CLEC12A antibody fragment sequences and methods

Abstract

Anti-CLEC12A polypeptides generally comprise an amino acid sequence having at least 90% amino acid similarity to SEQ ID NO: 14. In some embodiments, anti-CLEC12A polypeptides may be incorporated into anti-CLEC12A biologics. In some of these embodiments, the anti-CLEC12A biologic may be a bispecific killer engager molecule (BiKE), a trispecific killer engager molecule (TriKE), a tetraspecific killer engager molecule (TetraKE), a pentaspecific killer engager molecule (PentaKE), a bispecific T cell engager molecule (BiTE), a trispecific T cell engager molecule (TriTE), a tetraspecific T cell engager molecule (TetraTE), a pentaspecific T cell engager molecule (PentaTE), a chimeric antigen receptor, a complete antibody, an antibody-drug conjugate (ADC) molecule, a targeted delivery construct, or a labeling construct.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 916,340, filed October 17, 2019, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application is an ASCII text file entitled "0110-000633WO01" having a size of 39 KB and created on October 14, 2020. This document contains a sequence listing that has been electronically submitted to the U.S. Patent and Trademark Office via EFS-Web as "ST25.txt." For purposes of electronic filing of a sequence listing, the electronically submitted sequence listing serves as the written copy required by 37CRF §1.821(c) and 37CRF §1.821(e). The information contained in the sequence listing is incorporated herein by reference. Summary of the Invention

[0003] In some embodiments, the present disclosure describes anti-CLEC12A polypeptides having an amino acid sequence with at least 90% amino acid similarity to SEQ ID NO:14.

[0004] In some embodiments, the anti-CLEC12A polypeptide comprises the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 10, the amino acid sequence of SEQ ID NO: 11, the amino acid sequence of SEQ ID NO: 12, or the amino acid sequence of SEQ ID NO: 13.

[0005] In some embodiments, the anti-CLEC12A polypeptide comprises the amino acid sequence of SEQ ID NO:29, the amino acid sequence of SEQ ID NO:30, and the amino acid sequence of SEQ ID NO:31.

[0006] In some embodiments, the anti-CLEC12A polypeptides may be incorporated into an anti-CLEC12A biologic. In some of these embodiments, the anti-CLEC12A biologic may be a bispecific killer engager molecule (BiKE), a trispecific killer engager molecule (TriKE), a tetraspecific killer engager molecule (TetraKE), a pentaspecific killer engager molecule (PentaKE), a bispecific T cell engager molecule (BiTE), a trispecific T cell engager molecule (TriTE), a tetraspecific T cell engager molecule (TetraTE), a pentaspecific T cell engager molecule (PentaTE), a chimeric antigen receptor, a complete antibody, an antibody-drug conjugate (ADC) molecule, a targeted delivery construct, or a labeling construct.

[0007] In some embodiments, the anti-CLEC12A biologic can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition.

[0008] The above summary is not intended to describe each disclosed embodiment or every embodiment of the present invention. The following description more particularly exemplifies exemplary embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves merely as a representative group and should not be interpreted as an exclusive list. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows an amino acid sequence alignment of 13 unique anti-CLEC12A antibody variant clones (SEQ ID NOS: 1-13) identified by phage display and the anti-CLEC12A antibody consensus sequence (SEQ ID NOS: 14). CDR1, CDR2, and CDR3 sequences are underlined. [Figure 2]FIG. 2 shows SDS-PAGE of the His-tagged human CLEC12A extracellular domain used for screening. [Figure 3] Figure 3 shows the functional screening of bispecific compounds containing humanized camel (huCAM) anti-CLEC12A antibody fragments using PBMCs. Peripheral blood mononuclear cells (PBMCs) were incubated with the CLEC12A-expressing promyelocytic leukemia cell line HL60 in the presence of the described bispecific compounds containing different clones (SEQ ID NOs: 1-13). A trispecific compound (SEQ ID NO: 15) containing an anti-CLEC12A scFv and known to target CLEC12A was used as a positive control, while no treatment (NT) was used as a negative control. After 5 hours of culture, cells were harvested, stained for surface antigens, fixed, permeabilized, and stained for intracellular interferon-γ (IFNγ). Cells were run on a flow cytometer, and NK cell activation was measured via assessment of degranulation (CD107a) of CD56+CD3- NK cells. [Figure 4] Figure 4 shows the functional screening of bispecific compounds containing humanized camelid (huCAM) anti-CLEC12A antibody fragments using PBMCs. Peripheral blood mononuclear cells (PBMCs) were incubated with the CLEC12A-expressing promyelocytic leukemia cell line HL60 in the presence of the described bispecific compounds containing different clones (SEQ ID NOs: 1-13). A trispecific compound (SEQ ID NO: 15) containing an anti-CLEC12A scFv and known to target CLEC12A was used as a positive control, while no treatment (NT) was used as a negative control. After 5 hours of incubation, cells were harvested, stained for surface antigens, fixed, permeabilized, and stained for intracellular interferon-γ (IFNγ). Cells were run on a flow cytometer, and NK cell activation was measured via assessment of IFNγ production by CD56+CD3- NK cells. [Figure 5]Figure 5 shows the evaluation of a bispecific compound containing humanized camelid (huCAM) anti-CLEC12A clone 33. To determine background activation mediated by the clone 33 bispecific compound, enriched NK cells were incubated for 5 hours with a bispecific compound containing anti-CLEC12A clone 33 (clone 33 huCAM engager), a CLEC12A trispecific compound (SEQ ID NO: 15; scFv engager), or compound (NT). NK cell degranulation (CD107a, left) and cytokine production (IFNγ, right) were measured by flow cytometry. While the scFv showed some background activation of degranulation, clone 33 did not, highlighting its better specificity. [Figure 6] Figure 6 shows the evaluation of bispecific compounds containing humanized camel (huCAM) anti-CLEC12A clone 33. To determine activation mediated by bispecific compounds containing anti-CLEC12A clone 33 against CLEC12-expressing targets, enriched NK cells were incubated for 5 hours with HL60 cells and either anti-CLEC12A clone 33 (clone 33 huCAM engager), a CLEC12A trispecific compound (SEQ ID NO: 15; scFv engager), or a bispecific compound containing compound (NT) (SEQ ID NO: 34). NK cell degranulation (CD107a, left) and cytokine production (IFNγ, right) were measured by flow cytometry. The clone 33-containing avian-specific compound induced NK cell degranulation against HL60 targets and induced cytokine production at the same level as the scFv-containing positive control. [Figure 7]Figure 7 shows the evaluation of bispecific compounds containing humanized camel (huCAM) anti-CLEC12A clone 33. To determine activation mediated by bispecific compounds containing anti-CLEC12A clone 33 against CLEC12A-negative targets (nonspecific activation), enriched NK cells were incubated for 5 hours with Raji (Burkitt's lymphoma) cells and either anti-CLEC12A clone 33 (clone 33 huCAM engager), a CLEC12A trispecific compound (SEQ ID NO: 15; scFv engager), or a bispecific compound containing compound (NT) (SEQ ID NO: 34). NK cell degranulation (CD107a, left) and cytokine production (IFNγ, right) were measured by flow cytometry. Bispecific compounds containing clone 33 induced less nonspecific degranulation against CLEC12A-negative targets than trispecific compounds containing anti-CLEC12A scFv. [Figure 8] Figure 8 shows the evaluation of the binding specificity of clone 33. (A) Binding of the ClEC A12A scFv of clone 33-containing bispecific compound (SEQ ID NO: 34) and trispecific compound (SEQ ID NO: 15) containing anti-CLEC12A scFv to CLECA12A+ HL60 cells. (B) Binding of the Clone 33-containing bispecific compound (SEQ ID NO: 34) and trispecific compound (SEQ ID NO: 15) containing anti-CLEC12A scFv to CLEC12A-negative Raji cells. DETAILED DESCRIPTION OF THE INVENTION

[0010] C-type lectin domain family 12 member A (CLEC12A) is a protein encoded by the CLEC12A gene in humans. CLEC12A is a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. CLEC12A is an inhibitory C-type lectin-like receptor. It contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail that can associate with signaling phosphatases such as SHP-1 and SHP-2.

