, 27, 28, 29, 30, 95, 96, 97, or 98.

[0019] Embodiment 12. A polypeptide in any one of embodiments 1 to 8, wherein at least one VHH domain comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NOs 2, 6, 10, 14, 18, 22, 88, 89, 90, 91, 92, or 93.

[0020] Example 13. A polypeptide in any one of Examples 1 to 8, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NOs 2, 6, 10, 14, 18, 22, 88, 89, 90, 91, 92, or 93.

[0021] Embodiment 14. A polypeptide comprising two VHH domains in any one of embodiments 1 to 13.

[0022] Embodiment 15. A polypeptide comprising three VHH domains in any one of embodiments 1 to 13.

[0023] Embodiment 16. A polypeptide in any one of embodiments 1 to 15, wherein the polypeptide comprises at least one binding domain that binds to an antigen other than CLEC12a.

[0024] Example 17. The polypeptide of Example 16, wherein the polypeptide comprises at least one binding domain that binds to CD3, T cell receptor (TCR)α, TCRβ, CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D.

[0025] Embodiment 18. A polypeptide according to Embodiment 14 or 15, wherein each VHH domain is bound to CLEC12a.

[0026] Embodiment 19. A polypeptide according to Embodiment 18, wherein each VHH domain comprises the same CDR1, CDR2, and CDR3 amino acid sequence.

[0027] Embodiment 20. A polypeptide according to Embodiment 18, wherein each VHH domain comprises the same VHH sequence.

[0028] Embodiment 21. A polypeptide comprising one VHH domain in any one of embodiments 1 to 13.

[0029] Embodiment 22. A polypeptide in any one of embodiments 1 to 21, wherein the polypeptide comprises an Fc region.

[0030] Embodiment 23. A polypeptide according to Embodiment 22, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NOs 50 to 85.

[0031] Example 24. A polypeptide that forms a dimer under physiological conditions, in the manner of Example 22 or 23.

[0032] Embodiment 25. A polypeptide in any one of embodiments 1 to 24, wherein the CLEC12a is human CLEC12a.

[0033] Embodiment 26. A polypeptide according to Embodiment 25, wherein the human CLEC12a comprises the sequence of SEQ ID NO. 1.

[0034] Example 27. An immunoconjugate comprising a polypeptide of any one of Examples 1 to 26 and a cytotoxic agent.

[0035] Example 28. An immunoconjugate according to Example 27, wherein the cytotoxic agent is selected from calicheamicin, auristatin, dolastatin, tubulicin, maytansinoid, cryptophycin, duocarmycin, esperamicin, pyrrolobenzodiazepine, and enediyne antibiotics.

[0036] Example 29. A pharmaceutical composition comprising a polypeptide of any one of Examples 1 to 26 or an immunoconjugate of Example 27 or 28, and a pharmaceutically acceptable carrier.

[0037] Example 30. Isolated nucleic acid encoding a polypeptide of any one of Examples 1 to 26.

[0038] Example 31. A vector comprising the nucleic acid of Example 30.

[0039] Embodiment 32. A host cell comprising the nucleic acid of Embodiment 34 or the vector of Embodiment 31.

[0040] Example 33. A host cell expressing a polypeptide of any one of Examples 1 to 26.

[0041] Example 34. A method for preparing a polypeptide of any one of Examples 1 to 26, comprising the step of incubating a host cell of Example 32 or Example 33 under conditions suitable for the expression of the polypeptide.

[0042] Embodiment 35. The method of Embodiment 34, further comprising the step of isolating the polypeptide.

[0043] Example 36. A method for treating cancer comprising the step of administering a pharmaceutically effective amount of any one of the polypeptides of Examples 1 to 26, the immunoconjugate of Example 27 or Example 28, or the pharmaceutical composition of Example 29 to a subject having cancer.

[0044] Embodiment 37. The method of 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; medium-grade / follicular NHL; medium-grade diffuse NHL; high-grade immune blastocyst NHL; high-grade lymphoblastic NHL; high-grade small non-severing cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-associated lymphoma; Waldenstrom macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hair cell leukemia; and chronic myeloid leukemia.

[0045] Embodiment 38. The method of Embodiment 36 or 37, wherein the cancer is acute myeloid leukemia (AML).

[0046] Embodiment 39. A method comprising, in any one of embodiments 36 to 38, further comprising the step of administering an additional therapeutic agent.

[0047] Embodiment 40. The method of Embodiment 39, wherein the additional therapeutic agent is an anticancer agent.

[0048] Embodiment 41. The method of Embodiment 40, wherein the anticancer agent is selected from chemotherapy agents, anticancer biological agents, radiation therapy, CAR-T therapy, and oncolytic viruses.

[0049] Embodiment 42. A method in any one of embodiments 36 to 39, wherein the cancer is a CLEC12a-expressing cancer. Brief explanation of the drawing

[0050] Figures 1a and 1b show biolayer interferometer data for polypeptides containing a VHH domain that binds to CLEC12a. Figure 1a shows biolayer interferometer data for hzMB11v13 compared with another CLEC12a-binding sdAb described herein. Figure 1b shows biolayer interferometer data for hzME06v16 compared with another CLEC12a-binding sdAb described herein. Figures 2a-2l show the binding of specific single-domain antibodies (sdAbs) to CLEC12a expressed on HEK 293 cells. "CLEC12a-HEK-293" refers to HEK 293 cells transfected with a plasmid encoding CLEC12a as described in Example 2. "HEK 293" refers to untransfected HEK 293 cells. Figure 2a shows the binding of MB11-IgG1 to CLEC12a. Figure 2b shows the binding of hzMB11v13-IgG1 to CLEC12a. Figure 2c shows the binding of ME06-IgG1 to CLEC12a. Figure 2d shows the binding of hzME06v16-IgG1 to CLEC12a. Figure 2e shows the binding of MC02-IgG1 to CLEC12a. Figure 2f shows the binding of hzMC02v6-IgG1 to CLEC12a. Figure 2g shows the binding of MH07-IgG1 to CLEC12a. Figure 2h shows the binding of hzMH07v3-IgG1 to CLEC12a. Figure 2i shows the binding of MC06-IgG1 to CLEC12a. Figure 2j shows the binding of hzMC06v2-IgG1 to CLEC12a. Figure 2k shows the binding of ME02-IgG1 to CLEC12a. Figure 2l shows the binding of hzME02v4-IgG1 to CLEC12a. Specific details for implementing the invention

[0051] The embodiments provided herein relate to CLEC12a binding polypeptides and their use in various methods for treating cancer.

[0052] Definitions and various embodiments

[0053] The section titles used herein are for organizational purposes only and should not be interpreted as limiting the subject matter of the invention described herein.

[0054] All references, including patent applications, patent publications, and Genbank registration numbers, are incorporated herein by reference as each individual reference is specifically and individually indicated as being cited in its entirety.

[0055] The techniques and procedures described or referenced herein are generally well understood and are commonly used by those skilled in the art using conventional methodologies, such as the widely used methods described in the following literature: Literature [Sambrook et al. , Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel, et al. eds ., (2003)); Serials METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor eds . (1995)), Harlow and Lane, eds . (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed . (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney), ed ., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds .); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds ., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al. , eds ., 1994); Current Protocols in Immunology (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 JD Capra, eds ., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al. , eds ., JB Lippincott Company, 1993); and their revised editions].

[0056] Unless otherwise defined, all scientific and technical terms used in connection with the present invention have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. Additionally, unless otherwise required or specified by the context, singular terms shall include the plural, and plural terms shall include the singular. In the event of a conflict of definitions among various sources or references, the definitions provided herein shall prevail.

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

[0058] The embodiments of the invention described herein are understood to include embodiments that are "composed of" and / or "essentially composed of". The singular forms used herein (“a,” “an,” and “the”) include the corresponding objects in the plural form unless otherwise specified. The use of the term “or” herein does not imply that only one of the two options is selected.

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

[0060] The terms “reference sample,” “reference cell,” or “reference tissue” mean a sample having at least one known characteristic that can be used as a comparison for a sample having at least one unknown characteristic. In some embodiments, the reference sample may be used as a positive or negative indicator. The reference sample may be used to establish, for example, the levels of protein and / or mRNA present in healthy tissue with respect to the levels of protein and / or mRNA present in the sample having the unknown characteristic. In some embodiments, the reference sample is derived from the same subject but from a different part of that subject than the sample being tested. In some embodiments, the reference sample is derived from a tissue region surrounding or adjacent to the cancer. In some embodiments, the reference sample is a sample derived from a subject known to have or not have the disorder in question (e.g., a specific cancer or a CLEC12a-related disorder), rather than from the subject being tested. In some embodiments, the term reference sample is derived from the same subject, but from a point in time prior to the subject developing cancer. In some embodiments, the reference sample is derived from a positive cancer sample of the same or different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule in question in the negative reference sample represents a level at which a person skilled in the art can recognize that the molecule in question is absent or present at a low level when considering the present invention. When a positive reference sample is used for comparison, the expression level or amount of the molecule in question in the positive reference sample represents a level at which a person skilled in the art can recognize that the molecule in question is present at a certain level when considering the present invention.

[0061] The terms “benefit,” “clinical benefit,” “responsiveness,” and “therapeutic responsiveness” as used herein in relation to obtaining benefit from or responding to the administration of a therapeutic agent may be measured by evaluating various endpoints, such as: some degree of inhibition of disease progression, including slowing and complete cessation of disease progression; reduction in the number of disease episodes and / or symptoms; reduction in lesion size; inhibition of infiltration of disease cells into adjacent peripheral organs and / or tissues (i.e., reduction, slowing, or complete cessation); inhibition of disease spread (i.e., reduction, slowing, or complete cessation); some degree of alleviation of one or more symptoms associated with the disorder; period of disease-free status after treatment, e.g., increase in progression-free survival; increase in overall survival; higher response rate; and / or reduction in mortality at a given time point after treatment. “Non-responsive” or “unresponsive” subjects or cancers do not meet the qualification criteria for having “responsiveness” mentioned above.

[0062] "Nucleic acid molecule," "nucleic acid," and "polynucleotide" are interchangeable and refer to polymers 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. "Nucleic acid sequence" refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0063] The terms “polypeptide” and “protein” are used interchangeably to refer to polymers of amino acid residues, not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are included in the definition. Additionally, the terms include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Furthermore, for the purposes of the present invention, “polypeptide” refers to a protein that includes modifications to its natural sequence, such as deletions, additions, and (generally, substantially conservative) substitutions, provided that the protein maintains the desired activity. Such modifications may be intentional, such as through site-specific mutagenesis, or accidental, such as through mutations in the host producing the protein or errors resulting from PCR amplification.

[0064] As used herein, “CLEC12a” refers to any mature natural CLEC12a produced from the processing of a CLEC12a precursor in cells. Unless otherwise specified, the term includes CLEC12a from any vertebrate, including mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats). Additionally, the term includes naturally occurring variants of CLEC12a, such as splice variants or allelic variants. Non-limiting exemplary human CLEC12a amino acid sequences are shown, for example, in UniProt registry number Q5QGZ9.3. See SEQ ID NO. 1.

