EA046257B1 - CLEC12A-BINDING POLYPEPTIDES AND THEIR APPLICATIONS - Google Patents

Description

Cross reference to related applications

This application claims the benefit of U.S. Provisional Application No. 62/843411, filed May 4, 2019, and U.S. Provisional Application No. 62/844426, filed May 7, 2019, each of which is incorporated herein by reference for all purposes.

Field of technology to which the invention relates

The present invention relates to CLEC12a-binding polypeptides and methods of using CLEC12a-binding polypeptides to modulate the biological activity of CLEC12a. Such methods include, but are not limited to, methods for treating cancer.

State of the art

CLEC12a, also known as CLL-1, CLL1, DCAL-2, MICL, CD371 or C-type lectin domain family 12 member A, is a member of the C-type lectin domain/C-type lectin-like domain (CTL/CTLD) superfamily and regulates activation of myeloid cells. CLEC12a is also a marker of blast and leukemia stem cells in patients with acute myeloid leukemia (AML). In addition, CLEC12a has been used to detect minimal residual disease in patients after treatment for AML. Therefore, there is a therapeutic need for more effective treatments for CLEC12a-expressing cancers.

The essence of the invention

The present invention provides CLEC12a-binding polypeptides and methods of using CLEC12a-binding polypeptides for the treatment of, for example, leukemia. In some embodiments, the CLEC12a-binding polypeptide contains at least one VHH domain. Some embodiments are presented below.

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

Embodiment 2. The polypeptide of Embodiment 1, wherein at least one VHH domain contains a CDR1 containing the amino acid sequence of SEQ ID NO: 47 or 23; CDR2 containing the amino acid sequence SEQ ID NO: 48 or 24; and CDR3 containing the amino acid sequence of SEQ ID NO: 49 or 25.

Embodiment 3. The polypeptide of Embodiment 1 or Embodiment 2, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 32 or 3; CDR2 containing the amino acid sequence of SEQ ID NO: 33 or 4; and CDR3 containing the amino acid sequence of SEQ ID NO: 34 or 5.

Embodiment 4. The polypeptide of any one of embodiments 1-3, wherein at least one VHH domain contains a CDR1 containing the amino acid sequence of SEQ ID NO: 7 or 35; CDR2 containing the amino acid sequence of SEQ ID NO: 8 or 36; and CDR3 containing the amino acid sequence of SEQ ID NO: 9 or 37.

Embodiment 5. The polypeptide of any one of embodiments 1-4, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 11 or 38; CDR2 containing the amino acid sequence of SEQ ID NO: 12 or 39; and CDR3 containing the amino acid sequence of SEQ ID NO: 13 or 40.

Embodiment 6. The polypeptide of any one of embodiments 1-5, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 15 or 41; CDR2 containing the amino acid sequence of SEQ ID NO: 16 or 42; and CDR3 containing the amino acid sequence of SEQ ID NO: 17 or 43.

Embodiment 7. The polypeptide of any one of embodiments 1-6, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 19 or 44; CDR2 containing the amino acid sequence SEQ ID NO: 20 or 45; and CDR3 containing the amino acid sequence of SEQ ID NO: 21 or 46.

Embodiment 8. The polypeptide of any one of embodiments 1 to 7, wherein at least one VHH domain comprises CDR1, CDR2, and CDR3 containing, respectively, the amino acid sequences of SEQ ID NO: 32, 33, and 34; SEQ ID NO: 47, 48 and 49; SEQ ID NO: 3, 4 and 5; SEQ ID NO: 7, 8 and 9; SEQ ID NO: 11, 12 and 13; SEQ ID NO: 15, 16 and 17; SEQ ID NO: 19, 20 and 21; SEQ ID NO: 23, 24 and 25; SEQ ID NO: 35, 36 and 37; SEQ ID NO: 38, 39 and 40; SEQ ID NO: 41, 42 and 43; SEQ ID NO: 44, 45 and 46.

Embodiment 9. The polypeptide of any one of embodiments 1-8, wherein at least one VHH domain is humanized.

Embodiment 10. The polypeptide of any one of embodiments 1-9, wherein at least one VHH domain contains an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or at least 99 identical. % amino acid sequence SEQ ID NO: 26, 94, 31, 99, 27, 28, 29, 30, 95, 96, 97 or 98.

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Embodiment 11. The polypeptide of any one of embodiments 1-9, wherein at least one VHH domain contains the amino acid sequence SEQ ID NO: 26, 94, 31, 99, 27, 28, 29, 30, 95, 96, 97 or 98.

Embodiment 12. The polypeptide of any one of embodiments 1-8, wherein at least one VHH domain contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99 identical. % amino acid sequence SEQ ID NO: 2, 6, 10, 14, 18, 22, 88, 89, 90, 91, 92 or 93.

Embodiment 13. The polypeptide of any one of embodiments 1-8, wherein at least one VHH domain contains the amino acid sequence SEQ ID NO: 2, 6, 10, 14, 18, 22, 88, 89, 90, 91, 92 or 93.

Embodiment 14. The polypeptide of any one of embodiments 1-13, comprising two VHH domains.

Embodiment 15. The polypeptide of any one of embodiments 1-13, comprising three VHH domains.

Embodiment 16. The polypeptide of any one of embodiments 1-15, comprising at least one binding domain that binds to an antigen other than CLEC12a.

Embodiment 17. The polypeptide of Embodiment 16 comprising at least one binding domain that binds CD3, T-cell receptor (TCR)a, TCRe, CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D.

Embodiment 18. The polypeptide of embodiment 14 or 15 in which each VHH domain binds to CLEC12a.

Embodiment 19. The polypeptide of Embodiment 18, wherein each VHH domain contains the same amino acid sequences CDR1, CDR2 and CDR3.

Embodiment 20. The polypeptide of Embodiment 18, wherein each VHH domain contains the same VHH sequence.

Embodiment 21. The polypeptide of any one of embodiments 1-13, comprising a single VHH domain.

Embodiment 22. The polypeptide of any one of embodiments 1-21, comprising an Fc region.

Embodiment 23. The polypeptide of Embodiment 22, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NOs: 50-85.

Embodiment 24. The polypeptide of Embodiment 22 or Embodiment 23 that forms a dimer under physiological conditions.

Embodiment 25. The polypeptide of any one of embodiments 1-24, wherein CLEC12a is human CLEC12a.

Embodiment 26. The polypeptide of Embodiment 25, wherein human CLEC12a comprises the sequence SEQ ID NO: 1.

Embodiment 27. An immunoconjugate comprising the polypeptide of any one of embodiments 1-26 and a cytotoxic agent.

Embodiment 28. The immunoconjugate of Embodiment 27, wherein the cytotoxic agent is selected from calicheamicin, auristatin, dolastatin, tubulicin, maytansinoid, cryptophycin, duocarmycin, esperamycin, pyrrolobenzodiazepine, and enediyne antibiotic.

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

Embodiment 30. An isolated nucleic acid encoding the polypeptide of any one of embodiments 1-26.

Embodiment 31. A vector containing the nucleic acid of Embodiment 30.

Embodiment 32. A host cell containing the nucleic acid of Embodiment 34 or the vector of Embodiment 31.

Embodiment 33. A host cell that expresses the polypeptide of any one of embodiments 1-26.

Embodiment 34. The method for producing a polypeptide as in any one of Embodiments 1-26, comprising incubating a host cell as in Embodiment 32 or Embodiment 33 under conditions suitable for expression of the polypeptide.

Embodiment 35. The method of Embodiment 37, further comprising isolating the polypeptide.

Embodiment 36. A method of treating cancer, comprising administering to a subject suffering from cancer a pharmaceutically effective amount of the polypeptide of any one of Embodiments 1-26, the immunoconjugate of Embodiment 27 or Embodiment 28, or the pharmaceutical composition of Embodiment 29.

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Embodiment 37. The method of Embodiment 36, wherein the cancer is selected from lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; low-grade/follicular non-Hodgkin's lymphoma (NHL); NHL from small lymphocytes (ML); moderate malignancy/follicular NHL; diffuse NHL of moderate malignancy; immunoblastic NHL of high grade; high-grade lymphoblastic NHL; NHL from small non-divided cells with a high degree of malignancy; volumetric NHL; mantle cell lymphomas; AIDS-related lymphoma; Waldenström's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; and chronic myeloblastic leukemia.

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

Embodiment 39. The method of any one of embodiments 36-38, further comprising administering an additional therapeutic agent.

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

Embodiment 41. The method of Embodiment 40, wherein the anticancer agent is selected from a chemotherapeutic agent, a biological anticancer agent, radiation therapy, CAR-T therapy, and an oncolytic virus.

Embodiment 42. The method of any one of embodiments 36-39, wherein the cancer is a CLEC12a-expressing cancer.

Brief description of drawings

In fig. 1A and 1B show biolayer interferometry data for polypeptides containing a VHH domain that binds to CLEC12a. In fig. 1A shows biolayer interferometry data for hzMB11v13 compared to other CLEC12a-binding sdAbs disclosed in the present invention. In fig. 1B shows biolayer interferometry data for hzME06v16 compared to other CLEC12a-binding sdAbs disclosed in the present invention.

In fig. 2A-2L show the binding of certain single domain antibodies (sdAbs) to CLEC12a expressed on HEK 293 cells. CLEC12a-HEK-293 indicates HEK 293 cells that have been transfected with a plasmid encoding CLEC12a as described in Example 2. HEK-293 indicates untransfected cells HEK 293. In FIG. Figure 2A shows the binding of MB11-IgG1 to CLEC12a. In fig. 2B shows the binding of hzMB11v13-IgG1 to CLEC12a. In fig. Figure 2C shows the binding of ME06-IgG1 to CLEC12a. In fig. 2D shows the binding of hzME06v16-IgG1 to CLEC12a. In fig. Figure 2E shows the binding of MC02-IgG1 to CLEC12a. In fig. 2F shows the binding of hzMC02v6-IgG1 to CLEC12a. In fig. Figure 2G shows the binding of MH07-IgG1 to CLEC12a. In fig. 2H shows the binding of hzMH07v3-IgG1 to CLEC12a. In fig. 2I shows the binding of MC06-IgG1 to CLEC12a. In fig. Figure 2J shows the binding of hzMC06v2-IgG1 to CLEC12a. In fig. Figure 2K shows the binding of ME02-IgG1 to CLEC12a. In fig. Figure 2L shows the binding of hzME02v4-IgG1 to CLEC12a.

Detailed description

Embodiments provided herein relate to CLEC12a binding polypeptides and their use in various methods of treating cancer.

Definitions and various embodiments.

The section headings used in the present invention are for organizational purposes only and should not be construed as limiting the subject matter of the invention described.

All references cited in the present invention, including patent applications, patent publications and Genbank accession numbers, are incorporated herein by reference as if each individual reference were specifically and separately identified as being incorporated by reference in its entirety.

The methods and procedures disclosed or referred to in the present invention are generally well known and conventional methods commonly used by those skilled in the art, such as, for example, the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel et al., eds. (2003)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor, eds. (1995)), Harlow and Lane, eds. (1988), ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998), Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998), Plenum Press; Cell and Tissue Culture Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8), J. Wiley and Sons; Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in

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Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993); and their updated versions.

Unless otherwise specified, scientific and technical terms used in the context of the present invention have the meanings commonly used by those skilled in the art. In addition, unless the context otherwise requires or is expressly stated, terms in the singular are intended to include the plural and terms in the plural are intended to include the singular. In the event of any conflict in definitions between different sources or references, the definition given in the present invention prevails.

Typically, the numbering of residues in the immunoglobulin heavy chain corresponds to the EU index according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The Kabat EU index refers to the EU numbering of human IgG1 antibody residues.

It should be appreciated that the embodiments disclosed in the present invention include embodiments consisting of and/or essentially consisting of. As used herein, the singular forms include references to the plural unless otherwise indicated. The use of the term or in the present invention does not imply that the alternatives are mutually exclusive.

In the present invention, the use of or means and/or, unless otherwise explicitly stated or obvious to one skilled in the art. In the context of a multiple-dependency claim, the use of or refers to more than one preceding independent or dependent claim.

The phrase reference sample, reference cell, or reference tissue means a sample with at least one known characteristic that can be used as a comparison to a sample with at least one unknown characteristic. In some embodiments, a reference sample can be used as a positive or negative indicator. The reference sample can be used to determine the level of protein and/or mRNA observed, for example, in healthy tissue, as opposed to the level of protein and/or mRNA in a sample with unknown characteristics. In some embodiments, the reference sample is obtained from the same subject, but from a different part than the one being tested. In some embodiments, the reference sample is taken from an area of tissue surrounding or adjacent to the cancer. In some embodiments, the reference sample is obtained not from a subject, but from a subject known to have or not to have the disorder of interest (eg, a specific cancer or a CLEC12a-related disorder). In some embodiments, the reference sample is obtained from the same subject, but before the onset of cancer. In some embodiments, the reference sample is derived from a benign cancer sample from the same or a different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule of interest in the negative reference sample will indicate a level at which one skilled in the art would be able to determine, given the present disclosure, that the molecule is absent and/or at a low level. When a positive reference sample is used for comparison, the expression level or amount of the molecule of interest in the positive reference sample will indicate a level at which one of ordinary skill in the art would be able to determine, given the present disclosure, that the molecule is present at some level.