[0011] Human CLEC12A is a monomer expressed primarily on myeloid cells, such as granulocytes, monocytes, macrophages, and dendritic cells. Because CLEC12A is expressed in the majority of myeloblasts and leukemic stem cell (LSC) myeloid cells, but not in normal tissues or normal hematopoietic stem cells, CLEC12A is a target for immunotherapy to treat myeloid malignancies, such as acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS).

[0012] Human CLEC12 was subjected to phage-display single-domain antibody (sdAb) library screening. Three rounds of library panning were performed using a pre-generated single-domain antibody library for human CLEC12A. As shown in Figure 2, SDS-PAGE results indicated that the recombinant human CLEC12A protein was of high quality. Next, biopanning was performed to enrich for specific binders to the target human CLEC12A. As shown in Table 1, after three rounds of library screening, a strong enrichment effect was observed for human CLEC12A, with clear differences between the target screening group and the coating control group.

[0013] [Table 1]

[0014] Ninety-six clones were selected from the 3-P eluate and subjected to monoclonal phage ELISA. Eighty-two positive clones were identified and subjected to DNA sequencing. Seventy-seven clones were successfully sequenced, and 13 unique sequences were found (SEQ ID NOs: 1-13). The 13 unique sequences were aligned to generate a consensus sequence (SEQ ID NO: 14), as shown in Figure 1, and the complementarity-determining regions (CDRs) were identified.

[0015] After sequencing, 13 unique sequences were cloned into a soluble VHH-AP expression vector and subjected to soluble expression and soluble ELISA. As shown in Table 2, all 13 clones positively bound to human CLEC12A.

[0016] [Table 2]

[0017] Figures 3 and 4 show functional screening of humanized camel (huCAM) anti-CLEC12A clones using PBMCs. Humanized anti-CLEC12A camelized variants (SEQ ID NOs: 1-13) were cloned into a bispecific scaffold containing camelized anti-CD16 as the targeting domain and a linker. These bispecific compounds activated natural killer (NK) cells through the formation of a cytolytic bridge between NK cells and tumor cells (via binding of CD16) via CLEC12A binding. As a positive control, a trispecific killer engager (SEQ ID NO: 15) previously tested and shown to target CLEC12A via a single-chain variable fragment (scFv) was used. The data show that clone 33 exhibited the highest activity. Some clones were produced in lower amounts and therefore tested at lower concentrations than the scFv (trispecific positive control) or the clone 33 bispecific compound. Therefore, no conclusions can be drawn from results indicating that a clone appears to lack activity compared to the negative control.

[0018] To determine the functional specificity of the humanized CLEC12A camelized clone 33 antibody fragment, we used a CD16-Clone 33 bispecific compound (SEQ ID NO: 34). This bispecific compound has the ability to activate natural killer (NK) cells through the formation of a cytolytic bridge between NK cells (via binding of CD16) and tumor cells (via binding of CLEC12A). Figure 5 shows background activation of NK cells, as measured by NK cell degranulation (CD107a, left) and IFNγ induction (right). Bispecific compounds containing clone 33 exhibit minimal background NK cell activation. Figure 6 shows NK cell activation in the presence of CLEC12A-positive HL60 cells. Bispecific compounds containing anti-CLEC12A clone 33 antibody fragments induced both degranulation (left) and IFNγ (right), with IFNγ induction nearly identical to that of the positive control anti-CLEC12A-scFv-containing trispecific compound (SEQ ID NO: 15). Figure 7 shows NK activation in the presence of CLEC12A-negative Raji (Burkitt's lymphoma) cells. Clone-33-containing bispecific compounds induce less non-specific degranulation against CLEC12A-negative targets than anti-CLEC12A-scFv-containing trispecific compounds.

[0019] Furthermore, Figure 8 shows the binding specificity of anti-CLEC12A clone 33. Both bispecific compounds containing anti-CLEC12A clone 33 antibody fragments and trispecific compounds containing anti-CLEC12A scFv were constructed to contain a 10xHIS tag. Binding of these constructs to CLEC12A-positive HL60 cells (Figure 8A) and CLEC12A-negative Raji cells (Figure 8B) was assessed using an anti-HIS-phycoerythrin-labeled antibody. As a control, basal binding of the anti-HIS-PE antibody was measured without prior binding of an engager compound (gray bar). As the data show, both the clone 33 bispecific compound and the scFv trispecific compound induced minimal binding to CLEC12A-negative cells. The clone 33 bispecific compound induced greater binding to CLEC12A-positive cells than the anti-CLEC12A scFv-containing trispecific compound.

[0020] Thus, the present disclosure describes polypeptides that target CLEC12A on myeloid malignancies. Because the polypeptides target CLEC12A, the polypeptides are less likely to induce targeting of normal myeloid cells than polypeptides that target other antigens associated with myeloid malignancies, such as CD33. The present disclosure explicitly describes 12 unique humanized single-domain antibody (sdAb) sequences that target CLEC12A, which are consensus sequences derived from alignment of 12 unique anti-CLEC12A sdAb sequences, and provides guidance for further variants by identifying regions of high conservation and variability among all 13 sequences.

[0021] The anti-CLEC12A polypeptides described herein can be incorporated into biological constructs that can be used, for example, in connection with therapeutic, diagnostic, and / or detection methods. For example, the anti-CLEC12A polypeptides described herein can be incorporated into biological constructs that can be used, for example, in connection with therapeutic, diagnostic, and / or detection methods. For example, the anti-CLEC12A polypeptides described herein can be incorporated into bispecific killer engager molecules (BiKE, e.g., SEQ ID NO: 34), trispecific killer engager molecules (TriKE, e.g., SEQ ID NO: 35), tetraspecific killer engager molecules (TetraKE), pentaspecific killer engager molecules (PentaKE), bispecific T cell engager molecules (BiTE), trispecific T cell engager molecules (TriTE), tetraspecific T cell engager molecules (TetraTE), pentaspecific T cell engager molecules (PentaTE), chimeric antigen receptors (CARs, e.g., CAR T cells, CAR for expression in NK cells, CAR macrophage cells, etc.), complete antibody constructs (e.g., to induce antibody-dependent cellular cytotoxicity (ADCC)), antibody-drug conjugate (ADC) molecules (e.g., for delivery of toxins), labeled constructs (e.g., for commercial assessment of antigen expression), radiolabeled formats (e.g., for positron emission tomography (PET) imaging or directional radiation delivery), applications for delivery of cytokines and / or chemokines, or other common immunotherapeutic approaches.