[0065] The term "specifically binding" to an antigen or epitope is a well-known term 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" when it reacts with or associates with a specific cell or substance more frequently, more rapidly, for a longer duration, and / or with greater affinity than other cells or substances. A single-domain antibody (sdAb) or a VHH-containing polypeptide "specifically binds" or "preferentially binds" to a target when it binds with greater affinity, binding activity, more easily, and for a longer duration than it binds to other substances. For example, an sdAb or VHH-containing polypeptide that binds specifically or preferentially to a CLEC12a epitope is an sdAb or VHH-containing polypeptide that binds to the CLEC12a epitope more readily and / or for a longer duration, with greater affinity and binding activity, compared to binding to other CLEC12a epitopes or non-CLEC12a epitopes. Furthermore, if you have read these definitions, it will also be understood that, for example, an sdAb or VHH-containing polypeptide that binds specifically or preferentially to a first target may or may not bind specifically or preferentially to a second target. Therefore, "specific binding" or "preferential binding" does not necessarily require exclusive binding (although it may include it). Generally, though not always, references to binding mean preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0066] The terms “inhibition” or “inhibiting” mean a reduction or cessation of any phenotypic characteristic, or a reduction or cessation of the occurrence, degree, or likelihood of said characteristic. “Reduce” or “inhibit” means to lower, reduce, or stop activity, function, and / or amount relative to a reference. In some embodiments, “reduce” or “inhibit” means the ability to induce an overall reduction of 10% or more. In some embodiments, “reduce” or “inhibit” means the ability to induce an overall reduction of 50% or more. In some embodiments, “reduce” or “inhibit” means the ability to induce an overall reduction of 75%, 85%, 90%, 95%, or more. In some embodiments, the mentioned amount is inhibited or reduced compared to the control group after a certain period of time over the same duration. In relation to the activity of CLEC12a, the term “inhibit” as used herein refers to a reduction in CLEC12a activity, such as binding to monosodium urate. In some embodiments, “inhibit” refers to a decrease in CLEC12a activity compared to CLEC12a activity without a modifier. In some embodiments, the CLEC12a binding polypeptide blocks the binding of CLEC12a to monosodium urate.

[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) to which an antigen-binding molecule (e.g., an sdAb or VHH-containing polypeptide) binds. Epitopes often comprise a group of chemically active surface molecules, such as amino acids, polypeptides, or sugar side chains, and possess characteristic three-dimensional structural features as well as characteristic charge features. Epitopes can be formed from both adjacent residues of the target molecule and / or juxtaposed non-adjacent residues (e.g., amino acids, nucleotides, sugars, lipid moiety). Epitopes formed from adjacent residues (e.g., amino acids, nucleotides, sugars, lipid moiety) are generally retained upon exposure to a denaturing solvent, whereas epitopes formed by tertiary folding are generally lost upon treatment with a denaturing solvent. The epitope may comprise at least 3, at least 5, or 8 to 10 residues (e.g., amino acids or nucleotides), but is not limited thereto. In some embodiments, the epitope is a length of fewer than 20 residues (e.g., amino acids or nucleotides), fewer than 15, or fewer than 12 residues. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within that antigen. In some embodiments, the epitope may be identified by a specific minimum distance to a CDR residue on the antigen-binding molecule. In some embodiments, the epitope may be identified by said distance and is further limited to residues involved in binding (e.g., hydrogen bonding) between the residues of the antigen-binding molecule and the antigen residues. The epitope may also be identified by various scans, for example, an alanine or arginine scan may reveal one or more residues with which the antigen-binding molecule can interact. Unless otherwise specified, a set of residues as an epitope does not exclude other residues as part of the epitope for a specific antigen-binding molecule.Rather, the existence of such a set designates a minimum group (or species set) of epitopes. Thus, in some embodiments, a set of residues identified as epitopes designates a minimum number of epitopes associated with the antigen, rather than a list of residues exclusive to epitopes on the antigen.

[0068] "Non-linear epitope" or "stereoscopic epitope" comprises non-adjacent polypeptides, amino acids, and / or sugars within an antigenic protein to which an antigen-binding molecule specific to the epitope binds. In some embodiments, at least one of the residues will not be adjacent to other mentioned residues of the epitope; however, one or more of the residues may be adjacent to other residues.

[0069] A “linear epitope” comprises adjacent polypeptides, amino acids, and / or sugars within an antigenic protein to which an antigen-binding molecule specific to the epitope binds. It should be noted that in some embodiments, not all residues within the linear epitope need to be directly bound (or involved in binding) by the antigen-binding molecule. In some embodiments, the linear epitope may be derived from immunization using a peptide effectively composed of the sequence of the linear epitope, or it may be derived from structural parts of a protein relatively isolated from the remainder of the protein (so that the antigen-binding molecule can interact only with the sequence portions).

[0070] The term “antibody” is used in its broadest sense and includes various polypeptides comprising antibody-like antigen-binding domains, such as but not limited to conventional antibodies (generally comprising at least one heavy chain and at least one light chain), single-domain antibodies (sdAb, comprising at least one VHH domain and an Fc region), VHH-containing polypeptides (polypeptides comprising at least one VHH domain), and fragments of any of the above-mentioned ones as long as they exhibit the desired antigen-binding activity. In some embodiments, the antibody comprises a dimerization domain. Such dimerization domains include, but are not limited to, a heavy chain constant domain (comprising CH1, a hinge, CH2 and CH3, where CH1 is generally paired with a light chain constant domain CL, and the hinge mediates dimerization) and an Fc region (comprising a hinge, CH2 and CH3, where the hinge mediates dimerization).

[0071] In addition, the term antibody includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species, such as camelid (including llama), shark, mouse, human, cynomolgus monkey, etc.

[0072] As used herein, the term “antigen binding domain” refers to a portion of an antibody sufficient to bind to an antigen. In some embodiments, the antigen binding domain of a conventional antibody comprises three heavy chain CDRs and three light chain CDRs. Thus, in some embodiments, the antigen binding domain comprises a heavy chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 necessary to maintain binding to the antigen, and a light chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 necessary to maintain binding to the antigen. In some embodiments, the antigen binding domain of an sdAb or VHH-containing polypeptide comprises three CDRs of the VHH domain. Accordingly, in some embodiments, the antigen-binding domain of the sdAb or VHH-containing polypeptide comprises a VHH domain comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 necessary to maintain binding to the antigen.

[0073] As used herein, the terms “VHH,” “VHH domain,” or “VHH antigen-binding domain” refer to the antigen-binding portion of a single-domain antibody, such as a camelid antibody or a shark antibody. In some embodiments, VHH comprises three CDRs and four framework regions designated as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, VHH may be cleaved at the N-terminus or C-terminus to include only some of the FR1 and / or FR4, or one or both of these framework regions may be omitted as long as VHH substantially retains antigen binding and specificity.

[0074] The terms “single-domain antibody” and “sdAb” are used interchangeably herein to refer to antibodies comprising at least one monomeric domain, such as a VHH domain, which is absent from light chains and an Fc region. In some embodiments, an sdAb is a dimer of two polypeptides, each polypeptide comprising at least one VHH domain and an Fc region. As used herein, the terms “single-domain antibody” and “sdAb” encompass polypeptides comprising multiple VHH domains, such as polypeptides having a structure VHH1-VHH2-Fc or VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 may be the same or different from each other.

[0075] The term "VHH-containing polypeptide" refers to a polypeptide comprising at least one VHH domain. In some embodiments, the VHH polypeptide comprises two, three, or four or more VHH domains, wherein each VHH domain may be identical or different. In some embodiments, the VHH-containing polypeptide comprises an Fc region. In some of these embodiments, the VHH-containing polypeptide may be referred to as sdAb. In some of these embodiments, the VHH polypeptide may form a dimer. Non-limiting structures of the VHH-containing polypeptide that is also sdAb include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 may be identical or different from each other. In some embodiments of these structures, one VHH may be linked to another VHH by a linker, or one VHH may be linked to Fc by a linker. In some of these embodiments, the linker comprises 1 to 20 amino acids, preferably composed mainly of glycine and optionally serine. In some embodiments, if the VHH-containing polypeptide contains 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). Likewise, 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 antibodies of a substantially homogeneous antibody group (including sdAb or VHH-containing polypeptides), that is, each antibody in said group is identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and are induced for a single antigenic site. Furthermore, unlike polyclonal antibody preparations which generally contain different antibodies induced for different determinants (epitopes), each monoclonal antibody is induced for a single determinant on the antigen. Therefore, a sample of a monoclonal antibody can bind to the same epitope on the antigen. The modifier "monoclonal" means that the characteristics of the antibody are derived from a substantially homogeneous group of antibodies and are not interpreted as requiring the production of the antibody by any specific method. For example, monoclonal antibodies may be produced by a hybridoma method first described in the literature [Kohler and Milstein, 1975, Nature 256:495], or by a recombinant DNA method as described in U.S. Patent 4,816,567. Additionally, monoclonal antibodies, for example, [McCafferty et al It may also be isolated from a phage library generated using the technique described in [., 1990, Nature 348:552-554].

[0077] The term "CDR" indicates a complementarity determination region defined by at least one identification method to those skilled in the art. In some embodiments, the CDR may be defined according to any one of the following methods: the Chothia numbering method, the Kabat numbering method, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition. VHH includes three CDRs designated as CDR1, CDR2, and CDR3.

[0078] As used herein, the term "heavy chain invariant region" refers to at least three heavy chain invariant domains, C H 1, hinge, C H 2 and C H It refers to the region containing 3. Of course, deletions and modifications that modify non-functionality within the domains are also included within the scope of the term "heavy chain constant region" unless otherwise specified. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isoform. 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. Additionally, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε constant region is an IgE antibody. Specific isoforms may be further subdivided into subgroups. For example, IgG antibodies include, but are not limited to, IgG1 (containing a γ1 constant region), IgG2 (containing a γ2 constant region), IgG3 (containing a γ3 constant region), and IgG4 (containing a γ4 constant region); IgA antibodies include, but are not limited to, IgA1 (including α1 constant region) and IgA2 (including α2 constant region) antibodies; IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0079] As used herein, the “Fc region” refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. In various embodiments, where the Fc region comprises a hinge, said hinge mediates dimerization between two Fc-containing polypeptides. The Fc region may be isomorphic to any antibody heavy chain constant region discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0080] As used herein, the "acceptor human framework" refers to a heavy chain variable domain (V) derived from the human immunoglobulin framework or the human consensus framework, as discussed herein. H It is a framework containing the amino acid sequence of the ) framework. A receptor human framework derived from the human immunoglobulin framework or the human consensus framework may contain the same amino acid sequence as that, or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, or less than 3 across all human frameworks of a single antigen-binding domain such as VHH.

[0081] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody, e.g., sdAb, or a VHH-containing polypeptide) and its binding counterpart (e.g., an antigen). The affinity or apparent affinity of molecule X for counterpart Y is generally given by the dissociation constant (K), respectively. d ) or K d-겉보기 Affinity can be expressed as follows. Affinity can be expressed by general methods known in the art (e.g., ELISA K) including those described herein. d It can be measured by KinExA, flow cytometry and / or surface plasmon resonance devices. These methods include, but are not limited to, BIAcore®, Octet®, or methods involving flow cytometry.

