Clec12a antibody fragment sequences and methods

Abstract

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

Description

[0001] Cross-reference to related applications

[0002] 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.

[0003] sequence list

[0004] This application contains a sequence list electronically filed with the U.S. Patent and Trademark Office via EFS-Web. The sequence list is an ASCII text file entitled “0110-000633WO01_ST25.txt”, 39 KB in size, and created on October 14, 2020. Due to the electronic filing of the sequence list, the electronically filed sequence list serves as a paper copy as required by 37 CFR §1.821(c) and a CRF as required by §1.821(e). Information contained in the sequence list is incorporated herein by reference. Summary of the Invention

[0005] In one aspect, this disclosure describes an anti-CLEC12A polypeptide. The anti-CLEC12A polypeptide has an amino acid sequence that is at least 90% amino acid similar to SEQ ID NO:14.

[0006] In some embodiments, the anti-CLEC12A polypeptide comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

[0007] 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.

[0008] In some embodiments, the anti-CLEC12A peptide may be incorporated into an anti-CLEC12A biologic. In some of these embodiments, the anti-CLEC12A biologic may be a bi-specific killer engager molecule (BiKE), a tri-specific killer engager molecule (TriKE), a tetra-specific killer engager molecule (TetraKE), a penta-specific killer engager molecule (PentaKE), a bi-specific T-cell engager molecule (BiTE), a tri-specific T-cell engager molecule (TriTE), a tetra-specific T-cell engager molecule (TetraTE), a penta-specific T-cell engager molecule (PentaTE), a chimeric antigen receptor, a complete antibody, an antibody-drug conjugate (ADC) molecule, a targeted delivery construct, or a labeled construct.

[0009] In some embodiments, the anti-CLEC12A biologic can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition.

[0010] The foregoing description is not intended to depict every disclosed embodiment or implementation of the invention. The following description illustrates exemplary embodiments in more detail. Throughout this application, guidance is provided by a list of examples that can be used in various combinations. In each case, the enumerated list is intended only as a representative group and should not be construed as an exclusive list. Attached Figure Description

[0011] Figure 1 The amino acid sequences of 13 unique anti-CLEC12A antibody variant clones (SEQ ID NO:1-13) identified by phage display were compared with the common sequence of anti-CLEC12A antibody (SEQ ID NO:14). The CDR1, CDR2 and CDR3 sequences are underlined.

[0012] Figure 2 SDS-PAGE of His-tagged human CLEC12A extracellular domains for screening.

[0013] Figure 3Functional screening of bispecific compounds containing humanized huCAM anti-CLEC12A antibody fragments was performed using PBMCs. Peripheral blood mononuclear cells (PBMCs) were incubated with the HL60 promyelocytic leukemia cell line expressing CLEC12A in the presence of designated bispecific compounds (SEQ ID NO: 1-13) containing different clones. A trispecific compound (SEQ ID NO: 15) containing 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 with surface antigens, fixed, infiltrated, and stained with intracellular interferon-γ (IFNγ). Cells were run on a flow cytometer and evaluated for CD56. + CD3 - NK cell degranulation (CD107a) was used to measure NK cell activation.

[0014] Figure 4 Functional screening of bispecific compounds containing humanized huCAM anti-CLEC12A antibody fragments was performed using PBMCs. Peripheral blood mononuclear cells (PBMCs) were incubated with the HL60 promyelocytic leukemia cell line expressing CLEC12A in the presence of designated bispecific compounds (SEQ ID NO: 1-13) containing different clones. A trispecific compound (SEQ ID NO: 15) containing 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 with surface antigens, fixed, infiltrated, and stained with intracellular interferon-γ (IFNγ). Cells were run on a flow cytometer and evaluated for CD56. + CD3 - NK cell IFNγ production is used to measure NK cell activation.

[0015] Figure 5 Evaluation of a bispecific compound containing humanized 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 adaptor), a CLEC12A trispecific compound (SEQ ID NO: 15; scFv adaptor), or no compound (NT). NK cell degranulation (CD107a, left) and cytokine production (IFNγ, right) were measured by flow cytometry. Although scFv showed some background activation in degranulation, clone 33 did not, highlighting better specificity.