[0022] Other reagents—e.g., CD33-binding antibodies and / or antibody fragments—target myeloid malignancies that express CD33, but also target normal myeloid cells that express lower levels of the target. In contrast, the anti-CLEC12A polypeptides described herein target antigens that are more specifically expressed on myeloid malignancies (e.g., acute myeloid leukemia (AML) cells or myelodysplastic syndrome (MDS) cells) and are therefore much less likely to result in on-target, off-tumor toxicity. CLEC12A is also expressed in leukemia stem cells. Thus, targeting CLEC12A can limit the likelihood and / or severity of relapse. The anti-CLEC12A polypeptides described herein may also offer advantages over scFvs for forming constructs (e.g., BiKEs, TriKEs, BiTEs, CARs, etc.) because sdAbs are more stable than scFvs. Finally, the anti-CLEC12A sequences described herein are all from humanized libraries and are therefore unlikely to be rejected in human patients.

[0023] The present disclosure describes anti-CLEC12A polypeptides. Exemplary anti-CLEC12A polypeptides include polypeptides comprising, structurally similar to, or functional variants of the amino acid sequence of any one of SEQ ID NOS: 1-14 or 29-31. SEQ ID NOS: 1-13 are mutant single-domain antibody sequences selected via phage display screening for specific binding to CLEC12A. SEQ ID NOS: 14 is a consensus sequence derived from alignment analysis of SEQ ID NOS: 1-13 (FIG. 1). SEQ ID NOS: 29 is a consensus sequence for CDR1 determined from the alignment analysis shown in FIG. 1. SEQ ID NOS: 30 is a consensus sequence for CDR2 determined from the alignment analysis shown in FIG. 1. SEQ ID NOS: 31 is a consensus sequence for CDR3 determined from the alignment analysis shown in FIG. 1.

[0024] As used herein, an anti-CLEC12A polypeptide is "structurally similar" or a "functional variant" of a reference polypeptide if the amino acid sequence of the anti-CLEC12A polypeptide has a certain amount of identity compared to the reference polypeptide. An amino acid sequence is a "functional fragment" of a reference amino acid sequence if the "functional fragment" amino acid sequence is less than the full-length amino acid sequence of the reference amino acid sequence. A "functional fragment" may further have a certain amount of sequence identity or sequence specificity compared to the reference amino acid sequence.

[0025] Structural similarity and / or sequence identity of two polypeptides can be determined by aligning the amino acid residues of the two polypeptides (e.g., a candidate anti-CLEC12A polypeptide and, for example, a polypeptide of any one of SEQ ID NOS: 1-14 or 29-31) to optimize the number of identical amino acids along the length of their sequences. To optimize the number of identical amino acids, gaps in either or both sequences are allowed when performing the alignment, but the amino acids in each sequence must nevertheless maintain their proper order. A candidate anti-CLEC12A polypeptide is a polypeptide that is compared to a reference polypeptide (e.g., any one of SEQ ID NOS: 1-14 or 29-31). A candidate polypeptide can, for example, be isolated from an animal, produced using recombinant technology, or synthesized chemically or enzymatically.

[0026] Pairwise comparison analysis of amino acid sequences can be performed using the BESTFIT algorithm in the GCG package (version 10.2, Madison, WI). Alternatively, polypeptides can be compared using the Blastp program, a BLAST 2 search algorithm described by Tatiana et al. (FEMS Microbiol Lett, 174, 247-250 (1999)) and available on the National Center for Biotechnology Information (NCBI) website. The default values ​​for all BLAST 2 search parameters are: matrix = BLOSUM62, open gap penalty = 11, extended gap penalty = 1, gap x Includes dopoff=50, expectation=10, word size=3, and filter on.

[0027] In comparing two amino acid sequences, structural similarity can be referred to as percent "identity" or percent "similarity." "Identity" refers to the presence of identical amino acids. "Similarity" refers not only to the presence of identical amino acids but also to the presence of conservative substitutions. Conservative substitutions for amino acids in anti-CLEC12A polypeptides can be selected from other members of the class to which the amino acid belongs. For example, it is well known in the field of protein biochemistry that an amino acid belonging to a group of amino acids having a particular size or characteristic (such as charge, hydrophobicity, and hydrophilicity) can be substituted with another amino acid without altering the activity of the protein, particularly in regions of the protein not directly related to biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Lys for Arg to maintain a positive charge, Glu for Asp to maintain a positive charge, Glu to maintain a negative charge, Ser for Thr to maintain a free -OH, and Gln for Asn to maintain a free -NHOA. Similarly, biologically active analogs of polypeptides containing one or more consecutive or non-consecutive amino acid deletions or additions that do not eliminate the functional activity of the polypeptide are also contemplated.

[0028] In some embodiments, the anti-CLEC12A polypeptides described herein may include polypeptides having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to a reference amino acid sequence.

[0029] In some embodiments, the anti-CLEC12A polypeptides described herein may comprise at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to a reference amino acid sequence.

[0030] In some embodiments, the anti-CLEC12A polypeptides described herein can also be designed to provide additional sequences, such as, for example, amino acids added to the C-terminus or N-terminus of the anti-CLEC12A polypeptide. Such additional amino acids can include, for example, a signal sequence (e.g., SEQ ID NO: 32) or a tag that facilitates purification by trapping the tagged anti-CLEC12A polypeptide on a column or using an antibody. Exemplary tags include, for example, a histidine-rich tag (e.g., SEQ ID NO: 33), which allows for purification of the polypeptide on a nickel column.

[0031] The present disclosure also describes polynucleotides encoding anti-CLEC12A polypeptides. Exemplary polynucleotides encoding anti-CLEC12A polypeptides include polynucleotides comprising the nucleotide sequence of any one of SEQ ID NOS: 16-28. However, SEQ ID NOS: 16-28 are merely exemplary. Because the genetic code is well known, the present disclosure describes any polynucleotide that encodes an anti-CLEC12A polypeptide as described herein.

[0032] The anti-CLEC12A polypeptides described herein can be incorporated into biological preparations, which can then be formulated with a pharmaceutically acceptable carrier. As used herein, an "anti-CLEC12A biological preparation" is a biological compound containing an anti-CLEC12A polypeptide. Depending on the basic structural platform of the biological preparation (e.g., BiKE, TriKE, CAR, etc.), the anti-CLEC12A biological preparation may contain additional functional moieties. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating agent, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, etc. The use of such media and / or agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. As used herein, "pharmaceutically acceptable" refers to a biologically or otherwise undesirable substance, i.e., the substance may be administered to an individual in conjunction with an anti-CLEC12A biological agent without causing undesired biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0033] Therefore, anti-CLEC12A biological agents can be formulated into pharmaceutical compositions. Pharmaceutical compositions can be formulated in various forms compatible with the preferred route of administration. Thus, the compositions can be administered via known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, subcutaneous, rectal, etc.). Pharmaceutical compositions can be administered to mucosal surfaces, for example, by administration to the nasal or respiratory mucosa (e.g., by spray or aerosol). The compositions can also be administered via sustained or delayed release.

[0034] Therefore, the anti-CLEC12A biological preparation can be provided in any suitable form, including, but not limited to, a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture. The composition can be delivered in the formulation together with any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation can be delivered in a conventional topical administration form, such as a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, etc. The formulation can further include one or more additives, including, for example, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, etc.