[0082] "K" used in this document d The term "K" refers to the equilibrium dissociation constant of the antigen-binding molecule / antigen interaction. d Where the term "is used herein, it refers to K d or K d-겉보기 Includes

[0083] In some embodiments, the Kd of the antigen-binding molecule is measured by flow cytometry in which the average fluorescence intensity measured at each antibody concentration using an antigen-expressing cell line is fitted to a non-linear one-site binding equation (Prism Software graphpad). In some of these embodiments, K d is K d-겉보기 am.

[0084] The term "biological activity" refers to any one or more biological properties of a molecule (whether naturally occurring as found in vivo, or provided or made possible by recombinant methods).

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

[0086] "Antagonist," "blocker," or "neutralizing" antibody is an antibody that reduces and / or inactivates the biological activity of a target antigen. In some embodiments, the neutralizing antibody binds to the antigen and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 99%, or more.

[0087] "Affinity-enhanced" sdAb or VHH-containing polypeptides refer to sdAb or VHH-containing polypeptides having one or more modifications in one or more CDRs, such modifications induce an improvement in the affinity of the sdAb or VHH-containing polypeptide for an antigen when compared to a parent sdAb or VHH-containing polypeptide without such modifications.

[0088] As used herein, “humanized VHH” refers to a VHH in which one or more framework regions are substantially replaced by human framework regions. In some cases, specific framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. Additionally, the humanized VHH may contain residues not found in the original VHH or in the human framework sequence, but these are included to further improve and optimize the performance of the sdAb or VHH-containing polypeptide. In some embodiments, the humanized sdAb or VHH-containing polypeptide contains a human Fc region. As is understandable to anyone, the humanization sequence may be identified by its primary sequence and does not necessarily imply the process by which the antibody was generated.

[0089] "Effector-positive Fc regions" possess "effector functions" of the natural 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. These effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays.

[0090] The "natural sequence Fc region" contains an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. The natural sequence human Fc region includes the natural sequence human IgG1 Fc region (non-A and A homotypes); the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region, and their naturally occurring variants.

[0091] The “variant Fc region” comprises an amino acid sequence different from that of the natural sequence Fc region due to at least one amino acid modification. In some embodiments, the “variant Fc region” comprises an amino acid sequence that differs from that of the natural sequence Fc region due to at least one amino acid modification but retains at least one effector function of the natural sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution when compared to the natural sequence Fc region or the Fc region of the parent polypeptide, such as about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the natural sequence Fc region or the Fc region of the parent polypeptide. In some embodiments, the variant Fc region of the present invention will have at least about 80% sequence identity with the natural sequence Fc region and / or the Fc region of the parent polypeptide, at least about 90% sequence identity with it, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

[0092] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, FcγR is a natural human FcR. In some embodiments, FcR is one that binds to an IgG antibody (gamma receptor) and includes the FcγRI, FcγRII, and FcγRIII subclasses of receptors, as well as allelic variants and differently spliced ​​forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have different similar amino acid sequences, 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. (For example, see the literature [Daeron, Annu. Rev. Immunol. 15:203-234 (1997)]). FcR is, for example, the literature [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. It is outlined in [126:330-41 (1995)]. Other FcRs, including those to be identified in the future, are also included in the term “FcR” herein. For example, the terms “Fc receptor” or “FcR” include 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 FcRn, a neonatal receptor responsible for the homeostatic regulation of immunoglobulins. Methods for measuring binding to FcRn are known (e.g., Ghetie and Ward, Immunol. Today18(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. Refer to )]).

[0093] As used herein, "chimeric antigen receptor" refers to a genetically engineered polypeptide comprising 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” mean that there is a sufficiently high degree of similarity between two or more values ​​such that anyone skilled in the art would think that the difference between two or more values ​​has little or no biological and / or statistical significance within the scope of the content of the biological characteristics measured by those values. In some embodiments, the two or more substantially similar values ​​differ by about 5%, 10%, 15%, 20%, 25%, or 50% or less.

[0095] A polypeptide “variant” means a biologically active polypeptide having at least about 80% amino acid sequence identity with the natural sequence polypeptide, after aligning the sequence without considering any conservative substitutions as part of the sequence identity and introducing gaps if necessary to achieve the maximum sequence identity %. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N- 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 with the natural sequence polypeptide.

[0096] In this document, with respect to peptide, polypeptide, or antibody sequences, “amino acid sequence identity %” and “homology” are defined as the ratio of amino acid residues in the candidate sequence that are identical to the amino acid residues of the specific peptide or polypeptide sequence, after aligning the sequences without considering any conservative substitutions as part of the sequence identity and introducing gaps if necessary to achieve maximum sequence identity %. Alignment for measuring amino acid sequence identity % may be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Anyone skilled in the art can determine appropriate parameters for determining alignment, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared.

[0097] Amino acid substitutions may include, but are not limited to, replacing one amino acid within a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions may be introduced into antibodies of interest and their products screened for desired activity, such as maintained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0098]

[0099] Amino acids can be classified according to common side chain characteristics:

[0100] (1) Hydrophobicity: Norrusine, Met, Ala, Val, Leu, Ile;

[0101] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0102] (3) Acids: Asp, Glu;

[0103] (4) Basics: His, Lys, Arg;

[0104] (5) Residues affecting chain orientation: Gly, Pro;

[0105] (6) Aromatic: Trp, Tyr, Phe.

[0106] Non-conservative substitution involves exchanging a member of one of these classes for one of another class.

[0107] The term “vector” is used to describe a polynucleotide that can be genetically engineered to contain a cloned polynucleotide(s) that can be replicated in a host cell. A vector may contain one or more of the following components: a replication origin, one or more regulatory sequences that regulate the expression of the polypeptide of interest (e.g., a promoter and / or enhancer), and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used for colorimetric analysis, such as β-galactosidase). The term “expression vector” refers to a vector used to express the polypeptide of interest in a host cell.

[0108] "Host cell" refers to a cell that may or was a recipient of a vector or isolated polynucleotide. The host cell may be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Exemplary, non-limiting mammalian cells include NSO cells, PER.C6 ® Cells (Crucell), 293 and CHO cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells, are included but not limited thereto. Host cells include progenitor individuals of a single host cell, said progenitor individuals may not necessarily be completely identical to the original parent cell (in morphology or genomic DNA complement) due to natural, accidental, or intentional mutations. Host cells include cells in vivo transfected with the polynucleotide(s) provided herein.

[0109] As used herein, the term “isolated” generally refers to a molecule separated from at least some of the components found or produced in nature. For example, a polypeptide is said to be “isolated” when it is separated from at least some of the components of the cell in which it was produced. If a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered “isolating” the polypeptide. Likewise, a polynucleotide is said to be “isolated” when it is not part of a larger polynucleotide found in nature (e.g., genomic DNA or mitochondrial DNA in the case of DNA polynucleotides), or, for example in the case of RNA polynucleotides, when it is separated from at least some of the components of the cell in which it was produced. Thus, DNA polynucleotides contained in a vector inside a host cell may be referred to as “isolated.”

[0110] The terms “individual” and “subject” are used interchangeably herein to refer to animals; e.g., mammals. In some embodiments, a method for treating mammals is provided, including but not limited to humans, rodents, primates, cats, dogs, horses, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sports animals, and mammalian pets. In some examples, “individual” or “subject” refers to an individual or subject requiring treatment for a disease or disorder. In some embodiments, the subject to treatment may be a patient for whom it has been specified that the subject has a disorder related to the treatment or is at sufficient risk of developing such disorder.

[0111] As used herein, "disease" or "disorder" refers to a pathological condition that requires or necessitates treatment.

[0112] The terms “tumor cell,” “cancer cell,” “cancer,” “tumor,” and / or “neoplasm,” used interchangeably herein unless otherwise specified, refer to cell(s) exhibiting uncontrolled growth and / or an abnormal increase in cell viability and / or inhibition of apoptosis that interferes with the normal functioning of body organs and systems. The above definitions include benign and malignant cancers, blood cancers such as leukemia, lymphoma, and multiple myeloma, polyps, proliferations, hyperplasias, as well as dormant tumors or micrometastasis.

[0113] The terms “cancer” and “tumor” include solid tumors and hematological / lymphomas, and also include malignant, precancerous, and benign proliferations, such as dysplasia. Exemplary cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; gastrointestinal cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastrointestinal cancer (including gastrointestinal cancer); glioblastoma; liver carcinoma; hepatocellular carcinoma; neoplasm in situ; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); melanoma; multiple myeloma; neuroblastoma; oral cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; Rhabdomyosarcoma; rectal cancer; respiratory cancer; salivary gland carcinoma; sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urological cancer; vulvar cancer; lymphoma (including Hodgkin and non-Hodgkin lymphoma) as well as B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate / follicular NHL; intermediate diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small undifferentiated cell NHL; macrolesion NHL; mantle cell lymphoma; AIDS-associated lymphoma; and Waldenström macroglobulinemia; acute myeloid leukemia (AML); chronic lymphoblastic leukemia (CLL); acute lymphoblastic leukemia (ALL); hair cell leukemia; chronic myeloid leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal angiogenesis associated with follicular disease, edema (e.g., associated with brain tumors), and Meig syndrome.

[0114] As used herein, the terms “non-tumor cells” or “non-cancerous cells” refer to normal cells or tissues. Exemplary non-tumor cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, macrophages, epithelial cells, fibroblasts, hepatocytes, interstitial renal cells, fibroblast-like synovial cells, and osteoblasts; and cells located in the breast, skeletal muscle, pancreas, stomach, ovary, small intestine, placenta, uterus, testis, kidney, lung, heart, brain, liver, prostate, colon, lymphatic organs, bone, and mesenchymal stem cells derived from bone. As used herein, the term “peripherally located cells or tissues” refers to non-tumor cells that are not located near tumor cells and / or within the tumor microscopic environment.

[0115] As used herein, the term “cells or tissues within the tumor microenvironment” refers to cells, molecules, extracellular matrix, and / or blood vessels that surround or nourish tumor cells. Exemplary cells or tissues within the tumor microenvironment include, but are not limited to, the following: tumor vascular system; tumor-infiltrating lymphocytes; fibroblastic reticular cells; endothelial progenitor cells (EPCs); cancer-associated fibroblasts; perivascular cells; 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 proximal to the tumor. Methods for identifying tumor cells and / or cells / tissues located within the tumor microenvironment are well known in the art as described below in this document.

[0116] In some embodiments, “increase” or “decrease” each refers to a statistically significant increase or decrease. As will be apparent to those skilled in the art, the term “adjusting” may involve inducing a change (which may be an increase or a decrease) in the affinity, binding activity, specificity, and / or selectivity of one or more of the ligand, binding counterpart, counterpart for association in homopolymer or heteropolymer form, or substrate of the target or antigen; inducing a change (which may be an increase or a decrease) in the sensitivity of the said target or antigen to one or more conditions (e.g., pH, ion concentration, presence of cofactors, etc.) in the medium or surrounding environment where the target or antigen is present; and may involve cell proliferation or cytokine generation compared to the same conditions without the test agent. Depending on the relevant target, this may be measured in any suitable manner and / or using any suitable analytical method known in itself or described herein.