The terms benefit, clinical benefit, susceptibility and therapeutic susceptibility as used in the present invention in the context of the beneficial effect or response to administration of a therapeutic agent can be measured by assessing various endpoints, for example, inhibition to some extent of disease progression, including slowing and stopping; reducing the number of episodes and/or symptoms of the disease; reducing the size of the lesion; inhibiting (i.e. reducing, slowing or completely stopping) the infiltration of pathogenic cells into adjacent peripheral organs and/or tissues; inhibiting (i.e. reducing, slowing down or completely stopping) the spread of the disease; alleviating to some extent one or more symptoms associated with the disorder; increasing the duration of relapse-free course after treatment, for example progression-free survival; increased overall survival; higher response speed; and/or reduction in mortality at a certain point in time after treatment. A subject or cancer that does not respond or does not respond is a subject or cancer that does not meet the above susceptibility requirements.

The terms nucleic acid molecule, nucleic acid and polynucleotide can be used interchangeably and refer to a polymer of nucleotides. Such nucleotide polymers can

- 4 046257 contain natural and/or non-natural nucleotides and include, without limitation, DNA, RNA and PNA. Nucleic acid sequence refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

The terms polypeptide and protein are used interchangeably to refer to a polymer composed of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. This definition covers both full-length proteins and their fragments. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Moreover, for purposes of the present disclosure, a polypeptide refers to a protein that includes modifications such as deletions, additions and substitutions (generally conservative in nature) in the native sequence, provided that the protein retains the desired activity. These modifications may be intentional, such as the result of site-directed mutagenesis, or may be accidental, such as the result of mutations in the protein-producing hosts or errors in the PCR amplification process.

CLEC12a as used herein refers to any native mature CLEC12a that results from processing of a CLEC12a precursor in the cell. The term includes CLEC12a from any vertebrate source, including mammals such as primates (eg, humans and cynomolgus or rhesus monkeys) and rodents (eg, mice and rats), unless otherwise noted. The term also includes naturally occurring variants of CLEC12a, such as splice variants or allelic variants. A non-limiting exemplary amino acid sequence of human CLEC12a is shown, for example, in UniProt under accession number Q5QGZ9.3. See SEQ ID NO. 1.

The term specifically binds to an antigen or epitope is a term that is well known in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit specific binding or preferential binding if it reacts or binds more often, faster, stays bound longer, and/or binds with higher affinity to a particular cell or substance than to alternative cells or substances. A single-domain antibody (sdAb) or VHH-containing polypeptide specifically binds or binds preferentially to a target if it binds with higher affinity, avidity, is more readily bound, and/or remains bound longer than other substances. For example, a sdAb or VHH-containing polypeptide that specifically or preferentially binds to an epitope of CLEC12a is a sdAb or VHH-containing polypeptide that binds to that epitope with higher affinity, avidity, and is more easily and/or longer bound than with other CLEC12a epitopes or non-CLEC12a epitopes. It will also be understood by reading this definition that, for example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, specific binding or preferential binding does not necessarily require (although may include) exclusive binding. Typically, but not necessarily, a linking reference means a preferred binding. Specificity refers to the ability of a binding protein to selectively bind antigen.

The terms inhibition or inhibit refer to the reduction or elimination of a phenotypic trait, or to the reduction or elimination of the frequency, extent, or likelihood of that trait. To reduce or inhibit means to reduce, reduce or suppress activity, function and/or quantity compared to a reference. In some embodiments, reduction or inhibition means the ability to cause an overall reduction of 10% or more. In some embodiments, reduction or inhibition means the ability to cause an overall reduction of 50% or more. In some embodiments, reduction or inhibition means the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the above amount is suppressed or reduced over a period of time compared to a control over the same period of time. As used herein, the term inhibit in relation to CLEC12a activity refers to a decrease in the activity of CLEC12a, such as binding to monosodium urate. In some embodiments, inhibit refers to a decrease in the activity of CLEC12a compared to the activity of CLEC12a in the absence of the modulator. In some embodiments, the CLEC12a binding polypeptide inhibits the binding of CLEC12a to monosodium urate.

As used herein, the term epitope refers to a site on a target molecule (eg, an antigen, such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (eg, an sdAb or a VHH-containing polypeptide) binds. Epitopes often include chemically active surface groups of molecules, such as amino acids, polypeptides, or sugar side chains, and have certain three-dimensional structure characteristics as well as specific properties.

- 5 046257 technical characteristics of the charge. Epitopes can be formed from either contiguous and/or adjacent residues (eg, amino acids, nucleotides, sugars, lipid moiety) of the target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moiety) are typically retained when exposed to denaturing solvents, whereas epitopes formed from tertiary stacking are typically lost when treated with denaturing solvents. An epitope may include, without limitation, at least 3, at least 5, or 8-10 residues (eg, amino acids or nucleotides). In some embodiments, the epitope is less than 20 residues (eg, amino acids or nucleotides) in length, less than 15 residues, or less than 12 residues. Two antibodies can bind to the same epitope within an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope can be identified by a certain minimum distance to a CDR residue on an antigen-binding molecule. In some embodiments, an epitope may be identified by the above distance and further limited to those residues that participate in a bond (eg, a hydrogen bond) between the antigen binding molecule residue and the antigen residue. An epitope can also be identified using different types of scans, for example an alanine or arginine scan can indicate one or more residues with which an antigen binding molecule can interact. Unless explicitly stated otherwise, a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antigen-binding molecule. Rather, the presence of such a set means the presence of a minimal series (or set of species) of epitopes. Thus, in some embodiments, the set of residues identified as an epitope refers to the minimum epitope relevant to the antigen, rather than an exhaustive list of epitope residues on the antigen.

A non-linear epitope or conformational epitope includes non-contiguous polypeptides, amino acids and/or sugars in an antigenic protein to which an epitope-specific antigen binding molecule binds. In some embodiments, at least one of the residues will not be adjacent to other specified residues of the epitope; however, one or more residues may also be adjacent to other residues.

A linear epitope contains contiguous polypeptides, amino acids and/or sugars in an antigenic protein to which an epitope-specific antigen-binding molecule binds. It should be noted that in some embodiments, each of the residues in the linear epitope is not required to be directly associated with (or involved in the formation of a linkage with) an antigen binding molecule. In some embodiments, the linear epitopes may be from immunizations with a peptide actually consisting of the linear epitope sequence, or from structural regions of the protein that are relatively separate from the rest of the protein (such that the antigen binding molecule can interact at least substantially only with this region of the sequence ).

The term antibody is used in the broadest sense and covers a variety of polypeptides that contain antibody-like antigen-binding domains, including, but not limited to, traditional antibodies (typically containing at least one heavy chain and at least one light chain), single-domain antibodies (sdAbs, containing at least one VHH domain and Fc region), VHH-containing polypeptides (polypeptides containing at least one VHH domain) and fragments of any of the above, provided that 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, heavy chain constant domains (including CH1, hinge, CH2, and CH3, where CH1 is typically paired with a light chain constant domain, CL, while the hinge mediates dimerization) and Fc regions (including hinge, CH2, and CH3, where the hinge mediates dimerization).

The term antibody also includes, without limitation, chimeric antibodies, humanized antibodies, and antibodies from various species such as camel (including llama), shark, mouse, human, cynomolgus, etc.

As used herein, the term antigen binding domain refers to the portion of an antibody sufficient to bind 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 portions of FR1 and/or FR4 necessary to maintain antigen binding, and a light chain variable region comprising CDR1 -FR2CDR2-FR3-CDR3 and any parts of FR1 and/or FR4 necessary to maintain antigen binding. In some embodiments, the antigen binding domain of the sdAb or VHH-containing polypeptide contains three VHH CDR domains. Thus, in some embodiments, the antigen binding domain of an sdAb or VHH-containing polypeptide comprises a VHH domain containing CDR1-FR2-CDR2-FR3-CDR3, and any portions of FR1 and/or FR4 necessary to maintain binding to the antigen.

The term VHH, or VHH domain, or VHH antigen binding domain as used herein

- 6 046257 of the description refers to the antigen binding part of a single domain antibody, such as a camel antibody or a shark antibody. In some embodiments, the VHH contains three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In some embodiments, the VHH may be truncated at the N-terminus or C-terminus such that it contains only the FR1 and/or FR4 portion, or may lack one or both of these framework regions, provided that the VHH substantially retains antigen binding and specificity to it.

The terms single domain antibody and sdAb are used interchangeably as used herein to refer to an antibody comprising at least one monomeric domain, such as a VHH domain, without a light chain, and an Fc region. In some embodiments, the sdAb is a dimer of two polypeptides, where each polypeptide contains at least one VHH domain and an Fc region. As used herein, the terms single domain antibody and sdAb encompass polypeptides that contain multiple VHH domains, such as a polypeptide having the structure VHH1-VHH2-Fc or VHH1-VHH2-VHH3-Fc, where VHH1, VHH2 and VHH3 may be the same or different.

The term VHH-containing polypeptide refers to a polypeptide that contains at least one VHH domain. In some embodiments, the VHH polypeptide contains two, three, or four, or more VHH domains, where each VHH domain may be the same, or the VHH domains may be different. In some embodiments, the VHH-containing polypeptide contains an Fc region. In some such embodiments, the VHH-containing polypeptide may be referred to as a sdAb. Additionally, in some such embodiments, the VHH polypeptide may form a dimer. Non-limiting structures of VHH-containing polypeptides that are also sdAbs include VHH ₁ -Fc, VHH ₁ -VHH ₂ -Fc, and VHH ₁ -VHH ₂ -VHH ₃ -Fc, wherein VHH ₁ , VHH ₂ and VHH ₃ may be the same or different. In some embodiments of such structures, one VHH may be linked to another VHH via a linker, or one VHH may be linked to an Fc via a linker. In some such embodiments, the linker contains 1-20 amino acids, preferably 1-20 amino acids, predominantly consisting of glycine and optionally serine. In some embodiments, when the VHH-containing polypeptide contains an Fc, it forms a dimer. Thus, the VHHj-VHH ₂ -Fc structure, in the case of dimer formation, is considered tetravalent (ie, the dimer has four VHH domains). Similarly, the VHH ₁ -VHH ₂ -VHH ₃ -Fc structure, when formed as a dimer, is considered hexavalent (ie the dimer has six VHH domains).

The term monoclonal antibody refers to an antibody (including an sdAb or VHH-containing polypeptide) from a substantially homogeneous population of antibodies, i.e. the individual antibodies that make up such a population are identical, except for possible naturally occurring mutations that may be present in minute quantities. Monoclonal antibodies are highly specific and directed against a single antigenic site. Additionally, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. Thus, a sample of monoclonal antibodies can bind to the same epitope on an antigen. The modifier monoclonal indicates the nature of the antibody as being derived from an essentially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be produced by hybridoma technology, first described by Kohler and Milstein, 1975, Nature, 256:495, or can be produced by recombinant DNA methods described in US Pat. No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries, obtained by methods described, for example, in McCafferty et al., 1990, Nature, 348:552-554.

The term CDR means a complementarity determining region as determined by one skilled in the art by at least one identification method. In some embodiments, CDRs may be defined in accordance with any of the Chothia, Kabat, Kabat and Chotia combinations, AbM definition, and/or contact definition. VHH contains three CDRs, designated CDR1, CDR2 and CDR3.

As used herein, the term heavy chain constant region refers to a region containing at least three heavy chain constant domains, CH1, hinge, CH2 and CH3. The term heavy chain constant region certainly includes non-function-altering deletions and substitutions within domains unless otherwise noted. Non-limiting exemplary heavy chain constant regions include γ, δ and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy constant region corresponds to an antibody isotype. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, and an antibody containing an α constant region is an IgA antibody. Also, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε constant region is an IgE antibody. Some isotypes may be further subdivided into subclasses. For example,

- 7 046257 IgG antibodies include, but are not limited to, IgGl (γ1 constant region), IgG2 (γ ₂ constant region), IgG3 (γ ₃ constant region) and IgG4 (γ ₄ constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing the α1 constant region) and IgA2 (containing the _α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

As used herein, the term Fc region refers to the portion of the heavy chain constant region containing CH2 and CH3. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. In various embodiments, when the Fc region contains a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region may be any isotype of the antibody heavy chain constant region discussed in the present invention. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

An acceptor human framework as used herein is a framework comprising an amino acid sequence of a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as discussed herein. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence, or it may contain amino acid sequence substitutions. In some embodiments, the number of amino acid substitutions is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3 in all human framework regions in a single antigen binding domain, such as VHH .

Affinity refers to the strength of the aggregate non-covalent interactions between one binding site of a molecule (eg, an antibody such as an sdAb, or a VHH-containing polypeptide) and its binding partner (eg, an antigen). The affinity or apparent affinity of a molecule X for its partner Y can usually be represented by the dissociation constant (Kd) or _Kdapparent , respectively. Affinity can be measured by conventional methods known in the art (such as, for example, Kd ELISA, KinExA, flow cytometry and/or using a surface plasmon resonance device), including those presented in the present invention. Such methods include, but are not limited to, BIAcore®, Octet®, or flow cytometry.

The term Kd as used herein refers to the equilibrium dissociation constant of the antigen binding molecule/antigen interaction. The term Kd as used in the present invention includes Kd and Kd-apparent.

In some embodiments, the Kd of an antigen-binding molecule is measured by flow cytometry using an antigen-expressing cell line and by fitting the average fluorescence measured at each antibody concentration to a nonlinear single-site binding equation (Prism Software graphical panel). In some such embodiments _{, Kd} is Kd _-apparent .

The term biological activity refers to any one or more biological properties of a molecule (naturally occurring, found in vivo, or introduced or produced by recombinant means).