[0035] The formulations can be conveniently provided in unit dosage form and can be prepared by methods well known in the art of pharmacy. The method of preparing a composition using a pharmaceutically acceptable carrier includes combining the anti-CLEC12A biological agent with the carrier, which constitutes one or more accessory ingredients. In general, the formulations can be prepared by uniformly and / or intimately combining the active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

[0036] Thus, in another aspect, the present disclosure describes a method of treating any condition in which targeting cells that overexpress CLEC12A has therapeutic benefit. In many embodiments, the condition can be a myeloid malignancy. However, in other aspects, the condition can be an autoimmune condition, such as a condition in which myeloid cells infiltrate the central nervous system. Generally, the method includes administering to a subject an anti-CLEC12A biologic in an amount effective to treat the condition. "Treating" or variations thereof refers to reducing, limiting the progression of, ameliorating, or resolving, to any extent, symptoms or signs associated with a pathological condition. As used herein, "amelioration" refers to any decrease in the degree, severity, frequency, and / or likelihood of a symptom or clinical sign characteristic of a particular condition; "symptom" refers to any subjective evidence of a disease or a patient's condition; and "sign" or "clinical sign" refers to objective physical findings associated with a particular condition that may be detected by someone other than the patient.

[0037] "Treatment" can be therapeutic or prophylactic. "Therapeutic" and its variations refer to treatment that improves one or more existing symptoms or clinical signs associated with a condition. "Prophylactic" and its variations refer to treatment that limits, to some extent, the occurrence and / or emergence of a symptom or clinical signs of a condition. Generally, "therapeutic" treatment is initiated before a condition manifests in a subject, while "prophylactic" treatment is initiated before a condition manifests in a subject. Thus, in certain embodiments, the method can include prophylactic treatment of a subject at risk of developing a condition. "At risk" refers to a subject who may or may not actually have the stated risk. Thus, for example, a subject "at risk" of developing a particular condition is one who has one or more indicators of having or being at increased risk of developing a particular condition compared to an individual lacking one or more indicators, regardless of whether the subject exhibits symptoms or clinical signs of developing or having developed a symptom. Exemplary indicators of a condition can include, for example, genetic predisposition, ancestry, age, sex, geographic location, lifestyle, or medical history. Treatment can also be continued after symptoms have resolved, for example, to prevent or delay recurrence.

[0038] In some embodiments, "prophylactic" treatment also includes treatment in the setting of a relapse. In other words, a subject has once developed clinical signs of a symptom or condition but has received successful treatment such that the condition is considered to be in remission. Such patients may no longer develop symptoms or clinical signs of the remission state, but may be "at risk" of relapse. In such situations, treatment including an anti-CLEC12A agent may be considered "prophylactic" because it reduces the likelihood and / or severity of relapse.

[0039] Thus, an anti-CLEC12A biologic may be administered to a subject before, during, or after the subject first exhibits clinical signs of a symptom or condition. Treatment initiated before a subject first exhibits symptoms or clinical signs associated with the condition reduces the likelihood that the subject will experience clinical evidence of the condition, and / or completely resolve the symptoms and / or clinical signs of the condition, compared to subjects not administered the anti-CLEC12A biologic. Treatment initiated before a subject first exhibits symptoms or clinical signs associated with a recurrence of the condition reduces the likelihood that the subject will experience clinical evidence of recurrence, and / or completely resolve the symptoms and / or clinical signs of the recurrence, compared to subjects not administered the anti-CLEC12A biologic. Treatment initiated while a subject is exhibiting symptoms or clinical signs associated with the condition may result in a reduction in the severity of the symptoms and / or clinical signs of the condition, compared to subjects not administered the anti-CLEC12A biologic and / or whose condition is completely resolved.

[0040] The amount of anti-CLEC12A biological agent administered can vary depending on various factors, including, but not limited to, the specific anti-CLEC12A biological agent administered, the subject's weight, physical condition, and / or age, and / or the route of administration. Thus, the absolute weight of the anti-CLEC12A biological agent contained in a given unit dosage form can vary widely and can depend on factors such as the species, age, weight and physical condition of the subject, and / or the method of administration. Therefore, it is not practical to generally describe an amount that constitutes an amount of anti-CLEC12A biological agent effective for all possible uses. However, those skilled in the art can readily determine an appropriate amount, taking such factors into due consideration.

[0041] In some embodiments, the method can include administering an anti-CLEC12A biological agent sufficient to provide a dose of, for example, about 100 ng / kg / day to about 50 mg / kg / day to the subject, although in some embodiments, the method can be practiced by administering an anti-CLEC12A biological agent at a dose outside this range.

[0042] In some embodiments, the methods may include administering sufficient anti-CLEC12A biologic to provide a minimum dose of at least 100 ng / kg / day, e.g., at least 1 μg / kg / day, at least 5 μg / kg / day, at least 10 μg / kg / day, at least 25 μg / kg / day, at least 50 μg / kg / day, at least 100 μg / kg / day, at least 200 μg / kg / day, at least 300 μg / kg / day, at least 400 μg / kg / day, at least 500 μg / kg / day, at least 600 μg / kg / day, at least 700 μg / kg / day, at least 800 μg / kg / day, at least 900 μg / kg / day, or at least 1 mg / kg / day.

[0043] In some embodiments, the method provides a method for administering a dose of 10 mg / kg / day or less, e.g., 5 mg / kg / day or less, 4 mg / kg / day or less, 3 mg / kg / day or less, 2 mg / kg / day or less, 1 mg / kg / day or less, 900 μg / kg / day or less, 800 μg / kg / day or less, 700 μg / kg / day or less, 600 μg / kg / day or less, 500 μg / kg / day or less, 400 μg / kg / day or less, 300 μg / kg / day or less The present invention also includes administering sufficient anti-CLEC12A biologic to provide a maximum dose of up to 200 μg / kg / day, 100 μg / kg / day, 90 μg / kg / day, 80 μg / kg / day, 70 μg / kg / day, 60 μg / kg / day, 50 μg / kg / day, 40 μg / kg / day, 30 μg / kg / day, 20 μg / kg / day, or 10 μg / kg / day. The anti-CLEC12A biologic provides a dose "not exceeding" a specified amount when the anti-CLEC12A biologic is absent but present in an amount up to the specified amount.

[0044] In some embodiments, the methods involve administering sufficient anti-CLEC12A biologic to provide a dose characterized by a range having endpoints defined by any minimum dose identified above and any maximum dose greater than the selected minimum dose. For example, in some embodiments, the methods involve administering sufficient anti-CLEC12A biologic to provide a dose of about 10 μg / kg / day to about 10 mg / kg / day, a dose of about 100 μg / kg / day to about 1 mg / kg / day, a dose of 5 μg / kg / day to 100 μg / kg / day, etc.

[0045] In certain embodiments, the methods involve administering sufficient anti-CLEC12A biologic to provide a dose equal to any minimum dose or any maximum dose listed above. Thus, for example, in certain embodiments, the methods may involve administering sufficient anti-CLEC12A biologic to provide a dose of 1 μg / kg / day, 5 μg / kg / day, 10 μg / kg / day, 25 μg / kg / day, 50 μg / kg / day, 100 μg / kg / day, 200 μg / kg / day, 500 μg / kg / day, 1 mg / kg / day, 5 mg / kg / day, etc.

[0046] In some embodiments, the anti-CLEC12A biologic can be administered, for example, from a single dose to multiple times per week, although in some aspects, the method can be practiced by administering the anti-CLEC12A biologic at a frequency exceeding this range. In certain embodiments, the anti-CLEC12A biologic can be administered from about once per month to about five times per week. In some embodiments, the doses described above in terms of the amount of anti-CLEC12A biologic administered over a 24-hour period are administered in a 7-day cycle of 4 days on and 3 days off.