[0117] As used herein, the term "immune response" encompasses cellular and / or humoral immune responses sufficient to suppress the symptoms of a disease (e.g., cancer or cancer metastasis), prevent its onset, or improve its symptoms. The "immune response" may include aspects of both the innate immune system and the adaptive immune system.

[0118] As used herein, “treatment” refers to an approach to obtain a beneficial or desired clinical outcome. As used herein, “treatment” encompasses any administration or application of a therapeutic agent for a disease in mammals, including humans. For the purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, alleviation of one or more symptoms; reduction of the severity of the disease; prevention or delay of the spread of the disease (e.g., metastasis, e.g., to the lungs 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 disease progression; inhibition or slowing of the disease or its progression; cessation of its development; and any one or more of remission (partial or total). Additionally, the term “treatment” includes a reduction in the pathological consequences of a proliferative disease. The methods provided herein consider any one or more of these therapeutic aspects. As such, the term “treatment” does not require the 100% elimination of all aspects of the disorder in question.

[0119] The term "improving" means a reduction or improvement of one or more symptoms compared to the case where the treatment is not administered. Additionally, the term "improving" includes a shortening or reduction in the duration of symptoms.

[0120] The term “anticancer agent” is used herein in its broadest sense to refer to agents used for the treatment of one or more cancers. Exemplary classes of such agents include, but are not limited to, chemotherapy agents, anticancer biological agents (e.g., cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiation therapy, CAR-T therapy, therapeutic oligonucleotides (e.g., antisense oligonucleotides and siRNA), and tumor-disrupting viruses.

[0121] The term “biological sample” means a large amount of material derived from a living organism or a past organism. Such material includes, but is not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0122] The terms “control” or “reference” refer to a composition known to be free of the analyte (“negative control”) or to be containing the analyte (“positive control”). A positive control may contain an analyte at a known concentration. Additionally, a control or reference may refer to a preparation being tested, such as a control preparation known to lack antibody activity.

[0123] As used herein, the term "delaying the development of a disease" means postponing, inhibiting, slowing, delaying, stabilizing, suppressing, and / or prolonging the development of a disease (e.g., cancer). The duration of such delay may vary depending on the individual's history of the disease and / or the individual undergoing treatment. As will be apparent to those skilled in the art, a sufficient or substantial delay can effectively encompass prevention in that the individual does not develop the disease. For example, terminal cancer, such as the development of metastasis, may be delayed.

[0124] As used herein, the term “preventive” includes providing prevention against the occurrence or recurrence of the disease in subjects who may be susceptible to the disease but have not yet been diagnosed with it. Unless otherwise specified, the terms “reduce,” “inhibit,” or “prevent” do not imply or require complete prevention over the entire duration of time, but apply only during the measured period.

[0125] The "therapeutic effective dose" of a substance / molecule, agonist, or antagonist may vary depending on factors such as the individual's disease state, age, sex and body weight, and the ability of said substance / molecule, agonist, or antagonist to induce a desired response in said individual. Additionally, a therapeutic effective dose is an amount in which the therapeutically beneficial effect outweighs any toxic or adverse effect of said substance / molecule, agonist, or antagonist. The therapeutic effective dose may be delivered in a single or more administrations. The therapeutic effective dose refers to the effective amount over the dosage and duration required to achieve the desired therapeutic and / or prophylactic outcome.

[0126] The terms “pharmaceutical preparation” and “pharmaceutical composition” are used interchangeably to refer to a preparation in which the biological activity of the active ingredient(s) is allowed to be effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to whom the preparation is to be administered. Such preparations may be sterilized.

[0127] "Pharmaceuticalally acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art for use with a therapeutic agent comprising a "pharmaceutical composition" to be administered to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and is compatible with other components of the said formulation. A pharmaceutically acceptable carrier is suitable for the formulation used.

[0128] "Combination" administration with one or more additional therapeutic agents includes simultaneous (co) administration and sequential administration in any order.

[0129] The term “simultaneously” is used herein to refer to the administration of two or more therapeutic agents, in which case at least some of the administrations overlap in time, or one therapeutic agent is administered within a relatively short period of time compared to the administration of another therapeutic agent, or the therapeutic effects of both therapeutic agents overlap for at least a certain period.

[0130] The term “sequentially” is used herein to refer to the administration of two or more therapeutic agents that do not overlap in time or that do not overlap in therapeutic effects.

[0131] As used herein, "~in conjunction with (~in combination with)" refers to administering another treatment method in addition to a single treatment method. As such, "~in conjunction with" refers to administering one treatment method before, during, or after administering another treatment method to the subject.

[0132] The term "package insert" is used to refer to an instruction manual customarily included in the commercial package of a therapeutic drug, which contains information regarding indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings related to the use of the therapeutic drug.

[0133] The term “manufactured article” is any manufactured article (e.g., packaging or container) or kit comprising at least one reagent, e.g., a drug for treating a disease or disorder (e.g., cancer), or a probe for specifically detecting the biomarker described herein. In some embodiments, said manufactured article or kit is promoted, distributed, or sold as a unit for carrying out the method described herein.

[0134] The terms “label” and “detectable label” refer to a moiety attached, for example, to an antibody or antigen, which makes the reaction (e.g., binding) between the members of a specific binding pair detectable. The labeled member of a specific binding pair is said to be “detectablely labeled.” Accordingly, the term “labeled binding protein” refers to a protein incorporating a label that provides identification for the binding protein. In some embodiments, the label is a detectable marker capable of generating a signal detectable by visual or instrumental means, such as the incorporation of a radiolabeled amino acid or the attachment of a biotinyl moiety to a polypeptide detectable by a marked avidin (e.g., a fluorescent marker detectable by optical or colorimetric methods or streptavidin containing enzymatic activity). Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho or 153Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary 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 light-emitting moiety, such as acridinium compounds, and fluorescence-emitting moiety, such as fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with another moiety.

[0135] Exemplary CLEC12a-binding polypeptide

[0136] A CLEC12a binding polypeptide is provided herein. In various embodiments, the CLEC12a binding polypeptide comprises at least one VHH domain that binds to CLEC12a. In some embodiments, the CLEC12a is human CLEC12a. In some embodiments, the CLEC12a binding polypeptide blocks the binding of CLEC12a to a ligand. In some embodiments, the CLEC12a binding polypeptide provided herein comprises 1, 2, 3, 4, 5, 6, 7, or 8 VHH domains that bind to CLEC12a. In some embodiments, the CLEC12a binding polypeptide provided herein comprises 1, 2, 3, or 4 VHH domains that bind to CLEC12a. The CLEC12a binding polypeptide may comprise one or more VHH domains that bind to one or more target proteins other than CLEC12a. These polypeptides can be referred to as "multispecific" polypeptides.

[0137] In some embodiments, the CLEC12a binding polypeptide comprises at least one VHH domain that binds to the CLEC12a and Fc regions. In some embodiments, the CLEC12a binding polypeptide provided herein comprises one, two, three, or four VHH domains and an Fc region. In some embodiments, the Fc region mediates the dimerization of the CLEC12a binding polypeptide under physiological conditions to form a dimer that doubles the number of CLEC12a binding sites. For example, a CLEC12a binding polypeptide comprising three VHH domains and an Fc region that bind to CLEC12a is trivalent as a monomer, but since the Fc region can mediate dimerization under physiological conditions, the CLEC12a binding polypeptide exists as a hexavalent dimer under these conditions.

[0138] In some embodiments, the CLEC12a binding polypeptide comprises at least two VHH domains, wherein the first VHH domain binds to a first epitope of CLEC12a and the second VHH domain binds to a second epitope of CLEC12a. Where the CLEC12a binding polypeptide comprises a VHH domain that binds to a first epitope of CLEC12a and a VHH domain that binds to a second epitope of CLEC12a, the CLEC12a binding polypeptide may be referred to as "double-epitopic" or "double-specific." In some embodiments, the CLEC12a binding polypeptide comprises at least two VHH domains, wherein the first VHH domain binds to CLEC12a and the second VHH domain binds to an antigen other than CLEC12a. These polypeptides can be referred to as "bispecific" or "multispecific."

[0139] Non-limiting examples of CLEC12a-binding polypeptides are shown in Table 2. The sequences for the single-domain antibodies shown above are shown in the table of specific sequences in this invention. Polypeptide names starting with "hz" indicate that they are humanized versions of the corresponding parent polypeptide.

[0140]

[0141] CLEC12a-linked polypeptide

[0142] In various embodiments, the VHH domain binding to CLEC12a comprises a CDR1 sequence selected from SEQ NOs 3, 7, 11, 15, 19, 23, 32, 35, 38, 41, 44, and 47; a CDR2 sequence selected from SEQ NOs 4, 8, 12, 16, 20, 24, 33, 36, 39, 42, 45, and 48; and a CDR3 sequence selected from SEQ NOs 5, 9, 13, 17, 21, 25, 34, 37, 40, 43, 46, and 49. In various embodiments, the VHH domain binding to CLEC12a comprises SEQ NOs 3, 4, and 5; SEQ NOs 7, 8, and 9; SEQ NOs 11, 12, and 13; It includes CDR1, CDR2, and CDR3 sequences selected from SEQ ID NOs 15, 16, and 17; SEQ ID NOs 19, 20, and 21; SEQ ID NOs 23, 24, and 25; SEQ ID NOs 32, 33, and 34; SEQ ID NOs 35, 36, and 37; SEQ ID NOs 38, 39, and 40; SEQ ID NOs 41, 42, and 43; SEQ ID NOs 44, 45, and 46; and SEQ ID NOs 47, 48, and 49. In various embodiments, the VHH domain is humanized.

[0143] In some embodiments, the VHH domain binding to CLEC12a 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%, and at least 99% identical to an amino acid sequence selected from SEQ ID NOs 2, 6, 10, 14, 18, 22, 26, 27, 28, 29, 30, 31, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99. In some embodiments, the VHH domain that binds to CLEC12a comprises an amino acid sequence selected from SEQ ID NOs 2, 6, 10, 14, 18, 22, 26, 27, 28, 29, 30, 31, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99.

[0144] In some embodiments, the CLEC12a binding polypeptide comprises 1, 2, 3, or 4 VHH domains that bind to CLEC12a.

[0145] In various embodiments, the CLEC12a binding polypeptide comprises at least one VHH domain that binds to CLEC12a and at least one VHH domain that binds to a natural killer cell antigen or a T cell antigen. In some of these embodiments, the CLEC12a binding polypeptide may be referred to as a multispecific antibody.

[0146] In some embodiments, the CLEC12a binding polypeptide comprises at least one VHH domain described herein fused to the Fc region. In some embodiments, the Fc region has a sequence selected from SEQ ID NOs 50, 51, 52, 53, 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, and 85.

[0147] In some embodiments, the VHH domain binding to CLEC12a is humanized. Humanized antibodies (e.g., sdAb or VHH-containing polypeptides) are useful as therapeutic molecules because they reduce or eliminate the immune response to antibody therapeutics and the human immune response to non-human antibodies, which may result in reduced efficacy of said therapeutics. Generally, humanized antibodies comprise one or more variable domains in which the CDR (or part thereof) is derived from a non-human antibody and the FR (or part thereof) is derived from a human antibody sequence. The humanized antibody will optionally also comprise at least some human invariant regions. In some embodiments, some FR residues of the humanized antibody are substituted with corresponding residues of the non-human antibody (e.g., the antibody from which the corresponding CDR residues are derived) to restore or improve antibody specificity or affinity, for example.