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

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

An sdAb or VHH-containing polypeptide with affinity matured refers to a sdAb or VHH-containing polypeptide with one or more changes in one or more CDRs compared to a parent sdAb or VHH-containing polypeptide that does not have such changes, wherein such changes result in improved affinity sdAb or VHH-containing polypeptide to the antigen.

As used herein, the term humanized VHH refers to a VHH in which one or more framework regions are substantially replaced with human framework regions. In some cases, certain framework region (FR) residues of the human immunoglobulin are replaced by corresponding residues of non-human origin. In addition, the humanized VHH may contain residues not found in either the original VHH or the human framework sequences, but which are included to further refine and optimize the characteristics of the VHH-containing sdAb polypeptide. In some embodiments, the humanized sdAb or VHH-containing polypeptide comprises a human Fc region. It will be appreciated that the humanized sequence may be identified from the primary sequence and not necessarily

- 8 046257 means the process by which the antibody itself was created.

An effector-positive Fc region has the effector function of a native sequence Fc region. Examples of effector functions include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; inhibition of cell surface receptors (eg, B cell receptor); and activation of B cells, etc. Such effector functions typically require that the Fc region be combined with a binding domain (eg, an antibody variable domain), and these functions can be assessed using various assays.

The native sequence Fc region contains an amino acid sequence identical to the amino acid sequence of the naturally occurring Fc region. Native sequence human Fc region includes native sequence human IgG1 Fc region (non-A and A allotypes); Fc region of human IgG2 with native sequence; Fc region of human IgG3 with native sequence; and the native sequence Fc region of human IgG4, as well as naturally occurring variants thereof.

The Fc region variant contains an amino acid sequence that differs from the native sequence Fc region sequence due to at least one amino acid modification. In some embodiments, the variant Fc region comprises an amino acid sequence that differs from the native sequence Fc region by virtue of at least one amino acid modification, but retains at least one effector function of the native sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution relative to the native sequence Fc region or the Fc region of the parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the Fc region of the native sequence or in the Fc region of the parent polypeptide. In some embodiments, an Fc region variant provided herein may have at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the Fc region of the native sequence and/or the Fc region of the parent polypeptide.

Fc receptor or FcR describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcyR is a native human FcR. In some embodiments, the FcR is an FcR that binds to an IgG antibody (gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (activating receptor) and FcyRIIB (inhibitory receptor), which have similar amino acid sequences and differ mainly in their cytoplasmic domains. The activating receptor FcyRIIA contains an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine inhibition motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol., 15:203234 (1997)). A review of FcR is presented, for example, in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). The term FcR as used in the present invention includes other FcRs, including FcRs that may be identified in the future. For example, the term Fc receptor or FcR also includes the neonatal FcRn receptor, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587 (1976); and Kim et al., J. Immunol., 24 :249 (1994)) and regulation of immunoglobulin homeostasis. Methods for measuring FcRn binding are known (see, for example, Ghetie and Ward, Immunol. Today, 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997) );Hinton et al., J. Biol. Chem., 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

The term chimeric antigen receptor as used herein refers to an engineered polypeptide that includes an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular antigen recognition domain comprises a VHH domain.

As used herein, the term substantially similar or substantially the same means a sufficiently high degree of similarity between two or more numerical values such that, from the point of view of one skilled in the art, such difference between the two or more values is low or non-biological and/or non-biological. or statistical significance in the context of the biological characteristic measured by the specified value. In some embodiments, two or more substantially identical values differ by no more than about any of 5, 10, 15, 20, 25, or 50%.

- 9 046257

Variant polypeptide means a biologically active polypeptide having at least about 80% amino acid sequence identity with a native sequence polypeptide after sequence alignment and the introduction of gaps as necessary to achieve the maximum percentage of sequence identity without taking into account any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N- or C-terminus of the polypeptide. In some embodiments, the variant may have at least about 80% amino acid sequence identity. In some embodiments, the variant may have at least about 90% amino acid sequence identity. In some embodiments, the variant may have at least about 95% amino acid sequence identity to a native sequence polypeptide.

As used herein, percentage (%) amino acid sequence identity and homology to a peptide, polypeptide or antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence after sequence alignment and insertion as appropriate omissions to achieve the maximum percentage of sequence identity without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer programs such as the BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) programs. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

An amino acid substitution may include, but is not limited to, replacing one amino acid in a polypeptide with another amino acid. Typical substitutions are shown in table. 1. Amino acid substitutions can be introduced into the antibody of interest, and the products are screened for the desired activity, such as maintaining/improving antigen binding, reducing immunogenicity, or improving ADCC or CDC.

Table 1

Original balance Illustrative Substitutions Ala (A) Vai; Leu; He Arg(R) Lys; Gin; Asn Asn(N) Gin; His; Asp, Lys; Arg Asp (D) Glu; Asn Cys(C) Ser; Ala Gin (Q) Asn; Glu Glu(E) Asp; Gin Gly (G) Ala His(H) Asn; Gin; Lys; Arg He(I) Leu; Vai; Met; Ala; Phc; norleucine Leu (L) norleucine; He; Vai; Met; Ala: Phe Lys (K) Arg; Gin; Asn Met(M) Leu; Phc; lie

- 10 046257

Phe(F) Trp; Leu; Val; lie; Ala; Tyr Pro (P) Ala Ser (S) Thr Thr (T) Val; Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Ph; Thr; Ser Val(V) Ue; Leu; Met; Ph; Ala; norleucine

Amino acids can be grouped according to the general properties of their side chains:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues affecting chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions result in replacing a member of one of these classes with another class.

The term vector is used to describe a polynucleotide that can be constructed to contain a cloned polynucleotide or polynucleotides that can be propagated in a host cell. The vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (such such as antibiotic resistance genes and genes that can be used in colorimetric assays, such as β-galactosidases). The term expression vector refers to a vector that is used to express a polypeptide of interest in a host cell.

Host cell refers to a cell that can be or has been the recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic cells or eukaryotic cells. Examples of eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells such as yeast; plant cells; and insect cells. Non-limiting examples of mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell) and 293 and CHO cells, as well as derivatives thereof such as 293-6E, CHODG44, CHO-K1, CHO-S and CHO-DS cells. Host cells include the progeny of a single host cell, and these progeny may not necessarily be completely identical (in morphology or complementary genomic DNA) to the original parent cell due to a single random or intentional mutation. The host cell includes cells transfected in vivo with the polynucleotide(s) provided in the present invention.

As used herein, the term isolated refers to a molecule that has been separated from at least some of the components with which it is normally found in nature or produced. For example, a polypeptide is said to be isolated when it is separated from at least some components of the cell in which it was produced. When a polypeptide is secreted by a cell after expression, the physical separation of the supernatant containing the polypeptide from the cell producing it is considered release of the polypeptide. Similarly, a polynucleotide is said to be isolated if it is not part of a larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it normally occurs in nature, or is separated from at least some of its components the cell producing it, for example, in the case of an RNA polynucleotide. Thus, the DNA polynucleotide that is contained in the vector inside the host cell can be said to be isolated.

The terms individual and subject are used interchangeably in the present invention to refer to an animal, such as a mammal. In some embodiments, methods of treating mammals are provided, including, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sporting animals and domestic mammals. In some examples, the individual or subject refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject receiving treatment may be a patient, which indicates that the subject has been identified as having the disorder being treated or at significant risk of developing the disorder.

The term disease or disorder as used herein refers to a condition

- 11 046257 condition for which treatment is necessary and/or desirable.

The terms tumor cell, cancer cell, cancer, tumor and/or neoplasm, unless otherwise indicated, are used interchangeably in the present invention and refer to a cell (or cells) with uncontrolled growth and/or abnormal increase in cell survival and/or inhibition of apoptosis, interfering normal functioning of organs and systems of the body. This definition includes benign and malignant tumors, hematological cancers such as leukemias, lymphomas and multiple myelomas, polyps, hyperplasia, and latent tumors or micrometastases.

The terms cancer and tumor include solid and hematologic/lymphatic cancer, and also include malignant, premalignant, and benign growths such as dysplasia. Examples of cancer include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; cancer of the brain and central nervous system; mammary cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal carcinoma; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancer); glioblastoma; liver carcinoma; hepatoma; intraepithelial neoplasm; kidney or kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (eg, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell lung cancer); melanoma; myeloma; neuroblastoma; cancer of the oral cavity (lips, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland carcinoma; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocyte NHL; moderate-grade/follicular NHL; moderate-grade diffuse NHL; high-grade grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small cell non-cleaved cell NHL; massive NHL; mantle cell lymphoma; AIDS-associated lymphoma; and Waldenström macroglobulinemia; acute myeloid leukemia (AML); chronic lymphocytic leukemia ( CLL), acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (eg, associated with brain tumors brain) and Meigs syndrome.

The term non-tumor cell or non-cancerous cell as used herein refers to normal cells or tissue. Examples of non-neoplastic cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, macrophages, epithelial cells, fibroblasts, hepatocytes, renal interstitial cells, fibroblast-like synoviocytes , osteoblasts and cells located in the mammary gland, skeletal muscle, pancreas, stomach, ovary, small intestine, placenta, uterus, testes, kidneys, lungs, heart, brain, liver, prostate, colon, lymphoid organs, bone, and mesenchymal stem cells of bone origin. As used herein, the term peripherally located cell or tissue refers to non-tumor cells that are not located adjacent to tumor cells and/or in the tumor microenvironment.

As used herein, the term cells or tissue in the tumor microenvironment refers to the cells, molecules, extracellular matrix and/or blood vessels that surround and/or nourish the tumor cell. Examples of cells or tissue in the tumor microenvironment include, but are not limited to, tumor vasculature; tumor-infiltrating lymphocytes; fibroblastic reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; extracellular matrix (ECM) components; dendritic cells; antigen presenting cells; T cells; regulatory T cells (Treg cells); macrophages; neutrophils; myeloid-derived suppressor cells (MDSCs) and other immune cells located proximal to the tumor. Methods for identifying tumor cells and/or cells/tissues located in the tumor microenvironment are well known in the art, as disclosed below in the present invention.

In some embodiments, an increase or decrease refers to a statistically significant increase or decrease, respectively. As will be appreciated by one skilled in the art, modulation may also involve changing (which may be either an increase or decrease) the affinity, avidity, specificity and/or selectivity of a target or antigen for one or more of its ligands, binding partners, association into homomultimeric or heteromultimeric form or substrates; a change (which may be either an increase or decrease) in the sensitivity of a target or antigen to one or more environmental conditions in which the target or antigen is present (e.g., pH, ion strength, presence of concomitant

- 12 046257 current factors, etc.); and/or cell proliferation or cytokine production compared to the same conditions but in the absence of the test agent. This can be determined by any suitable method and/or any suitable assay known or disclosed herein depending on the target involved.

As used herein, the term immune response refers to cellular and/or humoral immune responses that are sufficient to suppress or prevent the onset or alleviation of symptoms of a disease (eg, cancer or cancer metastases). The immune response can involve aspects of both the innate and adaptive immune systems.

As used herein, the term treatment is an approach to obtain beneficial or desired clinical results. As used herein, the term treatment includes any administration or use of a therapeutic agent to treat a disease in a mammal, including a human. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of the following: relief of one or more symptoms, reduction in the extent of disease, prevention or delay of spread (e.g., metastasis, e.g., lung or lymph node metastasis) of disease, preventing or delaying the recurrence of a disease, delaying or slowing the progression of a disease, alleviating a disease state, suppressing a disease or its progression, suppressing or slowing a disease or its progression, arresting its development and remission (partial or complete). The term treatment also includes reducing the pathological consequences of a proliferative disease. The methods provided herein involve any one or more of these aspects of treatment. In keeping with the above, the term treatment does not require complete elimination of all aspects of the disorder.

Relief means a reduction or improvement of one or more symptoms compared to the absence of administration of the therapeutic agent. Relief also includes shortening or decreasing the duration of the symptom.

The term anticancer agent is used in the present invention in its broadest sense to refer to agents that are used to treat one or more types of cancer. Examples of classes of such agents include, but are not limited to, chemotherapeutic agents, anticancer biologic agents (such as cytokines, Fc-extracellular domain receptor fusion proteins, and antibodies), radiation therapy, CAR-T cell therapy, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNAs), and oncolytic agents. viruses.

The term biological sample means a quantity of a substance obtained from a living being or a previously living being. Such substances include, but are not limited to, blood (eg, whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, white blood cells, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

The term control or reference in the context of an experiment or comparison refers to a composition that is known to not contain an analyte (negative control) or to contain an analyte (positive control). A positive control may include a known concentration of the analyte. A control or standard can also refer to a control agent that is known to lack the activity of the test agent, such as an antibody.

As used herein, the term disease retardation means delaying, containing, slowing, inhibiting, stabilizing, suppressing, and/or delaying the development of a disease (such as cancer). This delay may vary in length depending on the medical history and/or person being treated. As will be apparent to one skilled in the art, a sufficient or significant delay may actually involve prevention in the sense that the individual does not develop the disease. For example, late stage cancer, such as the development of metastases, may be delayed.

Prevention as used herein includes preventing the occurrence or recurrence of a disease in a subject who may have a predisposition to the disease but has not yet been diagnosed with it. Unless otherwise specified, the terms reduce, inhibit or prevent do not mean or require complete prevention at all times, but only during the time period when the measurement occurs.

The therapeutically effective amount of a substance/molecule, agonist, or antagonist may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the substance/molecule, agonist, or antagonist to elicit a desired response in an individual. A therapeutically effective amount is also an amount at which the therapeutically beneficial effects outweigh any toxic or harmful effects of the substance/molecule, agonist or antagonist. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective in doses and for periods of time necessary to achieve the desired therapeutic and/or prophylactic result.