[0047] In some embodiments, the anti-CLEC12A biologic can be administered, for example, from a single dose to multiple cycles of treatment, although in some aspects, the method can be practiced by administering the anti-CLEC12A biologic for periods exceeding this range. In some aspects, the anti-CLEC12A biologic can be administered for three weeks. In such embodiments, each week can be a treatment cycle, such as the exemplary treatment cycle described above. In other embodiments, the anti-CLEC12A biologic can be administered for more than one treatment cycle without a gap between one set of treatment cycles and a subsequent set of treatment cycles. The gap between one set of treatment cycles can be one week or more, one month or more, or one year or more.

[0048] In some embodiments, the method further comprises administering one or more additional therapeutic agents. The one or more additional therapeutic agents (e.g., chemotherapeutic agents) may be administered before, after, and / or simultaneously with the administration of the anti-CLEC12A biologic. The anti-CLEC12A biologic and additional therapeutic agents may be used in combination. As used herein, "co-administration" refers to two or more components of a combination administered such that the therapeutic or prophylactic effect of the combination is greater than the therapeutic or prophylactic effect of either component administered alone. The two components may be co-administered simultaneously or sequentially. Components co-administered simultaneously may be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes when the components are administered such that each component is present at the treatment site at the same time. Alternatively, sequential co-administration of two components may include when at least one component is removed from the treatment site, but at least one cellular effect of administering the component (e.g., cytokine production, activation of a specific cell population, etc.) persists at the treatment site until one or more additional components are administered to the treatment site. Thus, co-administered combinations may, in certain circumstances, include components that are not present in chemical mixtures with each other. In other embodiments, the anti-CLEC12A biologic and additional therapeutic agent can be administered as part of a mixture or cocktail. In some aspects, administration of an anti-CLEC12A biologic may allow for the effectiveness of a lower dose of the other therapeutic agent or other therapeutic modality when compared to administration alone, thereby reducing the likelihood, severity, and / or extent of toxicity observed when higher doses of the other therapeutic agent or agent are administered.

[0049] Exemplary additional therapeutic agents include altretamine, amsacrine, L-asparaginase, colspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphan, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, fluorouracil, fludarabine, fotemustine, gancyclo These include benzodiazepine, benzodiazepine, benzocaine ...

[0050] In the foregoing description and in the claims that follow, the term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements. "Comprises," "comprising," and variations thereof are to be construed as open-ended, i.e., additional elements or steps are optional and may or may not be present. Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably to mean one or more. The recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0051] In the foregoing description, for clarity, a particular embodiment may be described in isolation. A particular embodiment may include any combination of compatible features described herein in connection with one or more embodiments, unless otherwise expressly specified that a feature of a particular embodiment is incompatible with a feature of another embodiment.

[0052] For any method disclosed herein that includes discrete steps, the steps can be performed in any practicable order, and, where appropriate, any combination of two or more steps can be performed simultaneously. [Example]

[0053] Construction of CD16-huCAMCLEC12A bispecific compound (SEQ ID NO: 34) Constructs encoding the CD16-huCAMCLEC12A bispecific compound were synthesized using PCR and HiFi cloning techniques as previously described (Vallera et al., 2016, Clin Cancer Res 22:3440-3450). Each fully assembled fragment contained an EcoRI restriction site, an ATG start codon, the coding sequence for a humanized camel anti-CD16 sdAb (Vincke et al., 2007, Protein Eng Des Sel 21:1-10), the coding sequence for a huCAMCLEC12A sdAb (one of SEQ ID NOS: 16-28), and a sequence encoding a 10xHis tag. The assembled fragments were cloned into a minicircle DNA vector (System Biosciences, LLC, Palo Alto, CA) under the control of the CMV promoter. The DNA sequence was verified to confirm the sequence and location of the gene insertion (Biomedical Genomics Center, University of Minnesota, Minneapolis, MN).

[0054] Construction of CD16-huCAMCLEC12A trispecific compound (SEQ ID NO: 35) Constructs encoding the CD16-huCAMCLEC12A trispecific compound were synthesized using PCR and HiFi cloning techniques as previously described (Vallera et al., 2016, Clin Cancer Res 22:3440-3450). Each fully assembled fragment contained an EcoRI restriction site, an ATG start codon, coding sequence for a humanized camel anti-CD16 sdAb (Vincke et al., 2007, Protein Eng Des Sel 21:1-10), a linker, coding sequence for a human IL-15 fragment (amino acids 162-175 of SEQ ID NO:35), a short amino acid linker (GSTSGSGKPGSGEGSTKG; SEQ ID NO:36), coding sequence for a huCAMCLEC12A sdAb (one of SEQ ID NOs:16-28), and a 10xHis tag. The assembled fragments were cloned into a minicircle DNA vector (System Biosciences, LLC, Palo Alto, CA) under the control of the CMV promoter, and the DNA sequence was verified to confirm the sequence and location of the gene insertion (Biomedical Genomics Center, University of Minnesota, Minneapolis, MN).

[0055] Production and isolation of CD16-huCAMCLEC12A bispecific and CD16-huCAMCLEC12A trispecific compounds Plasmids from all clones were transfected into Expi293 cells (Thermo Fisher Scientific, Inc., Waltham, MA) according to the manufacturer's protocol. Supernatants were collected, and proteins were purified using HisPur cobalt resin (Thermo Fisher Scientific, Inc., Waltham, MA) and Pierce centrifugal columns (Thermo Fisher Scientific, Inc., Waltham, MA). Proteins were eluted with 250 mM imidazole and desalted using prepacked disposable PD-10 columns (GE Healthcare Systems, Chicago, IL). Purity and size were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis using Simply Blue Life Stain (Invitrogen, Carlsbad, CA).

[0056] Cancer cell lines (HL60 and Raji) HL60 promyeloblast cells (ATCC CCL-240, American Type Culture Collection, Manasas, VA) were obtained from ATCC and used as a CLEC12A-expressing line. Raji Burkitt lymphoma lymphoblasts (ATCC CCL-86, American Type Culture Collection, Manasas, VA) were also obtained from ATCC and used as a CLECA12A-negative line.

[0057] Functional evaluation of different clones using PBMC against CLEC12A-positive HL60 cells Healthy donor blood was obtained from Memorial Blood Bank (Minneapolis, MN) and processed to obtain peripheral blood mononuclear cells (PBMCs) using a density gradient Ficoll-Paque (GE Healthcare Systems, Chicago, IL). NK cell function was assessed by flow cytometry as previously described (Vallera et al., 2016, Clin Cancer Res 22:3440-3450). PBMCs, HL60 cells, and treatment (30 nM) were co-cultured and stained with FITC-conjugated anti-CD107a (H4A3, BioLegend, San Diego, CA). One hour after addition of anti-CD107a, cells were loaded onto GolgiStop and GolgiPlug (BD Biosciences, San Jose, CA) and incubated for 3 hours. At the end of the incubation period, cells were stained with a Live / Dead Fixable Aqua staining kit (Thermo Fisher Scientific, Inc., Waltham, MA), PE-CY7-conjugated anti-CD56, PE-CF594-conjugated anti-CD3, and PE-conjugated anti-CD69 (FN50, BioLegend, San Diego, CA), fixed, and permeabilized. Permeabilized cells were stained with BV650-conjugated IFNγ (4S.B3, BioLegend, San Diego, CA), and expression was assessed by flow cytometry.