[0148] Humanized antibodies and methods for producing the same are, for example, [Almagro and Fransson, (2008) Front. Biosci It is outlined in . 13: 1619-1633], and in the literature [Riechmann et al ., (1988) Nature 332:323-329]; [Queen et al ., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033]; U.S. Patents No. 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] (Explaining "resurfacing"); [Dall'Acqua et al ., (2005) Methods 36:43-60] (explaining "FR shuffling"); and [Osbourn et al ., (2005) Methods36:61-68] and [Klimka et al ., (2000) Br. J. Cancer [ , 83:252-260] (describes the "induced selection" approach to FR shuffling).

[0149] The human framework domains that can be used for humanization are, but are not limited to, framework domains selected using the "best-fit" method (e.g., literature [Sims et al . (1993) J. Immunol. [Refer to 151:2296]); framework regions derived from the consensus sequences of human antibodies of specific subgroups of heavy chain variable regions (e.g., literature [Carter et al . (1992) Proc. Natl. Acad. Sci. USA , 89:4285]; and [Presta et al . (1993) J. Immunol [See , 151:2623]); human maturation (somatically mutated) framework region or human germline framework region (e.g., literature [Almagro and Fransson, (2008) Front. Biosci [Refer to . 13:1619-1633]); and framework areas derived from screening FR libraries (e.g., literature [Baca et al ., (1997) J. Biol. Chem . 272: 10678-10684] and [Rosok et al ., (1996) J. Biol. Chem. Includes [271 :22611-22618]). Typically, the FR region of the VHH is replaced with a human FR region to create a humanized VHH. In some embodiments, specific FR residues of the human FR are replaced to improve one or more characteristics of the humanized VHH. VHH domains having these replaced residues are also referred to herein as "humanized".

[0150] In various embodiments, the Fc region included in the CLEC12a binding polypeptide is a human Fc region or is derived from a human Fc region.

[0151] In some embodiments, the Fc region included in the CLEC12a binding polypeptide is derived from a human Fc region and includes a three-amino acid deletion in the lower hinge corresponding to IgG1 E233, L234, and L235, referred herein as "Fc xELL". The Fc xELL polypeptide is referred to as "effector silent" or "effector null" because it does not bind to FcγR, but in some embodiments, the xELL Fc region binds to FcRn and thus has an extended half-life and transcytosis associated with FcRn-mediated recycling.

[0152] In some embodiments, the Fc region included in the CLEC12a binding polypeptide is derived from a human Fc region and includes the mutants M252Y and M428V referred herein as “Fc-YV”. In some embodiments, these mutants enhance binding to FcRn at the acidic pH of the endosome (near 6.5), while losing detectable binding at a neutral pH (about 7.2), making it possible to have enhanced FcRn-mediated recycling and an extended half-life.

[0153] In some embodiments, the Fc region included in the CLEC12a binding polypeptide is derived from a human Fc region and includes mutations designed for heterodimerization, referred herein as "knob" and "hole". In some embodiments, the "knob" Fc region includes the mutation T366W. In some embodiments, the "hole" Fc region includes the mutations T366S, L368A, and Y407V. In some embodiments, the Fc region used for heterodimerization includes additional mutations, such as the mutation S354C on the first member of the heterodimer Fc pair forming an asymmetric disulfide, and the corresponding mutation Y349C on the second member of the heterodimer Fc pair. In some embodiments, one member of the heterodimer Fc pair includes a modification H435R or H435K to prevent protein A binding while maintaining FcRn binding. In some embodiments, one member of the heterodimer Fc pair contains a variant H435R or H435K, while the second member of the heterodimer Fc pair is not modified from H435. In various embodiments, the hole Fc region contains a variant H435R or H435K (where the variant is H435R, referred to as "hole-R" in some cases), while the nop Fc region does not. In some cases, the hole-R mutation improves the purification of the heterodimer for the homodimer hole Fc region that may be present.

[0154] Non-limiting exemplary Fc regions that can be used in CLEC12a binding polypeptides include Fc regions comprising the amino acid sequences of SEQ ID NOs 50 to 85.

[0155] Chimeric receptors and genetically engineered cells

[0156] A chimeric antigen receptor (CAR) having an extracellular domain comprising one or more CLEC12a-binding VHH domains provided herein is provided. The CAR construct provided herein comprises an extracellular domain containing one or more CLEC12a-binding VHH domains, a transmembrane domain, and an intracellular signaling domain. One or more CLEC12a-binding VHH domains forming the antigen-binding unit of the CAR can bind to, or bind to, i.e., target, the CLEC12a-binding with sufficient affinity to make the CAR therapeutically useful in targeting cells or tissues exhibiting CLEC12a binding.

[0157] CARs are synthetic receptors that typically contain extracellular targeting / binding moieties linked to one or more signaling domains within a single fusion molecule expressed on the surface of cells such as T cells. Thus, CARs combine antigen specificity and T cell activation properties within a single fusion molecule. First-generation CARs typically included the cytoplasmic domain of CD3 zeta or the Fc 1 receptor γ chain as their signaling domains. First-generation CARs were tested in Phase I clinical studies in patients with ovarian cancer, renal cancer, lymphoma, and neuroblastoma, and induced moderate responses (reviewed in [Sadelain et al., Curr Opin Immunol, 21 (2): 215-223, 2009]). Second-generation CARs containing signaling domains of co-stimulatory molecules, such as CD28 and CD3 zeta, provide dual signaling capabilities that induce a combination of activation and co-stimulatory signals. Third-generation CARs with three or more signaling domains are more complex and sophisticated (reviewed in [Sadelain et al., Cancer Discovery (3), 388-398, 2013] and [Dotti et al, Immuno. Rev, 257 (1), 1-36, 2014]).

[0158] In some embodiments, the provided CAR comprises a CLEC12a-binding VHH domain. In some embodiments, the CAR comprises at least two VHH domains that target one or more antigens. In one embodiment, the antigen-binding domain of the CAR comprises two or at least two CLEC12a-binding VHH domains, thereby providing a divalent molecule. In one embodiment, the antigen-binding domain comprises two or at least two CLEC12a-binding VHH domains but binds to different epitopes on CLEC12a. In this case, the antigen-binding domain comprises a first CLEC12a-binding VHH domain that binds to a first epitope of CLEC12a and a second VHH domain that binds to a second epitope of CLEC12a. The epitopes may overlap. Thus, in some embodiments, the antigen-binding domain is biparatopic and the CAR is a biparatopic CAR. In another embodiment, the antigen-binding domain comprises two CLEC12a-binding VHH domains that bind to the same epitope on CLEC12a.

[0159] The transmembrane domain of the CAR is generally a domain that crosses, or can cross, or spans the plasma membrane and is connected directly or indirectly (e.g., through a spacer such as an immunoglobulin hinge sequence) to a portion of the endoplasmic reticulum containing an extracellular antigen binding domain and an intracellular signaling domain. In some embodiments, the transmembrane domain of the CAR is a transmembrane region consisting of a transmembrane protein (e.g., a type I transmembrane protein), a hydrophobic artificial sequence, or a combination thereof. In some embodiments, the transmembrane domain comprises a CD3 zeta domain or a CD28 transmembrane domain. Other transmembrane domains will be apparent to those skilled in the art and may be used in connection with the embodiments of the CAR provided herein.

[0160] The intracellular signaling domain of the CAR provided herein comprises one or more intracellular signaling domains that transmit a signal to a T cell upon binding to the antigen-binding domain of the CAR, e.g., upon antigen binding. In some embodiments, the intracellular domain comprises an ITAM signaling domain or an intracellular signaling domain containing the same. Exemplary intracellular signaling domains include, for example, signaling domains derived from the ζ chain of the T cell receptor complex or any homologue thereof (e.g., η chain, FcsRIy and β chain, MB 1(Iga) chain, B29(Ig) chain, etc.), human CD3 zeta chain, CD3 polypeptide (Δ, δ and ε), syk-family tyrosine kinases (Syk, ZAP 70, etc.), src-family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, e.g., CD2, CD5, OX40 and CD28. In a specific embodiment, the intracellular signaling domain contains an intracellular signaling domain derived from the human CD3 zeta chain.

[0161] In some embodiments, the intracellular signaling domain of the CAR may additionally contain an intracellular signaling domain derived from a co-stimulatory molecule. As an example, such a signaling domain can enhance CAR-T cell activity, for instance, by improving the proliferation, survival, and / or development of memory cells after antigen-specific binding, compared to a CAR containing, for instance, an ITAM-containing signaling domain, such as CD3 zeta. In some embodiments, the co-stimulatory domain is a functional signaling domain obtained from a protein selected from CD28, CD137 (4-IBB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1 (CD1 la / 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-IBB).

[0162] In some embodiments, the co-stimulatory signaling domain is derived from CD28 or 4-IBB.

[0163] In certain embodiments, the CAR further comprises a hinge or spacer region connecting the extracellular antigen-binding domain and the transmembrane domain. By using said hinge or spacer region, various lengths and flexibility of the resulting CAR can be achieved. Examples of hinge or spacer regions that may be used include, but are not limited to, an Fc fragment of an antibody or a fragment or derivative thereof, a hinge region of an antibody or a fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, such as a peptide sequence, or a combination thereof. Other hinge or spacer regions will also be apparent to those skilled in the art and may be used. In one embodiment, the hinge is an IgG4 hinge or a CD8A hinge.

[0164] In some embodiments, the spacer and transmembrane domain are hinge and transmembrane domains derived from CD8.

[0165] Additionally, an isolated nucleic acid construct comprising at least one nucleic acid encoding the CAR provided herein is also provided herein. In some aspects, said construct is an expression vector for the expression of the CAR in cells. The expression vector may be a viral vector. Viral vector technology is well known in the art and is described, for example, in the literature [Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2013)]. Various virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses such as adenovirus vectors are used. In one embodiment, a lentivirus vector is used.

[0166] In a further aspect, isolated cells or cell populations comprising one or more nucleic acid constructs as described above are provided. Additionally, isolated cells or cell populations genetically modified to express the CAR provided herein are also provided. Thus, genetically engineered cells comprising the CAR provided herein, such as those stably expressing it, are provided herein. In one embodiment, the cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, hematopoietic stem cells, and / or pluripotent embryonic / induced stem cells. In some cases, the cells are T cells, such as CD4 and / or CD8. In some embodiments, the cells are autologous to the subject. For example, in some embodiments, T cells may be isolated from a patient for manipulation using the CAR nucleic acid construct, such as for transfection or transduction (also referred to as primary T cells).

[0167] In an exemplary method, primary T cells can be purified in vitro (CD4 or CD8 cells, or both) and stimulated with a TCR / CD28 agonist, 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 primary T cells via a standard lentiviral or retroviral transduction protocol or a plasmid electroporation strategy. Cells can be monitored for the secretion of CAR expression, for example, by flow cytometry using an anti-epitope tag or antibody that cross-links with the natural parent molecule. T cells expressing the CAR can be aggregated via sorting using an anti-epitope tag antibody, or aggregated for high expression or low expression depending on the application.