- 13 046257

The terms pharmaceutical composition and pharmaceutical composition are used interchangeably and refer to a preparation that is in a form that provides effective biological activity of the active ingredient(s) and that does not contain additional components that are unacceptably toxic to the subject to whom the composition will be administered. Such compositions may be sterile.

A pharmaceutically acceptable carrier refers to a non-toxic solid, semi-solid or liquid excipient, diluent, encapsulating material, excipient or carrier commonly used in the art with a therapeutic agent, which together constitute a pharmaceutical composition for administration to a subject. The pharmaceutically acceptable carrier is nontoxic to recipients at the dosages and concentrations used and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the composition used.

Administration in combination with one or more additional therapeutic agents includes simultaneous (parallel) and sequential administration in any order.

The term parallel is used in the present invention to refer to the administration of two or more therapeutic agents when the administrations at least partially overlap in time, or when the administration of one therapeutic agent occurs within a short period of time during the administration of another therapeutic agent, or when the therapeutic effects of both agents overlap. at least for some period of time.

The term sequentially is used in the present invention to mean the administration of two or more therapeutic agents that do not overlap in time or in which the therapeutic effects of the agents do not overlap.

As used herein, the term in combination with refers to the administration of one therapeutic agent in addition to another therapeutic agent. Thus, in combination with refers to the administration of one therapeutic agent before, during, or after the administration of another therapeutic agent to an individual.

The term package insert is used to refer to instructions typically included in commercial packaging of therapeutic products that contain information regarding the indications, uses, dosage, routes of administration, combination therapy, contraindications and/or warnings regarding the use of such therapeutic products.

An article of manufacture is any product (eg, package or container) or kit containing at least one reagent, such as a drug for treating a disease or disorder (eg, cancer), or a probe for specifically detecting a biomarker disclosed in the present invention. In some embodiments, a product or kit is advertised, distributed, or sold as a unit to implement the methods of the present invention.

The terms label and detectable label mean a moiety attached, for example, to an antibody or antigen to provide a detectable reaction (eg, binding) between members of a specific binding pair. The labeled member of a specific binding pair is called detection labeled. Thus, the term tagged binding protein refers to a protein with a tag included that allows identification of the binding protein. In some embodiments, the label is a detectable marker that can produce a visually or instrumentally detectable signal, for example, by incorporating a radiolabeled amino acid or by attaching biotinyl moieties to the polypeptide that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity, which can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (eg, ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, 'In. ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, or ¹⁵³ Sm); chromogens, fluorescent tags (eg, FITC, rhodamine, lanthanide phosphors), enzymatic tags (eg, horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predefined polypeptide epitopes recognized by the secondary reporter (eg, leucine zipper sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used for immunoassays include light-emitting moieties, such as acridinium compounds, and fluorescence-emitting moieties, such as fluorescein. In this case, the fragment itself may not be labeled with a detectable label, but may become detectable after reaction with another fragment.

Exemplary CHEC12a-binding polypeptides.

The present invention provides CHEC12a-binding polypeptides. In various embodiments, the CEC12a-binding polypeptides contain at least one VHH domain that binds to CLEC12a. In some embodiments, CLEC12a is human CLEC12a. In some embodiments, the CHEC12a-binding polypeptide

- 14 046257 blocks CLEC12a binding to ligand. In some embodiments, the CLEC12a binding polypeptide provided by the present invention contains one, two, three, four, five, six, seven, or eight VHH domains that bind to CLEC12a. In some embodiments, the CLEC12a-binding polypeptide provided by the present invention contains one, two, three, or four VHH domains that bind to CLEC12a. CLEC12a-binding polypeptides may contain one or more VHH domains that bind to one or more target proteins other than CLEC12a. Such polypeptides may be referred to as polyspecific polypeptides.

In some embodiments, the CLEC12a binding polypeptide comprises at least one VHH domain that binds CLEC12a and an Fc region. In some embodiments, the CLEC12a-binding polypeptide provided by the present invention contains one, two, three, or four VHH domains and an Fc region. In some embodiments, the Fc region mediates 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 containing three VHH domains that bind to CLEC12a and an Fc region is trivalent as a monomer, but under physiological conditions the Fc region may mediate dimerization such that the CLEC12a binding polypeptide will exist as a hexavalent polypeptide under such conditions. dimer.

In some embodiments, the CLEC12a-binding polypeptide contains at least two VHH domains, where a first VHH domain binds to a first CLEC12a epitope and a second VHH domain binds to a second CLEC12a epitope. When a CLEC12a-binding polypeptide contains 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 bi-epitopic or bispecific. In some embodiments, the CLEC12a-binding polypeptide contains at least two VHH domains, where the first VHH domain binds to CLEC12a and the second VHH domain binds to an antigen other than CLEC12a. Such polypeptides may be referred to as bispecific or polyspecific.

Non-limiting exemplary CLEC12a-binding polypeptides are shown in table. 2. The sequences of these single domain antibodies are shown in the table for some of the sequences provided in the present invention. A polypeptide name beginning with hz indicates that it is a humanized version of the corresponding parent polypeptide.

table 2

Polypeptides containing at least one VHH that binds CLEC12a

Name CDR VHH (or K option or KR option) MB11 SEQ ID NO: 3, 4 and 5 SEQ ID NO: 2 (or SEQ ID NO: 88) MC02 SEQ ID NO: 7, 8 and 9 SEQ ID NO: 6 (or SEQ ID NO: 89) MC06 SEQ ID NO: 11. 12 and 13 SEQ ID NO: 10 (or SEQ ID NO: 90) ME02 SEQ ID NO: 15, 16 and 17 SEQ ID NO: 14 (or SEQ ID NO: 91) ME06 SEQ ID NO: 19, 20 and 21 SEQ ID NO: 18 (or SEQ ID NO: 92) MH07 SEQ ID NO: 23, 24 and 25 SEQ ID NO: 22 (or SEQ ID NO: 93) hzMBllvl 3 SEQ ID NO: 32, 33 and 34 SEQ ID NO: 26 (or SEQ ID NO: 94) hzMC02v6 SEQ ID NO: 35, 36 and 37 SEQ ID NO: 27 (or SEQ ID NO: 95) hzMC06v2 SEQ ID NO: 38, 39 and 40 SEQ ID NO: 28 (or SEQ ID NO: 96) hzME02v4 SEQ ID NO: 41, 42 and 43 SEQ ID NO: 29 (or SEQ ID NO: 97) hzME06vl6 SEQ ID NO: 44, 45 and 46 SEQ ID NO: 30 (or SEQ ID NO: 98) haMH07v3 SEQ ID NO: 47, 48 and 49 SEQ ID NO: 31 (or SEQ ID NO: 99)

CLEC12a-binding polypeptides.

In various embodiments, the VHH domain that binds CLEC12a comprises a CDR1 sequence selected from SEQ ID NOs: 3, 7, 11, 15, 19, 23, 32, 35, 38, 41, 44, and 47; a CDR2 sequence selected from SEQ ID NOs: 4, 8, 12, 16, 20, 24, 33, 36, 39, 42, 45 and 48; and a CDR3 sequence selected from SEQ ID NOs: 5, 9, 13, 17, 21, 25, 34, 37, 40, 43, 46, and 49. In various embodiments, the VHH domain that binds CLEC12a comprises CDR1 sequences, CDR2 and CDR3 selected from: SEQ ID NO: 3, 4, and 5; SEQ ID NO: 7, 8 and 9; SEQ ID NO: 11, 12 and 13; SEQ ID NO: 15, 16 and 17; SEQ ID NO: 19, 20 and 21; SEQ ID NO: 23, 24 and 25; SEQ ID NO: 32, 33 and 34;

- 15 046257

SEQ ID NO: 35, 36 and 37; SEQ ID NO: 38, 39 and 40; SEQ ID NO: 41, 42 and 43; SEQ ID NO: 44, 45 and 46; and SEQ ID NOs: 47, 48 and 49. In various embodiments, the VHH domain is humanized.

In some embodiments, the VHH domain that binds CLEC12a contains an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98%, at least 99% of the amino acid sequence selected from SEQ ID NO: 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 CLEC12a comprises an amino acid sequence selected from SEQ ID NO: 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 various embodiments, the CLEC12a-binding polypeptide contains one, two, three, or four VHH domains that bind to CLEC12a.

In various embodiments, the CLEC12a-binding polypeptide contains at least one VHH domain that binds to CLEC12a and at least one VHH domain that binds to a natural killer cell antigen or T-cell antigen. In some such embodiments, the CLEC12a-binding polypeptide may be referred to as a multispecific antibody.

In some embodiments, the CLEC12a-binding polypeptide comprises at least one VHH domain provided herein fused to an Fc region. In some embodiments, the Fc region has a sequence selected from SEQ ID NO: 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.

In some embodiments, the VHH domain that binds to CLEC12a may be humanized. Humanized antibodies (such as sdAbs or VHH-containing polypeptides) are useful as therapeutic molecules because humanized antibodies can reduce or eliminate the occurrence of an immune response in a person to non-human antibodies, which may induce an immune response to the therapeutic antibody and reduce the effectiveness of the therapeutic antibody. Typically, a humanized antibody contains one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody may also optionally contain at least a portion of the human constant region. In some embodiments, certain FR residues in the humanized antibody are replaced with corresponding residues of a non-human antibody (eg, the antibody from which the CDR residues are derived), for example, to restore or improve the specificity or affinity of the antibody.

A review of humanized antibodies and methods for their production is presented, for example, in Almagro and Fransson (2008), Front. Biosci., 13:1619-1633, they are also described, for example, in Riechmann et al. (1988), Nature, 332:323-329; Queen et al. (1989), Proc. Natl. Acad. Sci., USA, 86:10029-10033; US patents No. 5821337, 7527791, 6982321 and 7087409; Kashmiri et al. (2005), Methods, 36:25-34; Padlan (1991), Mol. Immunol., 28:489-498 (describing surface restoration); Dall'Acqua et al. (2005), Methods, 36:43-60 (describing FR rearrangement); and Osbourn et al. (2005), Methods, 36:61-68; and Klimka et al. (2000), Br. J Cancer 83:252–260 (describing a guided selection approach for FR rearrangement).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the best-fit method (see, for example, Sims et al. (1993), J. Immunol., 151:2296); framework regions derived from the human antibody consensus sequence of a particular subset of heavy chain variable regions (see, for example, Carter et al. (1992), Proc. Natl. Acad. Sci., USA, 89:4285; and Presta et al. ( 1993), J. Immunol, 151:2623); mature (somatically mutated) human framework regions or human germline framework regions (see, for example, Almagro and Fransson (2008), Front. Biosci., 13:1619-1633); and framework regions obtained from FR screening libraries (see, for example, Baca et al. (1997), J. Biol. Chem., 272:10678-10684; and Rosok et al. (1996), J. Biol. Chem. ., 271:22611-22618). Typically, to obtain a humanized VHH, the FR regions in the VHH are replaced with human FR regions. In some embodiments, certain FR residues of human FR are replaced to improve one or more properties of the humanized VHH. VHH domains with such substituted residues are still referred to as humanized domains in the present invention.

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.

In some embodiments, the Fc region included in the CLEC12a binding polypeptide is derived from a human Fc region and contains a three amino acid deletion in the lower hinge corresponding to E233, L234 and L235 of IgG1, referred to herein as xELL Fc. xELL Fc polypeptides do not interact with FcyR and are thus referred to as effector silent or effector null, however, in some embodiments, xELL Fc regions bind to FcRn and therefore have increased half-life and transcytosis associated with FcRn-mediated recycling.

In some embodiments, the Fc region included in the CLEC12a-binding poly

- 16 046257 peptide, is derived from the human Fc region and contains mutations M252Y and M428V, referred to in the present invention as Fc-YV. In some embodiments, such mutations enhance FcRn binding at acidic endosomal pH (about 6.5) while losing detectable binding at neutral pH (about 7.2), resulting in enhanced FcRn-mediated recycling and increased half-life.

In some embodiments, the Fc region included in the CLEC12a binding polypeptide is derived from a human Fc region and contains heterodimerization mutations, referred to herein as knob and trench. In some embodiments, the Fc region of the protrusion contains the T366W mutation. In some embodiments, the Fc region depression contains mutations T366S, L368A, and Y407V. In some embodiments, the Fc regions used for heterodimerization contain additional mutations, such as the S354C mutation in the first member of the heterodimeric Fc pair, which forms an asymmetric disulfide with the corresponding Y349C mutation on the second member of the heterodimeric Fc pair. In some embodiments, one member of the heterodimeric Fc pair contains an H435R or H435K modification to prevent binding to protein A while maintaining binding to FcRn. In some embodiments, one member of the heterodimeric Fc pair contains an H435R or H435K modification, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the retained Fc region contains the H435R or H435K modification (in some cases where the H435R modification is referred to as recess-R), while the protrusion Fc region does not contain the modification. In some cases, the cavity-R mutation improves heterodimer purification compared to homodimeric cavity Fc regions that may be present.

Non-limiting exemplary Fc regions that can be used in a CLEC12a binding polypeptide include Fc regions containing the amino acid sequences of SEQ ID NO: 74-109.

Chimeric receptors and engineered cells.

The present invention provides chimeric antigen receptors (CARs) having an extracellular domain containing one or more CLEC12a-binding VHH domains provided in the present invention. The CAR constructs of the present invention include an extracellular domain containing one or more CLEC12a-binding VHH domains, a transmembrane domain, and an intracellular signaling region. One or more CLEC12a-binding VHH domains that form the CAR antigen binding unit bind or are capable of binding, i.e. target CLEC12a with sufficient affinity such that the CAR is useful in therapy for targeting a cell or tissue expressing CLEC12a.