[0058] Evaluation of the functional specificity of clone 33 for NK cells, CLEC12A-positive HL60 cells, or CLEC12A-negative Raji cells Healthy donor blood was obtained from Memorial Blood Bank (Minneapolis, MN) and processed to obtain peripheral blood mononuclear cells (PBMCs) using a density gradient Ficoll-Paque (GE Healthcare Systems, Chicago, IL). PBMCs were magnetically enriched for NK cells using the EasySep Human NK Cell Enrichment Kit (STEMCELL Technologies, Inc., Vancouver, BC). NK cell function was assessed by flow cytometry as previously described (Vallera et al., 2016, Clin Cancer Res 22:3440-3450). Enriched NK cells were incubated alone, with HL60 cells, or with Raji cells and the indicated treatments (30 nM). Cells and treatments were co-cultured and stained with FITC-conjugated anti-CD107a (H4A3, BioLegend, San Diego, CA). One hour after addition of anti-CD107a, cells were given Golgi Stop and Golgi Plug (BD Biosciences, San Jose, CA) and incubated for 3 hours. At the end of the incubation, cells were stained with a Live / Dead Fixable Aqua Staining Kit (Thermo Fisher Scientific, Inc., Waltham, MA), PE-CY7-conjugated anti-CD56, PE-CF594-conjugated anti-CD3, and PE-conjugated anti-CD69 (BioLegend, San Diego, CA), fixed, and permeabilized. Permeabilized cells were stained with BV650-conjugated IFNγ (BioLegend, San Diego, CA), and expression was assessed by flow cytometry.

[0059] Determination of binding specificity To measure binding specificity, 30 nM of the clone 33 bispecific compound or the anti-CLEC12A scFv-containing trispecific compound was incubated with CLEC12A-positive HL60 cells or CLEC12A-negative Raji cells for 30 minutes at 37°C. The cells were then centrifuged and washed twice. Anti-HIS, phycoerythrin (PE)-labeled antibody (OriGene Technologies, Inc., Rockville, MD) was added and incubated with the cells for 20 minutes at 4°C. The cells were washed twice, fixed with 2% paraformaldehyde, and run on a flow cytometer to assess the percentage of cells binding to the indicated constructs.

[0060] The complete disclosures of all patents, patent applications, publications, and electronically available materials (including, for example, nucleotide sequence submissions in GenBank or RefSeq, amino acid sequence submissions in SwissProt, PIR, PRF, PDB, etc.), and translations from the annotated coding regions of GenBank and RefSeq, cited herein, are incorporated by reference in their entirety. In the event of a conflict between the disclosure of this application and the disclosures of the documents incorporated herein by reference, the disclosure of this application shall control. The foregoing detailed description and examples are provided for ease of understanding only. No unnecessary limitations should be understood therefrom. The present invention is not limited to the exact details shown and described, as variations obvious to those skilled in the art are encompassed within the invention as defined by the claims.

[0061] Unless otherwise noted, all numbers expressing quantities of ingredients, molecular weights, and so forth used in the specification and claims should be understood to be modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending on the desired properties sought by the present invention. At the very least, and not as an attempt to limit the scope of the claims to the doctrine of equivalents, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0062] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviation found in their respective testing measurements.

[0063] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so designated.

[0064] Sequence-free text SEQ ID NO: 1: CLEC12A clone 1 1 QVQLQESGGG LVQPGGSLRL SCAASGDRFS NDVMAWVRQA PGKGLEWVSA 51 IVTQDGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAGEP 101 LDAEDLWYWG QGTLVTVSS

[0065] SEQ ID NO: 2: CLEC12A clone 3 1 QVQLLESGGG LVQPGGSLRL SCAASGVRVS AQFMSWVRQA PGKGLEWVSS 51 IYKKNGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAIDH 101 GEEHKELYSW GQGTLVTVSS

[0066] SEQ ID NO: 3: CLEC12A clone 9 1 QVQLLESGGG LVQPGGSLRL SCAASGYSIT DQDMSWVRQA PGKGLEWVSG 51 ILATSGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAGEV 101 EKSSQSMPFW GQGTLVTVSS

[0067] SEQ ID NO: 4: CLEC12A clone 10 1 QVQLLESGGG LVQPGGSLRL SCAASGVNVT ADDMSWVRQA PGKGLEWVST 51 IDGGDGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCATAF 101 TSHEAEVHSW GQGTLVTVSS

[0068] SEQ ID NO: 5: CLEC12A clone 11 1 QVQLLESGGG LVQPGGSLRL SCAASGYRLS NDIMAWVRQA PGKGLEWVSA 51 IVDTDGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDP 101 YNEDDVSYWG QGTLVTVSS

[0069] SEQ ID NO: 6: CLEC12A clone 17 1 QVQLLESGGG LVQPGGSLRL SCAASGFRFI AQDMSWVRQA TGKGLEWVST 51 IATNDGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCATDS 101 SWENDLWYWG QGTLVTVSS

[0070] SEQ ID NO: 7: CLEC12A clone 23 1 QVQLLESGGG LVQPGGSLRL SCAASGFRLT DEDMSWVRQA PGKGLEWVST 51 IATRDGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAGLR 101 DQLYGNEQLA FWGQGTLVTV SS

[0071] SEQ ID NO: 8: CLEC12A clone 29 1 QVQLLESGGG LVQPGGSLRL SCAASGYNVI YEDMGWVRQA PGKGLEWVSG 51 IDVPSGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAAVV 101 GQDWFQAEPS EMYYWGQGTL VTVSS

[0072] SEQ ID NO: 9: CLEC12A clone 33 1 QVQLLESGGG LVQPGGSLRL SCAASGDMFS YDDMGWVRQA PGKGLEWVSG 51 IQNTDGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCATLY 101 DRMVGKEEQL ASWGQGTLVT VSS

[0073] SEQ ID NO: 10: CLEC12A clone 40 1 QVQLLESGGG LVQPGGSLRL SCAASGDTIN PEDMGWVRQA PGKGLEWVSA 51 IEAQSGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARLT 101 AHQEEPVAYW GQGTLVTVSS

[0074] SEQ ID NO: 11: CLEC12A clone 56 1 QVQLLESGGG LVQPGGSLRL SCAASGFTVT DHDMGWVRQA PGKGLEWVSS 51 ITVGNGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCATEP 101 LPHQEVTYWG QGTLVTVSS

[0075] SEQ ID NO: 12: CLEC12A clone 60 1 QVQLLESGGG LVQPGGSLRL SCAASGYMFS ADVMSWVRQA PGKGLEWVSG 51 IWIPDGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAAQL 101 NERIASITKN MHSWGQGTLV TVSS

[0076] SEQ ID NO: 13: CLEC12A clone 86 1 QVQLLESGGG LVQPGGSLRL SCAASGDMLS AQDMGWVRQA PGKGLEWVSG 51 IDSDDGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAALW 101 GEDVIMQDSS VGSWGQGTLV TVSS

[0077] SEQ ID NO: 14: CLEC12A clone consensus sequence 1 QVQLLESGGG LVQPGGSLRL SCAASGYXVS XDDMSWVRQA PGKGLEWVSA 51 IXXXDGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAXXX 101 XXXXXXXXXX LXYWGQGTLV TVSS X = any amino acid or no amino acid