[0168] CAR-engineered T cells can be analyzed by various methods to determine whether they are functioning properly. In some cases, the function of the engineered T cells may be evaluated using in vitro cytotoxicity, proliferation, or cytokine analysis (e.g., IFN-gamma expression). Exemplary standard endpoints include the lysis rate of tumor cell lines, proliferation of genetically engineered T cells, or IFN-gamma protein expression in the culture supernatant. In some cases, for example, the ability of the CAR to stimulate T cell activation upon antigen stimulation can be evaluated by monitoring the expression of activation markers such as CD69, CD44, or CD62L, proliferation, and / or cytokine production.

[0169] polypeptide expression and generation

[0170] A nucleic acid molecule comprising a polynucleotide encoding a CLEC12a binding polypeptide is provided. In some embodiments, the nucleic acid molecule may code for a leader sequence that induces the secretion of the CLEC12a binding polypeptide, wherein such leader sequence is generally cleaved so as not to be present in said secreted polypeptide. said leader sequence may be a natural heavy chain (or VHH) leader sequence or another heterogeneous leader sequence.

[0171] Nucleic acid molecules can be constructed using recombinant DNA technology conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in selected host cells.

[0172] A vector comprising a nucleic acid encoding the CLEC12a binding polypeptide described herein is provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector optimized for the expression of the polypeptide in a desired cell type, e.g., CHO or CHO-derived cells, or NSO cells is selected. Examples of such vectors are, for example, from the literature [Running Deer et al ., Biotechnol. Prog It is listed in . 20:880-889 (2004).

[0173] In some embodiments, the CLEC12a binding polypeptide may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (e.g., yeast), plant cells, insect cells, and mammalian cells. Such expression may be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used to express the polypeptide include 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; and PER.C6 ® Cells (Crucell); and NSO cells, including but not limited to these. In some embodiments, the CLEC12a binding polypeptide may be expressed in yeast. For example, see U.S. Publication US 2006 / 0270045 A1. In some embodiments, a specific eukaryotic host cell is selected based on its ability to make a desired post-translation modification to the polypeptide. For example, in some embodiments, CHO cells produce a polypeptide with a higher sialylation level than the same polypeptide produced in 293 cells.

[0174] The introduction of one or more nucleic acids (e.g., vectors) into a desired host cell may be performed 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, for example, [Sambrook] in the literature et al ., Molecular Cloning, A Laboratory Manual, 3 rd It is described in [ed. Cold Spring Harbor Laboratory Press (2001)]. Nucleic acids can be transiently or stably transfected in a desired host cell according to any appropriate method.

[0175] Host cells comprising any nucleic acid or vector described herein are also provided. In some embodiments, host cells expressing the CLEC12a binding polypeptide described herein are provided. The CLEC12a binding polypeptide expressed in the host cells may be purified by any suitable method. Such methods include, but are not limited to, the use of an affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include ROR1 ECD and agents that bind to the Fc region. For example, a protein A, protein G, protein A / G, or antibody affinity column may be used to bind to the Fc region and to purify the CLEC12a binding polypeptide containing said Fc region. Hydrophobic interaction chromatography, such as butyl or phenyl columns, may also be suitable for purifying some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may also be suitable for purifying some polypeptides, such as antibodies. Mixed chromatography (e.g., reverse phase / anion exchange, reverse phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be suitable for purifying some polypeptides, such as antibodies. Various methods for purifying polypeptides are known in the art.

[0176] In some embodiments, the CLEC12a binding polypeptide is produced in a cell-free system. Non-limiting exemplary cell-free systems are, for example, the literature [Sitaraman et al ., Methods Mol. Biol. 498: 229-44 (2009)]; [Spirin, Trends Biotechnol. 22: 538-45 (2004)]; [Endo et al ., Biotechnol. Adv It is described in . 21: 695-713 (2003).

[0177] In some embodiments, a CLEC12a-binding polypeptide prepared by the methods described above is provided. In some embodiments, the CLEC12a-binding polypeptide is prepared in a host cell. In some embodiments, the CLEC12a-binding polypeptide is prepared in a cell-free system. In some embodiments, the CLEC12a-binding polypeptide is purified. In some embodiments, a cell culture medium containing the CLEC12a-binding polypeptide is provided.

[0178] In some embodiments, a composition comprising an antibody prepared by the methods described above is provided. In some embodiments, the composition comprises a CLEC12a binding polypeptide prepared in a host cell. In some embodiments, the composition comprises a CLEC12a binding polypeptide prepared in a cell-free system. In some embodiments, the composition comprises a purified CLEC12a binding polypeptide.

[0179] An exemplary method for treating a disease using CLEC12a binding polypeptide

[0180] In some embodiments, a method for treating a disease in an individual is provided, comprising the step of administering a CLEC12a binding polypeptide or cells expressing a CLEC12a binding polypeptide. In some embodiments, a method for treating cancer in an individual is provided. In some embodiments, a method for treating CLEC12a expression or CLEC12a-positive cancer in an individual is provided. The method comprises the step of administering an effective amount of a CLEC12a binding polypeptide or cells expressing the CLEC12a binding polypeptide provided herein to an individual. In some embodiments, a cytotoxic agent is introduced into CLEC12a-expressing cells using a CLEC12a binding polypeptide. In some of these embodiments, the CLEC12a binding polypeptide comprises a binding domain that binds to cytotoxic T cells or NK cells. In some of these embodiments, the binding domain binds to CD3, T cell receptor (TCR)α, TCRβ, CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D. In some embodiments, the binding domain may be 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.

[0181] In some embodiments, the CLEC12a binding polypeptide is linked to a cytotoxic agent to form an immunoconjugate. Various cytotoxic agents used in immunoconjugates are known in the art and include, but are not limited to, calichiamycin, oristatin, dolastatin, tubulicin, meitansinoid, cryptophycin, duocarmycin, esperamicin, pyrrolobenzodiazepine, and endyin antibiotics.

[0182] In some embodiments, the CLEC12a binding polypeptide is a chimeric antigen receptor expressed on cytotoxic cells such as T cells (CAR-T) or NK cells (CAR-NK). This therapeutic method may be performed on humans or animals. In some embodiments, a method for treating humans is provided.

[0183] Representative, non-limiting cancers that can be treated with the CLEC12a binding polypeptide or cells expressing the CLEC12a binding polypeptide provided herein include, but are not limited to, lymphoma; Hodgkin lymphoma; non-Hodgkin lymphoma; B-cell lymphoma; low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immune-blastic NHL; high-grade lymphoblastic NHL; high-grade small non-severing cell NHL; macrolesion NHL; mantle cell lymphoma; AIDS-associated lymphoma; Waldenstrom macroglobulinemia; acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hair cell leukemia; and chronic myeloid leukemia. In some embodiments, the cancer is a cancer that expresses CLEC12a (i.e., CLEC12a positive).

[0184] CLEC12a binding polypeptide or cells expressing CLEC12a binding polypeptide may be administered to a subject as needed. The frequency of administration may be determined by a person skilled in the art, such as an attending physician, taking into account the condition to be treated, the age of the subject to be treated, the severity of the condition to be treated, and the overall health condition of the subject to be treated. In some embodiments, an effective dose of CLEC12a binding polypeptide or cells expressing CLEC12a binding polypeptide is administered to the subject one or more times. In some embodiments, an effective dose of CLEC12a binding polypeptide or cells expressing CLEC12a binding polypeptide is administered to the subject daily, twice a week, weekly, every two weeks, once a month, etc. An effective amount of CLEC12a binding polypeptide is administered to the subject at least once. An effective dose of CLEC12a-binding polypeptide or cells expressing CLEC12a-binding polypeptide is administered to a subject at least once. In some embodiments, an effective dose of CLEC12a-binding polypeptide or cells expressing CLEC12a-binding polypeptide may be administered multiple times, including several times over a course of at least one month, at least six months, or at least one year.

[0185] In some embodiments, the pharmaceutical composition is administered in an amount effective for the treatment (including prevention) of cancer. The therapeutically effective amount generally depends on the body weight of the subject to be treated, the physical or health condition of the subject, the range of the condition to be treated, or the age of the subject to be treated. Generally, the antibody may be administered in an amount ranging from about 0.05 mg / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 10 μg / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 50 μg / kg body weight to about 5 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 100 μg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.5 mg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.05 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount ranging from about 0.05 mg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, the antibody may be administered in an amount in a range of about 5 mg / kg body weight or less, e.g., less than 4, less than 3, less than 2, or less than 1 mg / kg.

[0186] In some embodiments, the CLEC12a binding polypeptide or cells expressing the CLEC12a binding polypeptide may be administered in vivo by various routes including, but not limited to, intravenous, intra-arterial, parenteral, intraperitoneal, or subcutaneous. Appropriate formulations and routes of administration may be selected according to the intended use.

[0187] In some embodiments, therapeutic treatment using a CLEC12a binding polypeptide is achieved by targeting a cytotoxic agent to cells expressing CLEC12a, such as cancer cells expressing CLEC12a. In some of these embodiments, the CLEC12a binding polypeptide is a chimeric antigen receptor expressed on cytotoxic cells, such as T cells or NK cells.

[0188] Pharmaceutical composition

[0189] In some embodiments, compositions comprising a CLEC12a binding polypeptide are provided in formulations with a wide variety of pharmaceutically acceptable carriers (e.g., the literature [Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003)]; [Ansel et al ., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th ed., Lippencott Williams and Wilkins (2004)]; [Kibbe et al ., Handbook of Pharmaceutical Excipients, 3 rd(See ed., Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers, including vehicles, adjuvants, and diluents, may be used. Additionally, various pharmaceutically acceptable adjuvants, such as pH adjusters, buffers, isotonic adjusters, stabilizers, and wetting agents, may also be used. Non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

[0190] In some embodiments, the pharmaceutical composition comprises CLEC12a-binding polypeptide at a concentration of at least 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, 225 mg / mL, or 250 mg / mL.

[0191] Combination therapy

[0192] The CLEC12a binding polypeptide or the genetically engineered cells of the present invention may be administered alone or in combination with other therapeutic methods, such as other anticancer agents. They may be provided before, substantially simultaneously with, or after the other therapeutic method (i.e., simultaneously or sequentially). In some embodiments, the therapeutic method described herein may further include the step of performing or administering the following: radiation therapy, chemotherapy, vaccination, tumor-targeted therapy, CAR-T therapy, tumor-disintegrating virus therapy, cancer immunotherapy, cytokine therapy, surgical resection, chromatin modification, resection, cryotherapy, antisense agents for tumor targets, siRNA agents for tumor targets, microRNA agents for tumor targets or anticancer / antitumor agents, or biological agents, such as antibodies, cytokines, or receptor extracellular domain-Fc fusions.

[0193] In some embodiments, the CLEC12a binding polypeptide provided herein is provided concurrently with one or more chemotherapy agents, CAR-T (chimeric antigen receptor T cell) therapy, tumor-disrupting virus therapy, cytokine therapy, and / or agents targeting other checkpoint molecules such as VISTA, gpNMB, B7H4, HHLA2, CD73, CTLA4, TIGIT, etc.