CARs are synthetic receptors typically containing an extracellular targeting/binding moiety that is associated with one or more signaling domains in a single fusion molecule that is expressed on the surface of a cell, such as a T cell. Thus, CARs combine antigen-specificity and T-cell activation properties into a single fusion molecule. First-generation CARs typically included the cytoplasmic region of the CD3zeta or Fc1 receptor γ chain as a signaling domain. First-generation CARs were tested in phase I clinical trials in patients with ovarian cancer, kidney cancer, lymphoma, and neuroblastoma, where they produced modest responses (reviewed by Sadelain et al., Curr. Opin. Immunol., 21(2):215-223 , 2009). Second-generation CARs that contain costimulatory molecule signaling domains such as CD28 and CD3zeta provide dual signaling to direct combined activating and costimulatory signals. Third generation CARs are more complex with three or more signaling domains (reviewed by Sadelain et al., Cancer Discovery (3), 388-398, 2013; and Dotti et al., Immuno. Rev, 257(1), 1-36, 2014).

In some embodiments, the provided CAR comprises a CLEC12a VHH binding domain. In some embodiments, the CAR contains at least two VHH domains targeting one or more antigens. In one embodiment, the CAR antigen binding domain comprises two or at least two CLEC12a VHH binding domains, thereby providing a bivalent molecule. In one embodiment, the antigen binding domain comprises two or at least two CLEC12a VHH binding domains, but binds to different epitopes on CLEC12a. In such cases, the antigen binding domain comprises a first CLEC12a binding VHH domain that binds to a first CLEC12a epitope, and a second VHH domain that binds to a second CLEC12a epitope. 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 VHH binding domains that bind to the same epitopes on CLEC12a.

The transmembrane domain of the CAR provided in the present invention is a domain that typically crosses or is capable of crossing or passing through the plasma membrane and binds directly or indirectly (for example, through a spacer such as a hinge sequence

- 17 046257 immunoglobulin) with an extracellular antigen-binding domain and an endoplasmic region containing an intracellular signaling domain. In one embodiment, the CAR transmembrane domain is the transmembrane region of a transmembrane protein (eg, type I transmembrane proteins), an artificial hydrophobic sequence, or combinations thereof. In one embodiment, the transmembrane domain includes a CD3zeta domain or a CD28 transmembrane domain. Those skilled in the art will recognize other transmembrane domains that may be used in connection with the CAR embodiments of the present invention.

The CAR intracellular signaling region of the present invention comprises one or more intracellular signaling domains that convey a signal to a T cell upon engagement of the CAR antigen binding domain, for example upon binding of an antigen. In some embodiments, the intracellular region comprises an intracellular signaling domain that is or contains an ITAM signaling domain. Exemplary intracellular signaling domains include, for example, a signaling domain derived from the ζ chain of the T cell receptor complex or any of its homologues (e.g., η chain, FcsRIy and β chains, MB chain 1 (Iga), B29 (Ig) chain, etc.), human CDS chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) etc.) and other molecules involved in T cell transduction, such as CD2, CD5, OX40 and CD28. In specific embodiments, the intracellular signaling region comprises an intracellular signaling domain derived from the zeta chain of human CD3.

In some embodiments, the CAR intracellular signaling region may further comprise an intracellular signaling domain derived from a co-stimulatory molecule. In such examples, such a signaling domain may enhance the activity of CAR-T cells, for example, by increasing the proliferation, survival and/or development of memory cells, following an antigen-specific interaction, for example, compared to a CAR that contains only an ITAM containing the signaling domain, for example CD3 zeta. In some embodiments, the co-stimulatory domain is a functional signaling domain derived from a protein selected from CD28, CD137 (4-IBB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1 (CD1a/ CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof. In specific embodiments, the co-stimulatory signaling domain is derived from or derived from a human protein. In some aspects, the co-stimulatory signaling domain is derived from or derived from human CD28 or human CD137 (4-IBB).

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

In specific embodiments, the CAR further comprises a hinge or spacer region that connects the extracellular antigen binding domain and the transmembrane domain. This hinge or spacer region can be used to achieve varying lengths and flexibility of the resulting CAR. Examples of a hinge or spacer region that can be used include, but are not limited to, Fc portions of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences such as peptide sequences, or combinations thereof. . Those skilled in the art will recognize other hinge or spacer regions that can be used. In one embodiment, the hinge is an IgG4 hinge or a CD8A hinge.

In some embodiments, the spacer and transmembrane domain are a hinge and transmembrane domain derived from CD8.

The present invention also provides an isolated nucleic acid construct comprising at least one nucleic acid encoding a CAR as provided in the present invention. In some aspects, the construct is an expression vector for expressing a CAR in a cell. The expression vector may be a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2013). A number of viral systems have been developed to transfer genes into mammalian cells. For example, retroviruses such as adenoviral vectors are used. In one embodiment, a lentiviral vector is used.

In a further aspect, also provided is an isolated cell or population of cells containing one or more of the nucleic acid constructs described above. Also provided is an isolated cell or population of cells genetically modified to express the CAR of the present invention. Thus, the present invention provides genetically engineered cells that contain, for example, stably express the CAR provided in the present invention. In one embodiment, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, hematopoietic stem cells, and/or pluripotent embryonic/induced

- 18 046257 stem cells. In some cases, the cell is a T cell, such as a CD4 and/or CD8 T cell. In some embodiments, the cells are autologous to the subject. For example, in some embodiments, T cells may be isolated from a patient (also referred to as primary T cells) for engineering, such as transfection or transduction with a CAR nucleic acid construct.

In an illustrative example, primary T cells can be purified ex vivo (CD4 cells or CD8 cells or both) and stimulated with TCR/CD28 agonists, such as beads coated with anti-CD3 anti-CD28 antibodies. After a 2- or 3-day activation process, the recombinant expression vector encoding the CAR can be stably introduced into primary T cells using standard lentiviral or retroviral transduction protocols or plasmid electroporation strategies. Cells can be monitored for CAR expression, for example, by flow cytometry using an anti-epitope tag or antibodies that cross-react with the native parent molecule. T cells that express CAR can be enriched by sorting with tagged anti-epitope antibodies or can be enriched to achieve high or low levels of expression depending on the application.

CAR engineered T cells can be analyzed for appropriate function in a variety of ways. In some cases, in vitro cytotoxicity, proliferation, or cytokine (eg, IFN-gamma expression) assays may be used to assess the function of engineered T cells. Exemplary standard endpoints are the percentage of tumor line lysis, proliferation of engineered T cells, or expression of IFN-gamma protein in the culture supernatant. In some cases, the ability to stimulate T cell activation upon CAR stimulation, for example through an antigen, can be assessed, for example, by monitoring the expression of activation markers such as CD69, CD44 or CD62L, proliferation and/or cytokine production.

Expression and production of polypeptides.

Presented are nucleic acid molecules containing polynucleotides that encode a CLEC12a-binding polypeptide. In some embodiments, the nucleic acid molecule may also encode a leader sequence that directs secretion of a CLEC12a binding polypeptide, which leader sequence is typically cleaved off and therefore is not present in the secreted polypeptide. The leader sequence may be a native heavy chain (or VHH) leader sequence or may be another heterologous leader sequence.

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

Provided are vectors containing nucleic acids that encode the CLEC12a binding polypeptides of the present invention. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector is selected that is optimized for expression of polypeptides in a desired cell type, such as CHO cells or CHO-derived cells, or NSO cells. Examples of such vectors are described, for example, in Running Deer et al., Biotechnol. Prog., 20:880-889 (2004).

In some embodiments, the CLEC12a-binding polypeptide can be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells and mammalian cells. Such expression can be carried out, for example, in accordance with procedures known in the art. Examples of eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S cells, DG44.Lec13 CHO cells and FUT8 CHO cells; PER.C6® cells (Crucell); and HCO cells. In some embodiments, CLEC12a-binding polypeptides can be expressed in yeast. See, for example, US Patent Application Publication No. US 2006/0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make the desired post-translational modifications of the polypeptide. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

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

Also provided are host cells containing any of the nucleic acids or vectors

- 19 046257 disclosed in the present invention. In some embodiments, a host cell is provided that expresses a CLEC12a-binding polypeptide provided in the present invention. CLEC12a-binding polypeptides expressed in host cells can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include ROR1 ECD and agents that bind Fc regions. For example, a Protein A, Protein G, Protein A/G column, or an immobilized antibody column can be used to bind the Fc region and purify a CLEC12a binding polypeptide that contains the Fc region. For the purification of some polypeptides, such as antibodies, hydrophobic interactive chromatography, for example on a butyl or phenyl column, may also be suitable. Ion exchange chromatography (eg, anion exchange chromatography and/or cation exchange chromatography) may also be suitable for the purification of certain polypeptides, such as antibodies. Mixed chromatography (eg, reverse phase/anion exchange, reverse phase/cation exchange, hydrophilic interaction/anion exchange, hydrophilic interaction/cation exchange, etc.) may also be suitable for the purification of certain polypeptides, such as antibodies. Many methods for purifying polypeptides are known in the art.

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

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

In some embodiments, compositions are provided containing antibodies produced by the methods described above. In some embodiments, the composition comprises a CLEC12a-binding polypeptide produced in a host cell. In some embodiments, the composition comprises a CLEC12a-binding polypeptide produced in a cell-free system. In some embodiments, the composition comprises a purified CLEC12a-binding polypeptide.

Exemplary methods of treating diseases using CLEC12a-binding polypeptides.

In some embodiments, methods of treating a disease in an individual are provided, comprising administering a CLEC12a-binding polypeptide or cells expressing a CLEC12a-binding polypeptide. In some embodiments, methods of treating cancer in an individual are provided. In some embodiments, methods of treating CLEC12a-expressing or CLEC12a-positive cancer in an individual are provided. The method includes administering to an individual an effective amount of a CLEC12a-binding polypeptide or cells expressing a CLEC12a-binding polypeptide provided by the present invention. In some embodiments, a CLEC12a-binding polypeptide is used to transfer a cytotoxic agent into a CLEC12a-expressing cell. In some such embodiments, the CLEC12a binding polypeptide comprises a binding domain that binds to a cytotoxic T cell or NK cell. In some such embodiments, the binding domain binds to CD3, T cell receptor (TCR)a, TCRe, 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.

In some embodiments, the CLEC12a-binding polypeptide is coupled 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, calicheamicins, auristatins, dolastatins, tubulicins, maytansinoids, cryptophycins, duocarmycins, esperamycins, pyrrolobenzodiazepines, and enediyne antibiotics.

In some embodiments, the CLEC12a-binding polypeptide is a chimeric antigen receptor expressed on a cytotoxic cell, such as a T cell (CAR-T) or NK cell (CAR-NK). Such treatments can be used in humans or animals. In some embodiments, methods of treating humans are provided.

Non-limiting exemplary cancers that can be treated with CLEC12a-binding polypeptides or cells expressing CLEC12a-binding polypeptides provided in the present invention include, but are not limited to, lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; low grade/follicular neoplasm

- 20 046257 Zhkin lymphoma (NHL); NHL from small lymphocytes (ML); moderate grade/follicular NHL; diffuse NHL of moderate severity; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; NHL from small non-divided cells with a high degree of malignancy; volumetric NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenström's macroglobulinemia; acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia. In some embodiments, the cancer is a CLEC12a-expressing cancer (ie, a CLEC12a-positive cancer).

CLEC12a-binding polypeptides or cells expressing CLEC12a-binding polypeptides can be administered to subjects as needed. Determination of the frequency of administration can be made by those skilled in the art, such as a physician, taking into account the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the patient being treated, and the like. In some embodiments, an effective dose of CLEC12a-binding polypeptides or cells expressing CLEC12a-binding polypeptides is administered to a subject one or more times. In some embodiments, an effective dose of CLEC12a-binding polypeptides or cells expressing CLEC12a-binding polypeptides is administered to a subject daily, once a week, weekly, every two weeks, once a month, etc. 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 the CLEC12a-binding polypeptide or cells expressing the CLEC12a-binding polypeptide can be administered multiple times, including multiple times over the course of at least a month, at least six months, or at least a year.

In some embodiments, the pharmaceutical compositions are administered in an amount effective to treat (including prevent) cancer. The therapeutically effective amount will generally depend on the weight of the subject being treated, his or her physical or medical condition, the extent of the condition being treated, or the age of the subject being treated. Typically, antibodies can be administered in amounts of from about 0.05 mg/kg body weight to about 100 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 10 μg/kg body weight to about 100 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 50 μg/kg body weight to about 5 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 100 μg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 100 μg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 0.5 mg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 0.05 mg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 0.05 mg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of about 5 mg/kg body weight or less, such as less than 4, less than 3, less than 2, or less than 1 mg/kg of antibody.

In some embodiments, CLEC12a-binding polypeptides or cells expressing CLEC12a-binding polypeptides can be administered in vivo by various routes, including, but not limited to, intravenous, intra-arterial, parenteral, intraperitoneal, or subcutaneous. A suitable composition and route of administration can be selected according to the intended use.

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

Pharmaceutical compositions.

In some embodiments, compositions containing CLEC12a-binding polypeptides are formulated with a wide range of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparsions: Drugfacts Plus, 20th ed. (2003 ); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3 ed., Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers are available, which include carriers, adjuvants and diluents. In addition, various pharmaceutically acceptable excipients such as pH adjusters and buffering agents, tonicity agents, stabilizers, wetting agents and the like are also available. Non-limiting illustrative vehicles include saline, buffered saline, dextrose, water, glycerin

- 21 046257 rin, ethanol and their combinations.