[0078] SEQ ID NO: 15: CLEC12A TriKE amino acid sequence

[0079] [ka]

[0080] Underline: signal peptide Bold: anti-CLEC12A scFv

[0081] SEQ ID NO: 16: Clone 1 DNA sequence 1 CAGGTGCAGC TGCAGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC 61 TCCTGTGCAG CCTCCGGAGA TAGGTTTAGC AATGACGTTA TGGCCTGGGT CCGCCAGGCT 121 CCAGGGAAGG GTCTAGAGTG GGTATCAGCC ATTGTTACCC AAGACGGTAG CACATACTAC 181 GCAGACTCCG TGAAGGGCCG GTTCACCATC TCCCGTGACA ATTCCAAGAA CACGCTGTAT 241 CTGCAAATGA ACAGCCTGCG TGCCGAGGAC ACCGCGGTAT ATTATTGCGC GGGAGAGCCT 301 CTTGATGCCG AGGACTTGTG GTATTGGGGT CAGGGAACCC TGGTCACCGT CTCGAGC

[0082] SEQ ID NO: 17: Clone 3 DNA Sequence 1 CAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC 61 TCCTGTGCAG CCTCCGGAGT TAGGGTTAGC GCTCAGTTTA TGAGCTGGGT CCGCCAGGCT 121 CCAGGGAAGG GTCTAGAGTG GGTATCAAGC ATTTATAAGA AAAACGGTAG CACATACTAC 181 GCAGACTCCG TGAAGGGCCG GTTCACCATC TCCCGTGACA ATTCCAAGAA CACGCTGTAT 241 CTGCAAATGA ACAGCCTGCG TGCCGAGGAC ACCGCGGTAT ATTATTGCGC GATTGATCAT 301 GGGGAGGAGC ACAAGGAGCT GTACTCTTGG GGTCAGGGAA CCCTGGTCAC CGTCTCGAGC

[0083] SEQ ID NO: 18: Clone 9 DNA sequence 1 CAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC 61 TCCTGTGCAG CCTCCGGATA TAGCATTACC GATCAGGATA TGAGCTGGGT CCGCCAGGCT 121 CCAGGGAAGG GTCTAGAGTG GGTATCAGGC ATTCTGGCCA CAAGCGGTAG CACATACTAC 181 GCAGACTCCG TGAAGGGCCG GTTCACCATC TCCCGTGACA ATTCCAAGAA CACGCTGTAT 241 CTGCAAATGA ACAGCCTGCG TGCCGAGGAC ACCGCGGTAT ATTATTGCGC GGGAGAGGTT 301 GAGAAGAGTT CCCAGTCGAT GCCGTTTTGG GGTCAGGGAA CCCTGGTCAC CGTCTCGAGC

[0084] SEQ ID NO: 19: Clone 10 DNA sequence 1 CAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC 61 TCCTGTGCAG CCTCCGGAGT TAACGTTACC GCTGACGATA TGAGCTGGGT CCGCCAGGCT 121 CCAGGGAAGG GTCTAGAGTG GGTATCAACC ATTGATGGCG GTGACGGTAG CACATACTAC 181 GCAGACTCCG TGAAGGGCCG GTTCACCATC TCCCGTGACA ATTCCAAGAA CACGCTGTAT 241 CTGCAAATGA ACAGCCTGCG TGCCGAGGAC ACCGCGGTAT ATTATTGCGC GACTGCTTTT 301 ACGTCGCATG AGGCGGAGGT CCACTCTTGG GGTCAGGGAA CCCTGGTCAC CGTCTCGAGC

[0085] SEQ ID NO: 20: Clone 11 DNA sequence 1 CAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC 61 TCCTGTGCAG CCTCCGGATA TAGGCTTAGC AATGACATTA TGGCCTGGGT CCGCCAGGCT 121 CCAGGGAAGG GTCTAGAGTG GGTATCAGCC ATTGTGGACA CAGACGGTAG CACATACTAC 181 GCAGACTCCG TGAAGGGCCG GTTCACCATC TCCCGTGACA ATTCCAAGAA CACGCTGTAT 241 CTGCAAATGA ACAGCCTGCG TGCCGAGGAC ACCGCGGTAT ATTATTGCGC GAGAGATCCT 301 TATAATGAGG ACGACGTCAG CTATTGGGGT CAGGGAACCC TGGTCACCGT CTCGAGC

[0086] SEQ ID NO: 21: Clone 17 DNA sequence 1 CAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC 61 TCCTGTGCAG CCTCCGGATT TAGGTTTATC GCTCAGGATA TGAGCTGGGT CCGCCAGGCT 121 ACAGGGAAGG GTCTAGAGTG GGTATCAACC ATTGCTACGA ATGACGGTAG CACATACTAC 181 GCAGACTCCG TGAAGGGCCG GTTCACCATC TCCCGTGACA ATTCCAAGAA CACGCTGTAT 241 CTGCAAATGA ACAGCCTGCG TGCCGAGGAC ACCGCGGTAT ATTATTGCGC GACTGATTCG 301 TCTTGGGAGA ACGACTTGTG GTATTGGGGT CAGGGAACCC TGGTCACCGT CTCGAGC

[0087] Sequence number 22: Clone 23 DNA sequence 1 CAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC 61 TCCTGTGCAG CCTCCGGATT TAGGCTTACC GATGAGGATA TGAGCTGGGT CCGCCAGGCT 121 CCAGGGAAGG GTCTAGAGTG GGTATCAACC ATTGCTACCA GAGACGGTAG CACATACTAC 181 GCAGACTCCG TGAAGGGCCG GTTCACCATC TCCCGTGACA ATTCCAAGAA CACGCTGTAT 241 CTGCAAATGA ACAGCCTGCG TGCCGAGGAC ACCGCGGTAT ATTATTGCGC GGGACTTAGG 301 GATCAGTTGT ATGGGAACGA GCAGTTGGCC TTTTGGGGTC AGGGAACCCT GGTCACCGTC 361 TCGAGC

[0088] Sequence number 23: Clone 29 DNA sequence 1 CAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC 61 TCCTGTGCAG CCTCCGGATA TAACGTTATC TATGAGGATA TGGGCTGGGT CCGCCAGGCT 121 CCAGGGAAGG GTCTAGAGTG GGTATCAGGC ATTGATGTCC CTAGCGGTAG CACATACTAC 181 GCAGACTCCG TGAAGGGCCG GTTCACCATC TCCCGTGACA ATTCCAAGAA CACGCTGTAT 241 CTGCAAATGA ACAGCCTGCG TGCCGAGGAC ACCGCGGTAT ATTATTGCGC GGCTGTTGTT 301 GGTCAGGATT GGTTTCAGGC TGAGCCCTCG GAGATGTACT ATTGGGGTCA GGGAACCCTG 361 GTCACCGTCT CGAGC

[0089] SEQ ID NO: 24: Clone 33 DNA Sequence 1 CAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC 61 TCCTGTGCAG CCTCCGGAGA TATGTTTAGC TATGACGATA TGGGCTGGGT CCGCCAGGCT 121 CCAGGGAAGG GTCTAGAGTG GGTATCAGGC ATTCAGAACA CTGACGGTAG CACATACTAC 181 GCAGACTCCG TGAAGGGCCG GTTCACCATC TCCCGTGACA ATTCCAAGAA CACGCTGTAT 241 CTGCAAATGA ACAGCCTGCG TGCCGAGGAC ACCGCGGTAT ATTATTGCGC GACTCTGTAT 301 GATAGGATGG TTGGGAAGGA GGAGCAGTTG GCCTCTTGGG GTCAGGGAAC CCTGGTCACC 361 GTCTCGAGC