[0194] In some embodiments, the CLEC12a binding polypeptide or genetically engineered cells of the present invention may be other antitumor agents, e.g., anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (TARCEVA®), platelet-derived growth factor inhibitors (e.g., GLEEVEC® (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferon, CTLA4 inhibitors (e.g., anti-CTLA antibody ipilimumab (YERVOY®)), PD-1 inhibitors (e.g., anti-PD1 antibody, BMS-936558), PDL1 inhibitors (e.g., anti-PDL1 antibody, MPDL3280A), PDL2 inhibitors (e.g., anti-PDL2 antibody), cytokines, and the following targets: ErbB2, ErbB3, ErbB4, It is used in combination with PDGFR-beta, BlyS, APRIL, BCMA, PD-1, PDL1, PDL2, CTLA4, VEGF receptor(s), antagonists binding to one or more of TRAIL / Apo2 (e.g., neutralizing antibodies), and other bioactive and organic chemical agents.

[0195] In some embodiments, the CLEC12a binding polypeptide or genetically engineered cells provided herein are provided concurrently with PD-1 / PD-L1 therapy. Examples of PD-1 / PD-L1 therapy include nivolumab (BMS); fidilizumab (CureTech, CT-011), pembrolizumab (Merck); durvalumab (Medimmune / AstraZeneca); atezolizumab (Genentech / Roche); avelumab (Pfizer); AMP-224 (Amplimmune); BMS-936559; AMP-514 (Amplimmune); MDX-1105 (Merck); TSR-042 (Tesaro / AnaptysBio, ANB-011); STI-A1010 (Sorrento Therapeutics); STI-A1110 (Sorrento Therapeutics); and other agents that induce programmed death-1 (PD-1) or programmed death ligand 1 (PD-L1).

[0196] In some embodiments, the CLEC12a binding polypeptide of the present invention or genetically modified cells may be used in combination with a chemotherapy agent. Examples of chemotherapy agents include, but are not limited to, alkylating agents, such as thiotepa and CYTOXAN ®Cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan, and piposulfan; aziridines, e.g., benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoamide, triethylenethiophosphoamide, and trimethylolomelamine; acetogenins (particularly bulatacin and bulatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogs adozelesin, caselesin, and bizelesin); cryptophycin (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocamycin (including synthetic analogs KW-2189 and CB1-TM1); eleuterobin; pancratistatin; sarcodictin; Spongestatin; nitrogen mustard, e.g., chlorambucil, clonafazine, clophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novelichine, phenesterine, prednimustine, trophosphamide, uracil mustard; nitroureas, e.g., camustine, chlorozotocin, potemustine, lomustine, nimustine, and ranymnustine; antibiotics, e.g., endyin antibiotics [e.g., calichiamycin, particularly calichiamycin gamma 1I and calichiamycin omega 1I (e.g., literature [Agnew, Chem Intl. Ed. Engl [Refer to ., 33: 183-186 (1994)]; dynemycin including dynemycin A; bisphosphonates, e.g., clodronate; esperamicin; as well as neocasinostatin chromophores and related pigment proteins, enedin antibiotic chromophores, aclassinomycin, actinomycin, autranicin, azacerin, bleomycin, cactinomycin, carabicin, kaminomycin, casinophylline, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN ®Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, macelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogalanaicin, olibomycin, peflomycin, portpyromycin, furomycin, quellamycin, rhodorubicin, streptonigreen, streptozosin, tubecidin, uvenimex, genostatin, rhodorubicin; anmetatars, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogs, e.g., denopterin, methotrexate, pteropterin, trimetrexate; Purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiphrine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, camofur, cytarabine, dideoxyuridine, doxyfluridine, enositabine, floroxuridine; androgens, e.g., calosterone, dromostanolone propionate, epithiostanol, mepitiostan, testolactone; antiadrenal agents, e.g., aminoglutethimide, mitothan, trilostan; folic acid supplements, e.g., prolinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; enyluracil; amsacrine; vestrabusil; arsentren; edatlaxate; depopamine; demecolsin; diaziquone; eflornitine; Elliptinium acetate; Epotillone; Etogluside; Gallium nitrate; Hydroxyurea; Lentinan; Ronidinine; Meitancinoids, e.g., meitancin and ansamitosine; Mitoguazone; Mithoxantrone; Morpidanmol; Nitraerin; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-ethylhydrazide; Procarbazine; PSK ®Polysaccharide complexes (JHS Natural Products, Eugene, Oregon, USA); Razoxic acid; Lizoxin; Sizopyran; Spirogemanium; Tenuazonic acid; Triaziquone; 2,2',2"-Trichlorotriethylamine; Trichothecenes (specifically, T-2 toxin, Veraculin A, Roridin A, and Anguidin); Urethanes; Vindecin; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Acitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., TAXOL ® Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, New Jersey, USA), ABRAXANE ® Cremofor-free, Paclitaxel (American Pharmaceutical Partners, Schaumburg, Illinois, USA) and TAXOTERE ® Docetaxel (Rhone-Poulenc Rorer, Antony, France); Chlorambucil; GEMZAR ® Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, e.g., cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE ® Vinorelbine; Novantron; Tenifoside; Edatrexate; Daunomycin; Aminopterin; Exeloda; Ibandronate; Irinotecan (Camptosar, CPT-11) (including therapeutic regimens of irinotecan, 5-FU, and leucovorin); Topoisomerasase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids, e.g., retinoic acid; Capecitabine; Combretastatin; Leucovorin (LV); Oxaliplatin, including the oxaliplatin therapeutic regimen (FOLFOX); PKC-alpha, Raf, H-Ras, EGFR that reduce cell proliferation (e.g., Erlotinib (TARCEVA) ®Inhibitors of )) and VEGF-A, and pharmaceutically acceptable salts, acids, or derivatives of any of the aforementioned.

[0197] Additional non-restrictive exemplary chemotherapy agents include anti-hormonal agents that act to modulate or inhibit hormonal action against cancer, such as anti-estrogens, and selective estrogen receptor modulators (SERMs), such as tamoxifen (NOLVADEX ® (including tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and FARESTON ® Toremifene; aromatase inhibitors that inhibit aromatase, an enzyme that regulates estrogen production in the adrenal glands, e.g., 4(5)-imidazole, aminoglutethimide, MEGASE ® Megestrol Acetate, AROMASIN ® Exemestane, Formestani, Padrozol, RIVISOR ® Borozol, FEMARA ® Letrozole and ARIMIDEX ® Anastrozole; and anti-androgens, e.g., flutaamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as throxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways associated with abnormal cell proliferation, e.g., PKC-alpha, Ralf, and H-Ras; ribozymes, e.g., VEGF expression inhibitors (e.g., ANGIOZYME ® Ribozymes) and HER2 expression inhibitors; vaccines, e.g., gene therapy vaccines, e.g., ALLLOVECTIN ® Vaccine, LEUVECTIN ® Vaccines and VAXID ® Vaccine; PROLEUKIN ® (Aldesleukin) rIL-2; LURTOTECAN ® Topoisomerase 1 inhibitor; ABAELIX® GnRH agonists; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0198] In some embodiments, the CLEC12a binding polypeptide and additional agents are formulated into a single therapeutic composition, and the CLEC12a binding polypeptide and additional agents are administered simultaneously. Alternatively, the CLEC12a binding polypeptide or genetically modified cells and additional agents are separate from each other, for example, formulated into separate therapeutic compositions, and the CLEC12a binding polypeptide or genetically modified cells and additional agents are administered simultaneously, or the CLEC12a binding polypeptide or genetically modified cells and additional agents are administered at different times during the therapeutic regimen. For example, the CLEC12a binding polypeptide or genetically modified cells are administered before the administration of additional agents, the CLEC12a binding polypeptide or genetically modified cells are administered after the administration of additional agents, or the CLEC12a binding polypeptide or genetically modified cells and additional agents are administered alternately. CLEC12a binding polypeptide and additional preparations may be administered as a single dose or multiple doses.

[0199] In some embodiments, the CLEC12a binding polypeptide or genetically modified cells and additional preparation(s) are administered simultaneously. For example, the CLEC12a binding polypeptide and additional preparation(s) may be formulated as a single composition or administered as two or more separate compositions. In some embodiments, the CLEC12a binding polypeptide or genetically modified cells and additional preparation(s) are administered sequentially, or the CLEC12a binding polypeptide or genetically modified cells and additional preparation(s) are administered at different times during the therapeutic regimen.

[0200] Unlimited examples of diagnostic and treatment methods

[0201] In some embodiments, the method described herein is useful for evaluating a subject and / or a specimen of the subject (e.g., a cancer patient). In some embodiments, the evaluation is one or more of a diagnosis, prognosis, and / or response to treatment.

[0202] In some embodiments, the method described herein includes the step of evaluating the presence, absence, or level of a protein. In some embodiments, the method described herein includes the step of evaluating the presence, absence, or expression level of a nucleic acid. The compositions described herein may be used for such measurements. For example, in some embodiments, the method described herein includes the step of contacting a specimen of a tumor or cells cultured from a tumor with the therapeutic agent described herein.

[0203] In some embodiments, the evaluation may direct treatment (including treatment using the antibody described herein). In some embodiments, the evaluation may direct the use or withholding of adjuvant therapy after resection. Adjuvant therapy, also referred to as adjuvant medical care, is treatment provided in addition to primary, major, or initial treatment. As a non-limiting example, adjuvant therapy may be additional treatment typically provided after surgery when all detectable diseases have been eliminated but a statistical risk of recurrence remains due to latent disease. In some embodiments, the polypeptide is used as adjuvant therapy in cancer treatment. In some embodiments, the polypeptide is used as the sole adjuvant therapy in cancer treatment. In some embodiments, the polypeptide described herein is withheld as adjuvant therapy in cancer treatment. For example, if a patient is unlikely to respond to the antibody described herein or shows only a minimal response, treatment may be withheld for the sake of quality of life and to avoid unnecessary toxicity from ineffective chemotherapy. In such cases, a temporary prescription may be used.

[0204] In some embodiments, the polypeptide is administered as adjuvant therapy prior to resection. In some embodiments, adjuvant therapy refers to a therapy that shrinks and / or regresses a tumor prior to any surgery. In some embodiments, adjuvant therapy refers to chemotherapy administered to a cancer patient prior to surgery. In some embodiments, adjuvant therapy refers to administering an antibody to a cancer patient prior to surgery. Types of cancer for which adjuvant chemotherapy is generally considered include, for example, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, and lung cancer. In some embodiments, the polypeptide is used as adjuvant therapy in cancer treatment. In some embodiments, said use is performed prior to resection.

[0205] In some embodiments, the tumor microscopic environment considered in the method described herein is one or more of the tumor vascular system; tumor-infiltrating lymphocytes; fibroblastic reticular cells; endothelial progenitor cells (EPCs); cancer-associated fibroblasts; perivascular cells; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen-presenting cells; T cells; regulatory T cells; macrophages; other lymphoid cells; neutrophils; and other immune cells proximal to the tumor.