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

Combination therapy.

The CLEC12a-binding polypeptides or engineered cells of the present invention can be administered alone or in combination with other treatments, such as other anticancer agents. They may be provided before, substantially simultaneously with, or after other treatments (ie, simultaneously or sequentially). In some embodiments, the treatment method provided by the present invention may further include administering: radiation therapy, chemotherapy, vaccination, targeted cancer therapy, CAR T-cell therapy, oncolytic viral therapy, cancer immunotherapy, cytokine therapy, surgical resection, chromatin modification, ablation, cryotherapy, antisense agent against a tumor target, siRNA agent against a tumor target, microRNA agent against a tumor target or anticancer/tumor agent or biological agent such as an antibody, cytokine or Fc-extracellular receptor domain fusion protein.

In some embodiments, the CLEC12a-binding polypeptide provided in the present invention is administered concomitantly with one or more chemotherapeutic agents, CAR-T cell (chimeric antigen receptor T-cell) therapy, oncolytic viral therapy, cytokine therapy and/or agents targeting other checkpoint molecules such as VISTA, gpNMB, B7H4, HHLA2, CD73, CTLA4, TIGIT, etc.

In some embodiments, the CLEC12a-binding polypeptide or engineered cells of the present invention are used in combination with other anti-tumor agents, such as an anti-HER-2 antibody, an anti-CD20 antibody, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor) , HER1/EGFR inhibitor (eg, erlotinib (TARCEVA®), platelet-derived growth factor inhibitors (eg, GLEEVEC® (Imatinib Mesylate)), COX-2 inhibitor (eg, celecoxib), interferons, CTLA4 inhibitors (eg, anti-CTLA antibody ipilimumab (YERVOY®), PD-1 inhibitors (eg, anti-PD1 antibody, BMS936558), PDL1 inhibitors (eg, anti-PDL1 antibody, MPDL3280A), PDL2 inhibitors (eg, anti-PDL2 antibody), cytokines, antagonists ( e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA, PD-1, PDL1, PDL2, CTLA4 or VEGF receptor(s), TRAIL/Apo2 , and other bioactive and organic chemical agents, etc.

In some embodiments, a CLEC12a-binding polypeptide or engineered cell provided by the present invention is administered concomitantly with PD-1/PD-L1 therapy. Examples of PD-1/PD-L1 therapies include nivolumab (BMS); pidilizumab (CureTech, CT-011), pembrolizumab (Merck); durvalumab (Medimmune/AstraZeneca); atezolizumab (Genentech/Roche); avelumab (Pfizer); AMP-224 (Amplimmune); BMS-936559; AMP-514 (Amplimmune); MDX-1105 (Merck); TCP-042 (Tesaro/AnaptysBio, ANB-011); STI-A1010 (Sorrento Therapeutics); STI-A1110 (Sorrento Therapeutics); and other agents that are directed against programmed cell death protein 1 (PD-1) or programmed cell death ligand 1 (PD-L1).

In some embodiments, a CLEC12a-binding polypeptide or engineered cell of the present invention may be used in combination with a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbochon, meturedopa and uredopa; ethylene imines and methyl melamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethylol melamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including a synthetic analogue of topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, holophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorosotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (eg calicheamicin, especially calicheamicin gammaP and calicheamicin omegaP (see, for example, Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994)); dinemycin, including dinemycin A, bisphosphonates ; such as clodronate; esperamycin; as well as the neocarzinostatin chromophore and related chromoprotein enediine antibiotic chromophores), aclacinomysins, actinomycin, outramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophyllin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5 -oxo-b-norleucine, ADRIAMYCIN® doxorubi

- 22 046257 cin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, ezorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nagalamycin, olivomycins, peplomycin, potthirom icin, puromycin, kelamycin, rhodorubicin, streptonigrin , streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogues such as ancytabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepithiostane, testolactone; drugs that suppress adrenal function, such as aminoglutethimide, mitotane, trilostane; a folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquon; elfornitine; elliptinium acetate; epothilone; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenomet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurine A, roridin A and anhydrine); urethane; vindesine; dacarbazine; mannomustin; mitobronitol; mitolactol; pipobromance; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ non-creamaphore formulation containing albumin-bound nanoparticle paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine GEMZAR®; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16;); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; Novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including irinotecan regimen with 5-FU and leucovorin); topoisomerase inhibitors RFS 2000; difluoromethylornithine (DFMO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin regimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (eg, erlotinib (TARCEVA®)) and VEGF-A that reduce cell proliferation, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

Additional non-limiting illustrative chemotherapeutic agents include antihormonal agents that act by regulating or inhibiting the action of hormones on cancer, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4- hydroxytamoxifene, trioxifene, keoxifene, LY117018, onapristone and toremifene FARESTON®; aromatase inhibitors, which inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestane, fadrozole, RIVISOR® vorozole, FEMARA® letrozole and ARIMIDEX ® anastrozole; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as troxacitabine (cytosine analogue of 1,3-dioxolane nucleoside); antisense oligonucleotides, in particular those that inhibit the expression of genes in signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGF expression inhibitor (eg ANGIOZYME® ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapy vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; PROLEUKIN® (aldesleukin) rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® GnRH agonist; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

In some embodiments, the CL·EC12a-binding polypeptide and the additional agent constitute a single therapeutic composition, and the CL·EC12a-binding polypeptide and the additional agent are administered simultaneously. Alternatively, the CL·EC12a-binding polypeptide or engineered cell and the additional agent are separated from each other, for example, each formulated as a separate therapeutic composition, and the CL·EC12a-binding polypeptide or engineered cell and the additional agent are administered simultaneously, or CL·EC12a- the binding polypeptide or engineered cell and the additional agent are administered at different times according to the treatment regimen. For example, the CL·EC12a-binding polypeptide or engineered cell is administered before administration of the additional agent, the CL·EC12a-binding polypeptide or engineered cell is administered after administration of the additional agent, or the CL·EC12a-binding polypeptide or engineered cell and the additional agent are administered alternately. The CLEC12a-binding polypeptide and additional agent can be administered in single or multiple doses.

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In some embodiments, the C.bEC.'12a-binding polypeptide or engineered cell and the additional agent(s) are administered simultaneously. For example, the CLEC12a-binding polypeptide and additional agent(s) may be formulated as a single composition or may be administered as two or more separate compositions. In some embodiments, the CLEC12a-binding polypeptide or engineered cell and additional agent(s) are administered sequentially, or the CLEC12a-binding polypeptide or engineered cell and additional agent are administered at different times according to the treatment regimen.

Non-limiting illustrative methods of diagnosis and treatment.

In some embodiments, the methods provided in the present invention are useful for evaluating a subject and/or a sample obtained from a subject (eg, a cancer patient). In some embodiments, the assessment is one or more of the following: diagnosis, prognosis, and/or response to treatment.

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

In some embodiments, the assessment allows treatment to be directed (including treatment with antibodies provided in the present invention). In some embodiments, the assessment allows the use or avoidance of adjuvant therapy after resection to be determined. Adjuvant therapy, also called adjuvant treatment, is treatment that is given in addition to primary, primary, or initial treatment. By way of non-limiting example, adjuvant therapy may be an additional treatment typically given after surgery that has removed all detectable disease but remains at statistical risk of recurrence due to occult disease. In some embodiments, the polypeptides are used as adjuvant therapy in the treatment of cancer. In some embodiments, the polypeptides are used as the only adjuvant therapy in the treatment of cancer. In some embodiments, the polypeptides provided herein are not used as adjuvant therapy in the treatment of cancer. For example, if a patient's response to an antibody of the present invention is unlikely or minimal, then in the interests of quality of life and to avoid unnecessary toxicity from ineffective chemotherapy drugs, treatment may be withheld. In such cases, palliative care can be used.

In some embodiments, the polypeptides are administered as neoadjuvant therapy before resection. In some embodiments, neoadjuvant therapy refers to therapy aimed at shrinking and/or downgrading the tumor before any surgery. In some embodiments, neoadjuvant therapy refers to chemotherapy given to cancer patients before surgery. In some embodiments, neoadjuvant therapy means that the antibody is administered to cancer patients prior to surgery. Types of cancer for which neoadjuvant chemotherapy is usually prescribed include, for example, breast, colon, ovarian, cervical, bladder and lung cancer. In some embodiments, the polypeptides are used as neoadjuvant therapy in the treatment of cancer. In some embodiments, such application is performed prior to resection.

In some embodiments, the tumor microenvironment considered in the methods presented in the present invention is one or more of: the tumor vasculature; tumor-infiltrating lymphocytes; fibroblastic reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; extracellular matrix (ECM) components; dendritic cells; antigen presenting cells; T cells; regulatory T cells; macrophages; other lymphoid cells; neutrophils; and other immune cells located proximal to the tumor.

Sets.

Also provided are articles and kits that include any of the CLEC12a-binding polypeptides provided in the present invention and suitable packaging. In some embodiments, the invention includes a kit with (i) a CLEC12a-binding polypeptide and (ii) instructions for using the kit to administer the CLEC12a-binding polypeptide to an individual.

Suitable packaging for the compositions of the present invention is known in the art and includes, for example, vials (eg, sealed vials), vials, ampoules, bottles, jars, flexible packaging (eg, sealed Mylar or plastic bags), and the like. These products can be additionally sterilized and/or sealed. Unit dosage forms containing the compositions of the present invention are also provided. These units

- 24 046257 The dosage forms may be stored in suitable packaging in one or more unit doses and may be further sterilized and sealed. The instructions provided in the kits of the invention are typically written instructions on a label or package insert (eg, on a piece of paper included in the kit), but machine-readable instructions (eg, instructions on a magnetic or optical storage disk) are also possible. Instructions related to the use of antibodies typically include information on dosage, dosing schedule, and route of administration for targeted treatment or industrial use. The kit may further comprise a description of selecting a suitable individual or treatment.

Containers may be unit doses, bulk units (eg, multi-dose units), or subunit units. For example, kits may also be provided that contain sufficient doses of the molecules provided herein to provide effective treatment to an individual over an extended period, e.g., about 1, 2, 3, 4, 6, 8 weeks, 3, 4, 5 , 6, 7, 8, 9 months or more. The kits may also include multiple unit doses of the molecules and instructions for use and may be packaged in quantities sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies. In some embodiments, the kit includes a dry (eg, lyophilized) composition that can be reconstituted, resuspended, or rehydrated to form a generally stable aqueous suspension of the antibody.

Examples

The examples discussed below are intended solely to illustrate the invention and should not be construed as limiting the invention in any way. The examples are not intended to demonstrate that the experiments below are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numbers used (e.g. quantity, temperature, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

Example 1. Anti-CLEC12a single domain antibodies.

Single domain antibodies targeting human CLEC12a were generated by immunizing llamas and alpacas with a recombinant version of the extracellular domain of human CLEC12a.

After generating specific anti-CLEC12a antibody titers, llama/alpaca peripheral blood mononuclear cells (PBMCs) were isolated from 500 ml of immunized animal blood and total mRNA was isolated using the Qiagen RNeasy Maxi Kit and then transformed into first-strand cDNA using Thermo Superscript reverse transcriptase IV and oligo-dT priming. VHH sequences were specifically amplified by PCR using cDNA as a template and cloned into a vector into yeast cells as VHH-Fc-AGA2 fusion proteins for display on the yeast surface.

Yeast clone libraries displaying VHH-Fc-AGA2 fusion proteins were enriched using recombinant forms of ECD CLEC12a by magnetic bead isolation followed by fluorescence-activated cell sorting (FACS). Sorted yeast cells were plated and isolated colonies were collected into 96-well blocks and grown in a medium that switched expression from VHH-Fc being displayed on the surface to being secreted into the medium. Supernatants from 96-well yeast secretions were applied to 293F cells transiently transfected with CLEC12a (CLEC12a positive) or untransfected 293F cells (CLEC12a negative), washed, treated with fluorophore-labeled secondary antibodies to human IgG1 Fc, and analyzed by flow cytometry in a 96-well tablet.

Nucleic acid sequences encoding VHH that bound CLEC12a positive cells rather than CLEC12a negative cells were cloned in frame with the human Fc coding region into mammalian expression vectors and expressed by transient transfection in HEK293 Freestyle cells (293F cells) or CHO cells using polyethylenimine. The supernatant was collected after 3-7 days, the secreted recombinant protein was purified by protein A chromatography, and the concentration was calculated from the absorbance at 280 nm and the extinction coefficient.

Single domain antibody (sdAb) epitopes containing VHH domains that bound CLEC12a were compared using biolayer interferometry. 7 μg/ml mFc-CLEC12a-ECD (SEQ ID NO: 87) was immobilized on capture sensors coated with anti-mFC antibodies. 100 nM of sdAb alone was then loaded onto the CLEC12a antigen and allowed to reach equilibrium. The sensors were then transferred to 100 nM of the second sdAb. An increase in the assay signal indicated binding, indicating that the second sdAb targets a different epitope than the first sdAb.

Camel-derived VHH CLEC12a was humanized using the human germline VH3-23 as a scaffold. Camelite residues that affect solubility, specificity, stability, and/or affinity were left unmodified. In addition, where possible and according to

- 25 046257 as necessary, amino acid sequences that had potential exhibiting ability were changed to reduce the risk of this event. In addition, all humanized variants contained the Leu11Glu modification (L11E), as described in US 20160207981.