[0090] SEQ ID NO: 25: Clone 40 DNA sequence 1 CAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC 61 TCCTGTGCAG CCTCCGGAGA TACCATTAAC CCTGAGGATA TGGGCTGGGT CCGCCAGGCT 121 CCAGGGAAGG GTCTAGAGTG GGTATCAGCC ATTGAGGCGC AAAGCGGTAG CACATACTAC 181 GCAGACTCCG TGAAGGGCCG GTTCACCATC TCCCGTGACA ATTCCAAGAA CACGCTGTAT 241 CTGCAAATGA ACAGCCTGCG TGCCGAGGAC ACCGCGGTAT ATTATTGCGC GAGATTGACT 301 GCTCATCAGG AGGAGCCGGT CGCGTATTGG GGTCAGGGAA CCCTGGTCAC CGTCTCGAGC

[0091] SEQ ID NO: 26: Clone 56 DNA sequence 1 CAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC 61 TCCTGTGCAG CCTCCGGATT TACGGTTACC GATCACGATA TGGGCTGGGT CCGCCAGGCT 121 CCAGGGAAGG GTCTAGAGTG GGTATCAAGC ATTACTGTGG GAAACGGTAG CACATACTAC 181 GCAGACTCCG TGAAGGGCCG GTTCACCATC TCCCGTGACA ATTCCAAGAA CACGCTGTAT 241 CTGCAAATGA ACAGCCTGCG TGCCGAGGAC ACCGCGGTAT ATTATTGCGC GACAGAGCCT 301 TTGCCGCACC AGGAGGTCAC GTATTGGGGT CAGGGAACCC TGGTCACCGT CTCGAGC

[0092] SEQ ID NO: 27: Clone 60 DNA Sequence 1 CAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC 61 TCCTGTGCAG CCTCCGGATA TATGTTTAGC GCTGACGTTA TGAGCTGGGT CCGCCAGGCT 121 CCAGGGAAGG GTCTAGAGTG GGTATCAGGC ATTTGGATCC CTGACGGTAG CACATACTAC 181 GCAGACTCCG TGAAGGGCCG GTTCACCATC TCCCGTGACA ATTCCAAGAA CACGCTGTAT 241 CTGCAAATGA ACAGCCTGCG TGCCGAGGAC ACCGCGGTAT ATTATTGCGC GGCTCAGCTG 301 AATGAGAGGA TTGCTTCTAT TACGAAGAAC ATGCACTCTT GGGGTCAGGG AACCCTGGTC 361 ACCGTCTCGA GC

[0093] SEQ ID NO: 28: Clone 86 DNA Sequence<​​61 TCCTGTGCAG CCTCCGGAGA TATGCTTAGC GCTCAGGATA TGGGCTGGGT CCGCCAGGCT 121 CCAGGGAAGG GTCTAGAGTG GGTATCAGGC ATTGATAGCG ATGACGGTAG CACATACTAC 181 GCAGACTCCG TGAAGGGCCG GTTCACCATC TCCCGTGACA ATTCCAAGAA CACGCTGTAT 241 CTGCAAATGA ACAGCCTGCG TGCCGAGGAC ACCGCGGTAT ATTATTGCGC GGCATTGTGG 301 GGGGAGGATG TGATTATGCA GGACTCGTCG GTGGGGTCTT GGGGTCAGGG AACCCTGGTC 361 ACCGTCTCGA GC

[0094] SEQ ID NO: 29: CDR1 X1X2X3MX4 X1 = N, A, D, Y, or P X​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​15 X 16 X1 = any amino acid or deletion X2 = any amino acid or deletion X3 = any amino acid or deletion X4 = P, E, S, Q, R, or H X5 = any amino acid or deletion X6 = any amino acid or deletion X7 = any amino acid or deletion X8 = any amino acid or deletion X9 = any amino acid or deletion X 10 = any amino acid or deletion X 11 = any amino acid or deletion X 12 = any amino acid or deletion X 13 = any amino acid or deletion X 14 = L, M, or V X 15 =W, Y, P, H, S, A, T, or G X 16 = Y, S, or F

[0097] SEQ ID NO: 32: signal peptide MKWVTFISLL FLFSSAYS

[0098] SEQ ID NO: 33: Histidine tag with spacer VDEHHHHHHH HHH

[0099] SEQ ID NO: 34: CD16-huCAM clone 33 bispecific compound

[0100] [ka]

[0101] Underline: signal sequence Double underline: HuEF91 Bold: anti-CLEC12A clone 33

[0102] SEQ ID NO: 35: CD16-huCAM clone 33 trispecific compound

[0103] [ka]

[0104] Underline: signal sequence Double underline: HuEF91 Dashed line: human IL-15 fragment Bold: anti-CLEC12A clone 33

[0105] SEQ ID NO: 36: Whitlow linker GSTSGSGKPG SGEGSTKG

Claims

1. An anti-CLEC12A single domain antibody (sdAb) comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 13.

2. A biological therapeutic compound comprising the anti-CLEC12 AsdAb of claim 1.

3. bispecific killer engager molecules (BiKE); Trispecific killer engager molecule (TriKE); Quadruple specific killer engager molecule (TetraKE); Penta-specific killer engager molecule (PentaKE); bispecific T cell engager molecules (BiTEs); Trispecific T cell engager molecule (TriTE); Quadruspecific T cell engager molecule (TetraTE); Pentaspecific T cell engager molecule (PentaTE); chimeric antigen receptors; complete antibody; antibody-drug conjugate (ADC) molecules; a targeted delivery construct; or Labeled constructs 3. The biological therapeutic compound of claim 2, wherein:

4. The biotherapeutic compound of claim 2; and Pharmaceutically acceptable carrier 10. A pharmaceutical composition comprising:

5. 10. The biological therapeutic compound of claim 2 for use in therapy.

6. 10. The biotherapeutic compound of claim 2 for use in ameliorating at least one symptom or clinical sign of cancer in a subject having a cancer characterized by expression of CLEC12A.

7. The biological therapeutic compound is bispecific killer engager molecules (BiKE); Trispecific killer engager molecule (TriKE); Quadruple specific killer engager molecule (TetraKE); Penta-specific killer engager molecule (PentaKE); bispecific T cell engager molecules (BiTEs); Trispecific T cell engager molecule (TriTE); Quadruspecific T cell engager molecule (TetraTE); Pentaspecific T cell engager molecule (PentaTE); chimeric antigen receptors; complete antibody; an antibody-drug conjugate (ADC) molecule; or Targeted delivery construct 7. The biological therapeutic compound of claim 6, wherein:

8. An anti-CLEC12 AsdAb as described in claim 1, wherein the sdAb comprises the amino acid sequence of SEQ ID NO:

9.

9. 3. The biological therapeutic compound of claim 2, wherein the biological therapeutic compound comprises the amino acid sequence of SEQ ID NO: 34 or 35.

10. 7. The biotherapeutic compound of claim 6, wherein the cancer is a myeloid malignancy.

11. 11. The biotherapeutic compound of claim 10, wherein the malignant tumor is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).

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