[0206] Kit

[0207] Additionally, manufactured articles and kits comprising any CLEC12a binding polypeptide described herein and suitable packaging are also provided. In some embodiments, the present invention comprises a kit comprising (i) a CLEC12a binding polypeptide, and (ii) instructions for use of the kit for administering said CLEC12a binding polypeptide to an individual.

[0208] Packaging suitable for the compositions described herein is known in the art and includes, for example, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. These products may be further sterilized and / or sealed. Additionally, unit formulations comprising the compositions described herein are also provided. These unit formulations may be stored in suitable packaging in single or multiple unit doses and may be further sterilized and sealed. Instructions provided in the kit of the present invention are generally instructions written on a label or packaging insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions included on a magnetic or optical storage disc) are also acceptable. Instructions regarding the use of the antibody generally include information on the dosage, administration schedule, and route of administration for the intended therapeutic or industrial use. The kit may further include instructions for selecting the appropriate subject for treatment.

[0209] The container may be a unit dose, bulk packaging (e.g., multi-dose packaging), or small unit dose. For example, a kit containing a sufficient dose of the molecule disclosed herein to provide effective treatment to an individual for a long period, such as 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, may also be provided. Additionally, the kit may include a multi-dose unit dose of the molecule and instructions for use, and is packaged in an amount sufficient for storage and use in pharmacies, such as hospital pharmacies and dispensing pharmacies. In some embodiments, the kit generally comprises a dry (e.g., lyophilized) composition that may be reconstituted, resuspended, or rehydrated to form a stable aqueous suspension of the antibody.

[0210] Examples

[0211] The embodiments discussed below are purely for illustrating the invention and should not be construed as limiting the invention in any way. These embodiments are not intended to represent all or only experiments performed. While efforts have been made to ensure accuracy regarding the figures used (e.g., amounts, temperatures, etc.), some experimental errors and deviations must be taken into account. Unless otherwise indicated, parts are by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0212] Example 1: CLEC12a single-domain antibody

[0213] A single-domain antibody targeting human CLEC12a was produced in the form of a recombinant human CLEC12a extracellular domain through the immunization of llamas and alpacas.

[0214] After the development of specific anti-CLEC12a antibody titers, peripheral blood mononuclear cells (PBMCs) from 500 mL of blood from immunized animals were isolated, and total mRNA was isolated using the Qiagen RNeasy Maxi kit and converted into first-strand cDNA using Thermo Superscript IV reverse transcriptase and oligo-dT priming. VHH sequences were specifically amplified via PCR using the cDNA as a template and cloned into a yeast surface expression vector as a VHH-Fc-AGA2 fusion protein.

[0215] A yeast library expressing the VHH-Fc-AGA2 fusion protein was aggregated using a recombinant CLEC12a ECD via magnetic bead separation, followed by fluorescence-activated cell sorting (FACS). The sorted yeasts were plated, and isolated colonies were selected, placed in a 96-well block, and grown in medium to switch expression from surface-expressed VHH-Fc to secretion into the medium. The supernatant of the 96-well yeast secretion culture was applied to 293F cells transiently transfected with CLEC12a (CLEC12a positive) or untransfected 293F cells (CLEC12a negative). After washing, the cells were treated with a fluorescently labeled anti-human IgG1 Fc secondary antibody and analyzed by 96-well flow cytometry.

[0216] A nucleic acid sequence encoding VHH that binds to CLEC12a-positive cells but not to CLEC12a-negative cells was cloned in-frame into a mammalian expression vector along with a human Fc coding region and expressed by transient transfection in HEK293 Freestyle cells (293F cells) or CHO cells using polyethyleneimine. After 3 to 7 days, the supernatant was collected, and the secreted recombinant protein was purified by protein A chromatography, after which the concentration was calculated from the absorbance and extinction coefficient at 280 nm.

[0217] The epitopes of a single-domain antibody (sdAb) containing a VHH domain that binds to CLEC12a were compared using biolayer interferorefractography. 7 µg / mL of mFc-CLEC12a-ECD (SEQ No. 87) was immobilized on an anti-mFc-coated capture sensor. Then, 100 nM of one sdAb was loaded onto the CLEC12a antigen and allowed to equilibrate. Subsequently, the sensor was transferred to 100 nM of a second sdAb. An increase in the analysis signal indicates binding, which means that the second sdAb targeted an epitope distinct from the epitope of the first sdAb.

[0218] CLEC12a VHH of camelid origin was humanized using the human VH3-23 germline line as a scaffold. Camelid residues contributing to solubility, specificity, stability, and / or affinity were not modified. Additionally, where possible and necessary, amino acid sequences with potential for development were modified to mitigate these risks. Furthermore, all humanized variants contained the Leu11Glu (L11E) modification as described in US20160207981.

[0219] As a result, three distinct epitopes were found among the humanized forms of sdAb MB11, ME06, MC02, MH07, MC06, and ME02. As shown in Fig. 1a, the sdAb hzME02v4 and hzME06v16 have epitopes different from the sdAb hzMB11v13, hzMC02v6, hzMH07v3, and hzMC06v2. As shown in Fig. 1b, none of the sdAb hzME06v16, hzME02v4, and hzMB11v13 had a common epitope. A summary of the above results is shown in Table 3 below.

[0220]

[0221] Example 2: Binding of polypeptide to CLEC12a

[0222] The binding of sdAbs to human CLEC12a was evaluated by flow cytometry. Each sdAb contained a human IgG1 xELL Fc region with a VHH domain as shown in Table 4 below, and amino acids Glu233, Leu234, and Leu235 deleted according to EU numbering (SEQ No. 51). HEK 293 cells were transiently transfected with a plasmid encoding CLEC12a (UniProt registry No. Q5QGZ9.3; SEQ No. 1) and used as a CLEC12a-positive cell line, while untransfected HEK 293 cells were used as CLEC12a-negative cells. 30,000 cells per well of each cell type were plated in FACS buffer (PBS 1% BSA, 0.1% NaN3, pH 7.4) on 96-well round-bottom plates. sdAb was diluted in FACS buffer by a 3-fold, 11-point serial dilution. The diluted sdAb solution was added to the plated cells, and the analysis plate was incubated at 4°C for 30 minutes. After washing twice in 150 μL of FACS buffer, the cells in each well were resuspended in 100 μL of 1:2000 Alexa Fluor 647-conjugated anti-human IgG secondary antibody diluted in FACS buffer and incubated at 4°C for 30 minutes. After washing the cells two more times, the conjugated antibody was detected by flow cytometry.

[0223] Flow cytometry was performed on Intellicyte iQue Plus, and fluorescence was plotted against median fluorescence intensity. Apparent affinity (K d , nM) was measured.

[0224] As shown in Figures 2a to 2l, the tested sdAb exhibited CLEC12a binding, and did not bind or showed minimal binding to untransfected HEK 293 cells that do not express CLEC12a. Apparent binding affinities are shown in Table 4 below.

[0225]

[0226] The present invention may be embodied in other specific forms without departing from its spirit or essential nature. Accordingly, the embodiments described above should be regarded as all exemplary aspects rather than limiting the invention. Accordingly, since the scope of the invention is defined by the appended claims rather than by the description above, all variations falling within the meaning and scope of equivalents of such claims are to be included herein.

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

Claims

Claim 1 A polypeptide comprising at least one VHH domain that binds to CLEC12a, comprising CDR1 having the amino acid sequence of SEQ ID NO. 32; CDR2 having the amino acid sequence of SEQ ID NO. 33; and CDR3 having the amino acid sequence of SEQ ID NO. 34 or 5. Claim 2 A polypeptide according to claim 1, wherein at least one VHH domain is humanized. Claim 3 A polypeptide according to claim 1, wherein at least one VHH domain comprises an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO. 26 or 94. Claim 4 A polypeptide according to claim 1, wherein at least one VHH domain comprises the amino acid sequence of SEQ ID NO. 26 or 94. Claim 5 A polypeptide comprising two or three VHH domains in claim 1. Claim 6 A polypeptide according to claim 1, wherein the polypeptide comprises at least one binding domain that binds to an antigen other than CLEC12a. Claim 7 A polypeptide according to claim 6, wherein the polypeptide comprises at least one binding domain that binds to CD3, T cell receptor (TCR)α, TCRβ, CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D. Claim 8 In paragraph 5, a polypeptide in which each VHH domain binds to CLEC12a. Claim 9 In claim 8, a polypeptide in which each VHH domain comprises the same CDR1, CDR2, and CDR3 amino acid sequence. Claim 10 In paragraph 8, a polypeptide in which each VHH domain contains the same VHH sequence. Claim 11 A polypeptide comprising one VHH domain in claim 1. Claim 12 A polypeptide according to claim 1, wherein the polypeptide comprises an Fc region. Claim 13 A polypeptide according to claim 12, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NOs 50 to 85. Claim 14 In paragraph 12, a polypeptide that forms a dimer under physiological conditions. Claim 15 In claim 1, the polypeptide wherein the CLEC12a is human CLEC12a. Claim 16 In claim 15, the polypeptide wherein the human CLEC12a comprises the sequence of SEQ ID NO.

1. Claim 17 An immunoconjugate comprising the polypeptide of claim 1 and a cytotoxic agent. Claim 18 An immunoconjugate according to claim 17, wherein the cytotoxic agent is selected from the antibiotics chalichemycin, oristatin, dolastatin, tubulicin, meitansinoid, cryptophycin, duocarmycin, esperamicin, pyrrolobenzodiazepine, and enediyne. Claim 19 Isolated nucleic acid encoding the polypeptide of any one of claims 1 to 16. Claim 20 A vector containing the nucleic acid of claim 19. Claim 21 Isolated host cell containing the nucleic acid of paragraph 19. Claim 22 An isolated host cell expressing the polypeptide of any one of claims 1 to 16. Claim 23 A method for producing a polypeptide according to any one of claims 1 to 16, comprising the step of incubating a host cell containing a nucleic acid encoding said polypeptide under conditions suitable for the expression of said polypeptide. Claim 24 A method according to claim 23, further comprising the step of isolating the polypeptide. Claim 25 A pharmaceutical composition for treating cancer in a subject requiring treatment for cancer, comprising a pharmaceutically effective amount of the polypeptide of any one of claims 1 to 16, or the immunoconjugate of claim 17 or 18. Claim 26 A pharmaceutical composition according to claim 25, 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; medium-grade / follicular NHL; medium-grade diffuse NHL; high-grade immune blastocyst NHL; high-grade lymphoblastic NHL; high-grade small non-severing cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-associated lymphoma; Waldenstrom macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hair cell leukemia; and chronic myeloid leukemia. Claim 27 A pharmaceutical composition according to claim 26, wherein the cancer is acute myeloid leukemia (AML). Claim 28 A pharmaceutical composition according to claim 25, wherein an additional therapeutic agent is further administered to the subject. Claim 29 A pharmaceutical composition according to claim 28, wherein the additional therapeutic agent is an anticancer agent. Claim 30 A pharmaceutical composition according to claim 29, wherein the anticancer agent is selected from chemotherapy agents, anticancer biological agents, radiation therapy, CAR-T therapy, and oncolytic viruses. Claim 31 A pharmaceutical composition according to claim 25, wherein the cancer is a CLEC12a expressing cancer. Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete

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