The results show that three different epitopes were detected among the humanized versions of sdAbs MB11, ME06, MC02, MH07, MC06, and ME02. As shown in FIG. 1A, sdAbs hzME02v4 and hzME06v16 have different epitopes from those for sdAbs hzMB11v13, hzMC02v6, hzMH07v3 and hzMC06v2. As shown in FIG. 1B, none of the sdAbs hzME06v16, hzME02v4, and hzMB11v13 share a common epitope. The summary results are presented in the table below. 3.

Binding of sdAb to human CLEC12a was assessed by flow cytometry. Each sdAb contained a VHH domain, as shown in Table. 4 below, and the xELL Fc region of human IgG1 in which the amino acids Glu233, Leu234 and Leu235 have been deleted according to EU numbering (SEQ ID NO: 1). HEK 293 cells were transiently transfected with a plasmid encoding CLEC12a (UniProt accession number Q5QGZ9.3; SEQ ID NO: 1) and used as a positive cell line, and untransfected HEK 293 cells were used as CLEC12a-negative cells. Each cell type was seeded at 30,000 cells/well in FACS buffer (PBS, 1% BSA, 0.1% NaN ₃ , pH 7.4) in a 96-well round bottom plate. SdAb was diluted in FACS buffer by a 3-fold 11-point serial dilution. Dilutions of sdAb were added to the seeded cells, and the assay plates were incubated for 30 min at 4°C. After washing twice in 150 μl of FACS buffer, the cells in each well were resuspended in 100 μl of Alexa Fluor 647-conjugated secondary anti-human IgG antibody diluted 1:2000 in FACS buffer and incubated at 4°C for 30 min. Cells were washed twice, then bound antibody was detected by flow cytometry.

Flow cytometry analysis was performed on an Intellicyte iQue Plus, and fluorescence values were plotted as median fluorescence intensity. Apparent affinity (K _d , nM) was determined by nonlinear single-site binding regression using PRISM graphics software.

As seen in FIG. 2A-2L, the sdAbs tested showed binding to CLEC12a but showed no or minimal binding to untransfected HEK 293 cells not expressing CLEC12a. The apparent binding affinity is given in table. 4 below.

Table 4

sdAb Apparent _Kd (nM) SEQ ID NO of VHH domain M.B.H. 58.6 2 hzMBllvl3 0.3 26 ME06 0.05 18 hzME06vl6 0.06 thirty MS02 0.37 6 hzMC02v6 0.35 27 MH07 0.10 22 hzMH07v3 0.28 31 MC06 1.58 10 hzMC06v2 13.98 28 ME02 1.2 14 hzME02v4 0.87 29

The invention may be embodied in other specific forms without departing from its essence or essential characteristics. Accordingly, the foregoing embodiments are to be considered in all respects as illustrative and not limiting of the invention. The scope of the invention is thus defined by the appended claims and not by the preceding description, and therefore all changes covered by the meaning and range of equivalence of the claims are included within the scope of the invention.

Table of some sequences

SEQID NO Description Subsequence 1 Human CLEC12a MSEEVTYADL QFQNSSEMEK IPEIGKFGEK APPAPSHVWR PAALFLTLLC LLLLIGLGVL ASMFHVTLKI EMKKMNKLQN 1SEELQRN1S LQLMSNMN1S NK1RNLSTTL QT1ATKLCRE LYSKEQEHKC KPCPRRWIWH KDSCYFLSDD VQTWQESKMA CAAQNASLLK INNKNALEFI KSQSRSYDYW LGLSPEEDST RGMRVDNIIN SSAWVIRNAP DLNNMYCGYI NRLYVQYYHC TYKKRMICEK MANPVQLGST YFREA 2 profit center QVQLQQSGGGLVQPGGSLRLSCVASGFLFSIYDMNWYRQA PGKEREWIAGITNNGYSTAYTDSVQGRFTISRDNAKNTIYL QMTSLKPEDTAVYYCHADLTKCYDGEYAWGQGTQVTV 88 profit center to option QVQLQQSGGGLVQPGGSLRLSCVASGFLFSIYDMNWYRQA PGKEREW1AG1TNNGYSTAYTDSVQGRFT1SRDNAKNT1YL QMTSLKPEDTAVYYCHADLTKCYDGEYAWGQGTQVTVK 3 Profit center CDR1 GFLFSIYDMN 4 Profit center CDR2 GITNNGYSTA 5 Profit center CDR3 DLTKCYDGEYA 6 MS02 QVQLVQSGGGLAQPGGSLRLSCTASGFTFRINDMGWYRQA PGKECEMVAGISNNGF STAYAD S VKGRFTISRDNAKNTVSL QMD SLKSEDTAVYYCHRTDDGAAHAC SRGQGTQ VT V 89 MS02 K option QVQLVQSGGGLAQPGGSLRLSCTASGFTFRINDMGWYRQA PGKECEMVAGISNNGF STAYAD S VKGRFTISRDNAKNTVSL QMDSLKSEDTAVYYCHRTDDGAAHACSRGQGTQVTVK 7 MC02 CDR1 GFTFRINDMG 8 MC02 CDR2 GISNNGFSTA 9 MC02 CDR3 TDDGAAAHACS 10 MS06 QVQLQESGGGLVQPGGSLRLSCAASGFTFSVYDMNWYRQ TPGKEREWVAGISNNGFSTAYAGSVKGRFTISRDNAENTVY LQMNSLKSEDTAVYYCNLDVHGRVGAQGDYWGQGTQVT V 90 MS06 k option QVQLQESGGGLVQPGGSLRLSCAASGFTFSVYDMNWYRQ TPGKEREWVAGISNNGFSTAYAGSVKGRFTISRDNAENTVY LQMNSLKSEDTAVYYCNLDVHGRVGAQGDYWGQGTQVT VK eleven MC06 CDR1 GFTFSVYDMN 12 MC06 CDR2 GISNNGFSTA 13 MC06 CDR3 DVHGRVGAQGDY

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14 ME02 QVQLQQSGGGLVQVGGSQRLSCAASGRTFEYDDMVWFRQ APGI<EREEVAGISWSGGSIYYADSVI<GRFSISRDNAI<NTVY LEMNRLKPEDTAVYYCNVGSYWGQGTQVTV 91 ME02 K option QVQLQQSGGGLVQVGGSQRLSCAASGRTFEYDDMVWFRQ APGKEREEVAG1SWSGGS1YYADSVKGRFS1SRDNAKNTVY LEMNRLKPEDTAVYYCNVGSYWGQGTQVTVK 15 ME02 CDR1 GRTFEYDDMV 16 ME02 CDR2 GISWSGGSIY 17 ME02 CDR3 GSY 18 ME06 QVQLVQSGGGLVQAGGSLRLSCVASGFTFDDYAIGWFRQA PGKEREGVSCISNSDGSTYYADSVKGRFTISSDNAKNTVYL HVNSLNPEDTAVYYCALREGLPLLLDSLWRQPVEYDYWGQ GTQVTV 92 ME06 K option QVQLVQSGGGLVQAGGSLRLSCVASGFTFDDYAIGWFRQA PGKEREGVSCISNSDGSTYYADSVKGRFTISSDNAKNTVYL HVNSLNPEDTAVYYCALREGLPLLLDSLWRQPVEYDYWGQ GTQVTVK 19 ME06 CDR1 GFTFDDYAIG 20 ME06 CDR2 CISNSDGSTY 21 ME06 CDR3 REGLPLLLD SLWRQP VEYD Y 22 MH07 QVQLVQSGGGLVQPGGSLRLSCVASGLLFSIYDMNWYRQA PGKEREWIAGITNNGYSTAYTDSVQGRFTISRDNAKNTVYL QMNSLKPEDTAVYYCHTDEWGREYWGQGTQVTV 93 MH07 K option QVQLVQSGGGLVQPGGSLRLSCVASGLLFSIYDMNWYRQA PGKEREWIAGITNNGYSTAYTDSVQGRFTISRDNAKNTVYL QMNSLKPEDTAVYYCHTDEWGREYWGQGTQVTVK 23 МН07 CDR1 GLLFSIYDMN 24 МН07 CDR2 GITNNGYSTA 25 МН07 CDR3 DEWGRAY 26 hzMBllvl3 EVQLVESGGGEVQPGGSLRLSCAASGFLFSIYDMNWYRQA PGKEREWVAGITNNGYSTAYAESVKGRFTISRDNAKNTIYL QMS SLRAEDTAV Y YCHADLTKAYD VE YAWGQGTLVT V 94 hzMBllvl3 KR option EVQLVESGGGEVQPGGSLRLSCAASGFLFSIYDMNWYRQA PGKEREWVAGITNNGYSTAYAESVKGRFTISRDNAKNTIYL QMSSLRAEDTAVYYCHADLTKAYDVEYAWGQGTLVTVKP 27 hzMC02v6 EVQLVESGGGEVQPGGSLRLSCAASGFTFRINDMGWYRQA PGKECEMVAGISNNGFSTAYAESVKGRFTISRDNAKNTVYL QMSSLRAEDTAVYYCHRTDEGAAHACSRGQGTLVTV 95 hzMC02v6 KR option EVQLVESGGGEVQPGGSLRLSCAASGFTFRINDMGWYRQA PGKECEMVAGISNNGFSTAYAESVKGRFTISRDNAKNTVYL QMSSLRAEDTAVYYCHRTDEGAAHACSRGQGTLVTVKP 28 hzMC06v2 EVQLVESGGGEVQPGGSLRLSCAASGFTFSVYDMNWYRQ APGI<EREWVSGISNNGFSTAYAESVI<GRFTISRDNAI<NTLY LQMSSLRAEDTAVYYCNLDVHGRVGAQGDYWGQGTLVTV 96 hzMC06v2 KR option EVQLVESGGGEVQPGGSLRLSCAASGFTFSVYDMNWYRQ APGI<EREWVSGISNNGFSTAYAESVI<GRFTISRDNAI<NTLY LQMSSLRAEDTAVYYCNLDVHGRVGAQGDYWGQGTLVTV KP

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29 hzME02v4 EVQLVESGGGEVQPGGSLRLSCAASGRTFEYDDMVWFRQA PGKEREEVAGISWSGGSIYYAESVKGRFSISRDNAKNTVYLE MNRLKPEDTAVYYCNVGSYWGQGTLVTV 97 hzME02v4 KP option EVQLVESGGGEVQPGGSLRLSCAASGRTFEYDDMVWFRQA PGKEREEVAG1S W SGGS1Y YAES VKGRF S1SRDN AKNT V YLE MNRLKPEDTAVYYCNVGSYWGQGTLVTVKP thirty hzME06vl6 EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAIGWFRQAP GKEREGVSTISSSDGSTYYAESVKGRFTISRDNAKNTVYLQ MSSLRAEDTAVYYCALREGLPLLLDTLWRQPVEYDYWGQ GTQVTV 98 hzME06vl6 KP option EVQLVESGGGEVQPGGSLRLSCAASGFTFDDYAIGWFRQAP GKEREGVSTISSSDGSTYYAESVKGRFTISRDNAKNTVYLQ MSSLRAEDTAVYYCALREGLPLLLDTLWRQPVEYDYWGQ GTQVTVKP 31 hzMH07v3 EVQLVESGGGEVQPGGSLRLSCAASGLLFSIYDMNWYRQA PGKEREWVAGITNNGYSTAYAESVKGRFTISRDNAKNTVYL QMS SLRAEDTAVYYCHTDEWGREYWGQGTLVTV 99 hzMH07v3 KP option EVQLVESGGGEVQPGGSLRLSCAASGLLFSIYDMNWYRQA PGKEREWVAGITNNGYSTAYAESVKGRFTISRDNAKNTVYL QMS SLRAEDTAVYYCHTDEWGREYWGQGTLVTVKP 32 hzMBllvl3 CDR1 GFLFSIYDMN 33 hzMBllv!3 CDR2 GITNNGYSTA 34 hzMBllvl3 CDR3 DLTKAYDVEYA 35 hzMC02v6 CDR1 GFTFRINDMG 36 hzMC02v6 CDR2 GISNNGFSTA 37 hzMC02v6 CDR3 TDEGAAHACS 38 hzMC06v2 CDR1 GFTFSVYDMN 39 hzMC06v2 CDR2 GISNNGFSTA 40 hzMC06v2 CDR3 DVHGRVGAQGDY 41 hzME02v4 CDR1 GRTFEYDDMV 42 hzME02v4 CDR2 GISWSGGSIY 43 hzME02v4 CDR3 GSY 44 hzME06vl6 CDR1 GFTFDDYAIG 45 hzME06vl6 CDR2 TISSSDGSTY 46 hzME06vl6 CDR3 REGLPLLLDTLWRQPVEYDY

- 29 046257

47 hzMH07v3 CDR1 GLLFSTYDMN 48 hzMH07v3 CDR2 GITNNGYSTA 49 hzMH07v3 CDR3 DEWGRAY 50 Human Fc region IgGl DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVE WES NGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVF SC S VMHEALHNHYTQKSLSLSPGK 51 xELL Fc- human IgGl region DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQ VYTLPP SRDELTKNQ VSLTCLVKGF YP SDIAVEWESNG QPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 52 M252Y and M428V (YV) S354C T366W lug Fc section DKTHTCPPCP APELLGGPSV FLFPPKPKDT LYISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPCRDELTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVVHE ALHNHYTQKS LSLSPGK 53 M252Y, M428V, H435R (YVR) T366S, L368A, Y407V depression Fcsection DKTHTCPPCP APELLGGPSV FLFPPKPKDT LYISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVCT LPPSRDELTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVVHE ALHNRYTQKS LSLSPGK 54 H435R xELL Fc section DKTHTC PPCPAPGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 55 M252Y and M428V (YV) xELL Fc- plot DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNHYTQK SLSLSPGK

- 30 046257

56 M252Y and M428L (YL) xELL Fc- plot DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNHYTQK SLSLSPGK 57 M252Y, M428L, H435R (YLR) xELL Fc- plot DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNRYTQK SLSLSPGK 58 M252Y, M428V, H435R (YVR) xELL Fc- plot DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNRYTQK SLSLSPGK 59 S354C T366W xELL Fc-section projection DKTHTC PPCPAPGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 60 H435R S354C T366W xELL Fc-section projection DKTHTC PPCPAPGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 61 M252Y and M428V (YV) S354C T366W xELL Fc-section projection DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCS VVHEALHNHYTQKSL SLSPGK 62 M252Y and M428L (YL) S354C T366W xELL Fc-section projection DKTHTCPPCPAPGGPSVFLFPPKPKDTLY1SRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCS VLHEALHNHYTQKSLSLSPGK

- 31 046257

63 M252Y, M428L, H435R (YLR) S354C T366W xELL Fc-section projection DKTHTCPPCPAPGGPSVFLFPPKPKDTLYTSRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCS VLHEALHNRYTQKSLSLSPGK 64 M252Y, M428V, H435R (YVR) S354C T366W xELL Fc-section projection DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCS VVHEALHNRYTQKSLSLSPGK 65 T366S, L368A, Y407V depression xELL Fc-section DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLVS KLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 66 H435R, T366S, L368A, Y407V xELL cavity of Fc region DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLVS KLTVDKSRWQQGNVFSCS VMHEALHNRYTQKSLSLSPGK 67 M252Y and M428V (YV) T366S, L368A, Y407V depression xELL Fc-section DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNG QPENNYKTTPP VLD SDGSFFLVS KLTVDKSRWQQGNVF SC S VVHEALHNHYTQKSL SLSPGK 68 M252Y and M428L (YL) T366S, L368A, Y407V depression xELL Fc-section DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNG QPENNYKTTPP VLD SDGSFFLVS KLTVDKSRWQQGNVF SC S VLHEALHNHYTQKSLSLSPGK 69 M252Y, M428L, H435R (YLR) T366S, L368A, Y407V xELL cavity Fc-section DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLVS KLTVDKSRWQQGNVFSCS VLHEALHNRYTQKSLSLSPGK

- 32 046257

70 M252Y, M428V, H435R (YVR) T366S, L368A, Y407V recess projection xELL Fc section DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLVS KLTVDKSRWQQGNVFSCS VVHEALHNRYTQKSLSLSPGK 71 H435R Fcsection DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 72 M252Y and M428V (YV) Fc section DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNHYTQK SLSLSPGK 73 M252Y and M428L (YL) Fc region DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNHYTQK SLSLSPGK 74 M252Y, M428L, H435R (YLR) Fc section DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNRYTQK SLSLSPGK 75 M252Y, M428V, H435R (YVR) Fc section DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNRYTQK SLSLSPGK 76 S354C T366W lug Fc section DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK

- 33 046257

77 H435R S354C T366W Fc section lug DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMTSRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 78 M252Y and M428L (YL) S354C T366W lug Fc section DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVV VDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVF SC S VLHEALHNHYTQKSLSLSPGK 79 M252Y, M428L, H435R (YLR) S354C T366W Fc section projection DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVV VDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVF SCSVLHEALHNRYTQKSLSLSPGK 80 M252Y, M428V, H435R (YVR) S354C T366W Fc section projection DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVV VDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVF SC S VVHEALHNRYTQKSLSLSPGK 81 T366S, L368A, Y407V cavity Fc section DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLVSK LTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 82 H435R, T366S, L368A, Y407V cavity Fc section DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLVSK LTVDKSRWQQGNVF SCSVMHEALHNRYTQKSLSLSPGK 83 M252Y and M428V (YV) T366S, L368A, Y407V depression of Fc section DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVV VDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESN GQPENN YKTTPP VLD SDGSFFLV SK LT VDKSRWQQGN VF S C S VVHEALHNHYTQKSLSLSPGK 84 M252Y and M428L (YL) T366S, L368A, Y407V depression of Fc section DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVV VDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLVSK LTVDKSRWQQGNVFS WITH S VLHEALHNHYTQKSLSLSPGK

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Claims (37)

85 M252Y, M428L, H435R (YLR) T366S, L368A, Y407V depression Fcsection DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYTSRTPEVTCVV VDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLVSK LTVDKSRWQQGNVFS C S VLHEALHNRYTQKSLSLSPGK 86 CLEC12aECD TLKIEMI<I<MNI<LQNISEELQRNISLQLMSNMNISNI<IRNLS TTLQTIATKLCRELYSKEQEHKCKPCPRRWIWHKDSCYFLS DDVQTWQESKMACAAQNASLLKINNKNALEFIKSQSRSYD YWLGLSPEEDSTRGMRVDNIINSSAWVIRNAPDLNNMYCG YINRLYVQYYHCTYKKRMICEKPVQ LGSTYFREA 87 mFcCLEC12aECD LEVLFQGPGSPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMI SLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHR EDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEK TISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEI AVDWTSNGRTEQNYKNTATVLD SDGSYFMYSKLRVQKST WERGSLFACSVVHEGLHNHLTTKT1SRSLGKGGTGGSTLKI EMKKMNKLQNISEELQRNISLQLMSNMNISNKIRNLSTTLQ TIATKLCRELYSKEQEHKCKPCPRRWIWHKDSCYFLSDDVQ TWQESKMACAAQNASLLKINNKNALEFIKSQSRSYDYWLG LSPEEDSTRG MRVDNIINSSAWVIRNAPDLNNMYCGYINRL YVQYYHCTYKKRMICEKMANPVQLGSTYFREA CLAIM 1. A polypeptide containing at least one VHH domain that binds to CLEC12a and which contains (a) a CDR1 containing the amino acid sequence of SEQ ID NO: 47; CDR2 containing the amino acid sequence SEQ ID NO: 48; and CDR3 containing the amino acid sequence of SEQ ID NO: 49; (b) CDR1 containing the amino acid sequence of SEQ ID NO: 32; CDR2 containing the amino acid sequence SEQ ID NO: 33; and CDR3 containing the amino acid sequence of SEQ ID NO: 34 or 5; (c) CDR1 containing the amino acid sequence of SEQ ID NO: 7; CDR2 containing the amino acid sequence SEQ ID NO: 8; and CDR3 containing the amino acid sequence of SEQ ID NO: 9 or 37; (d) CDR1 containing the amino acid sequence of SEQ ID NO: 11; CDR2 containing the amino acid sequence SEQ ID NO: 12; and CDR3 containing the amino acid sequence of SEQ ID NO: 13; (e) CDR1 containing the amino acid sequence of SEQ ID NO: 15; CDR2 containing the amino acid sequence SEQ ID NO: 16; and CDR3 containing the amino acid sequence SEQ ID NO: 17; and/or (f) a CDR1 containing the amino acid sequence of SEQ ID NO: 19; CDR2 containing the amino acid sequence SEQ ID NO: 20 or 45; and CDR3 containing the amino acid sequence of SEQ ID NO: 21 or 46. 2. The polypeptide according to claim 1, in which at least one VHH domain contains a CDR1 containing the amino acid sequence SEQ ID NO: 47; CDR2 containing the amino acid sequence SEQ ID NO: 48; and CDR3 containing the amino acid sequence of SEQ ID NO: 49. 3. The polypeptide according to claim 1 or 2, in which at least one VHH domain contains a CDR1 containing the amino acid sequence SEQ ID NO: 32; CDR2 containing the amino acid sequence SEQ ID NO: 33; and CDR3 containing the amino acid sequence of SEQ ID NO: 34 or 5. 4. The polypeptide according to any one of claims 1 to 3, in which at least one VHH domain contains a CDR1 containing the amino acid sequence SEQ ID NO: 7; CDR2 containing the amino acid sequence SEQ ID NO: 8; and CDR3 containing the amino acid sequence of SEQ ID NO: 9 or 37. 5. The polypeptide according to any one of claims 1 to 4, in which at least one VHH domain contains a CDR1 containing the amino acid sequence SEQ ID NO: 11; CDR2 containing the amino acid sequence SEQ ID NO: 12; and CDR3 containing the amino acid sequence of SEQ ID NO: 13. 6. The polypeptide according to any one of claims 1 to 5, in which at least one VHH domain contains a CDR1 containing the amino acid sequence SEQ ID NO: 15; CDR2 containing the amino acid sequence SEQ ID NO: 16; and CDR3 containing the amino acid sequence - 35 046257 activity SEQ ID NO: 17. 7. The polypeptide according to any one of claims 1 to 6, in which at least one VHH domain contains a CDR1 containing the amino acid sequence SEQ ID NO: 19; CDR2 containing the amino acid sequence SEQ ID NO: 20 or 45; and CDR3 containing the amino acid sequence of SEQ ID NO: 21 or 46. 8. The polypeptide according to any one of claims 1 to 7, in which at least one VHH domain contains CDR1, CDR2 and CDR3, respectively, containing the amino acid sequences of SEQ ID NO: 32, 33 and 34; SEQ ID NO: 47, 48 and 49; SEQ ID NO: 32, 33 and 5; SEQ ID NO: 7, 8 and 9; SEQ ID NO: 11, 12 and 13; SEQ ID NO: 15, 16 and 17; SEQ ID NO: 19,20 and 21; SEQ ID NO: 7, 8 and 37; SEQ ID NO: 19, 45 and 46. 9. The polypeptide according to any one of claims 1 to 8, wherein at least one VHH domain is humanized. 10. The polypeptide according to any one of claims 1 to 9, in which at least one VHH domain contains an amino acid sequence that is at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequences SEQ ID NO: 26, 94, 31, 99, 27, 28, 29, 30, 95, 96, 97 or 98. 11. A polypeptide according to any one of claims 1 to 9, wherein at least one VHH domain contains the amino acid sequence SEQ ID NO: 26, 94, 31, 99, 27, 28, 29, 30, 95, 96, 97 or 98 . 12. The polypeptide according to any one of claims 1 to 8, in which at least one VHH domain contains an amino acid sequence that is at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequences SEQ ID NO: 2, 6, 10, 14, 18, 22, 88, 89, 90, 91, 92 or 93. 13. A polypeptide according to any one of claims 1 to 8, wherein at least one VHH domain contains the amino acid sequence SEQ ID NO: 2, 6, 10, 14, 18, 22, 88, 89, 90, 91, 92 or 93 . 14. A polypeptide according to any one of claims 1 to 13, containing two VHH domains. 15. A polypeptide according to any one of claims 1 to 13, containing three VHH domains. 16. A polypeptide according to any one of claims 1 to 15, comprising at least one binding domain that binds to an antigen other than CLEC12a. 17. The polypeptide of claim 16, comprising at least one binding domain that binds CD3, T cell receptor (TCR)a, TCRe, CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D. 18. The polypeptide of claim 14 or 15, wherein each VHH domain binds to CLEC12a. 19. The polypeptide of claim 18, wherein each VHH domain contains the same amino acid sequences CDR1, CDR2 and CDR3. 20. The polypeptide of claim 18, wherein each VHH domain contains the same VHH sequence. 21. A polypeptide according to any one of claims 1 to 13, containing one VHH domain. 22. A polypeptide according to any one of claims 1 to 21, containing an Fc region. 23. The polypeptide according to claim 22, wherein the Fc region contains an amino acid sequence selected from SEQ ID NO: 50-85. 24. The polypeptide according to claim 22 or 23, which forms a dimer under physiological conditions. 25. The polypeptide according to any one of claims 1 to 24, wherein CLEC12a is human CLEC12a. 26. The polypeptide of claim 25, wherein human CLEC12a comprises the sequence SEQ ID NO: 1. 27. An immunoconjugate containing a polypeptide according to any one of claims 1 to 26 and a cytotoxic agent. 28. The immunoconjugate of claim 27, wherein the cytotoxic agent is selected from calicheamicin, auristatin, dolastatin, tubulicin, maytansinoid, cryptophycin, duocarmycin, esperamycin, pyrrolobenzodiazepine and enediyne antibiotic. 29. A pharmaceutical composition containing a polypeptide according to any one of claims 1 to 26 or an immunoconjugate according to claim 27 or 28 and a pharmaceutically acceptable carrier. 30. An isolated nucleic acid encoding a polypeptide according to any one of claims 1 to 26. 31. A vector containing the nucleic acid according to claim 30. 32. A host cell containing the nucleic acid of claim 30 or the vector of claim 31. 33. A host cell that expresses the polypeptide according to any one of claims 1 to 26. 34. A method for producing a polypeptide according to any one of claims 1 to 26, comprising incubating a host cell according to claim 32 or 33 under conditions suitable for expression of the polypeptide. 35. The method of claim 34, further comprising isolating the polypeptide. 36. A method of treating cancer, comprising administering to a subject suffering from cancer a pharmaceutically effective amount of a polypeptide according to any one of claims 1 to 26, an immunoconjugate according to claim 27 or 28, or a pharmaceutical composition according to claim 29. 37. The method according to claim 36, where the cancer is selected from lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; low-grade/follicular non-Hodgkin's lymphoma (NHL); NHL from small lymphocytes (ML); moderate malignancy/follicular NHL; diffuse NHL of moderate malignancy; immunoblastic NHL high grade malignancy -