[0016] Figure 6 Evaluation of a bispecific compound containing humanized huCAM anti-CLEC12A clone 33. To determine activation against CLEC12-expressing targets mediated by a bispecific compound containing anti-CLEC12A clone 33, enriched NK cells were incubated with HL60 cells and a bispecific compound containing anti-CLEC12A clone 33 (clone 33 huCAM adaptor) (SEQ ID NO:34), a CLEC12A trispecific compound (SEQ ID NO:15; scFv adaptor), or no compound (NT) for 5 hours. NK cell degranulation (CD107a, left) and cytokine production (IFNγ, right) were measured by flow cytometry. The trispecific compound containing clone 33 induced NK cell degranulation against the HL60 target and induced the same amount of cytokine production as the positive control containing scFv.

[0017] Figure 7 Evaluation of a bispecific compound containing humanized huCAM anti-CLEC12A clone 33. To determine activation (non-specific activation) against CLEC12-negative targets mediated by a bispecific compound containing anti-CLEC12A clone 33, enriched NK cells were incubated for 5 hours with Raji (Burkitt lymphoma) cells and a bispecific compound containing anti-CLEC12A clone 33 (clone 33 huCAM adaptor) (SEQ ID NO:34), a CLEC12A trispecific compound (SEQ ID NO:15; scFv adaptor), or no compound (NT). NK cell degranulation (CD107a, left) and cytokine production (IFNγ, right) were measured by flow cytometry. Compared with the trispecific compound containing anti-CLEC12A scFv, the bispecific compound containing clone 33 induced less non-specific degranulation against CLEC12A-negative targets.

[0018] Figure 8 Evaluation of the binding specificity of clone 33. (A) Bispecific compounds containing clone 33 (SEQ ID NO: 34) and trispecific compounds containing anti-CLEC12A scFv (SEQ ID NO: 15) against CLEC12A. + (B) Binding of HL60 cells. Binding of CLEC12A-negative Raji cells to a bispecific compound containing clone 33 (SEQ ID NO:34) and a trispecific compound containing anti-CLEC12A scFv (SEQ ID NO:15). Detailed Implementation

[0019] C-type lectin domain family 12 member A (CLEC12A) is a human lectin domain composed of... CLEC12A A gene-encoded protein. CLEC12A is a member of the C-lectin / C-lectin-like domain (CTL / CTLD) superfamily. CLEC12A is an inhibitory C-lectin-like receptor. It contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail, which can bind to signal transduction phosphatases such as SHP-1 and SHP-2.

[0020] Human CLEC12A is a monomer primarily expressed on myeloid cells such as granulocytes, monocytes, macrophages, and dendritic cells. CLEC12A is a target for immunotherapy in the treatment of myeloid malignancies such as acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) because it is expressed on most medulloblasts and leukemia stem cells (LSCs) but not on normal hematopoietic stem cells or normal tissue cells.

[0021] Phage display single-domain antibody (sdAb) libraries for human CLEC12 were screened. Three rounds of library panning were performed using a pre-prepared human CLEC12A single-domain antibody library. (For example, in...) Figure 2 As shown in Table 1, SDS-PAGE results indicate that the recombinant human CLEC12A protein is of high quality. Biopanning was then performed to enrich the specific binders to the target human CLEC12A. As shown in Table 1, after three rounds of library screening, strong enrichment of human CLEC12A was observed, and a significant difference was found between the target screening group and the uncoated control group.

[0022] Table 1

[0023] wheel enter Output Enrichment factor 1 <![CDATA[2.00×10 11 ]]> <![CDATA[5.84×10 4 ]]> <![CDATA[3.42×10 6 ]]> 2 - P <![CDATA[2.44×10 11 ]]> <![CDATA[2.13×10 5 ]]> <![CDATA[1.15×10 6 ]]> 2 - N <![CDATA[3.05×10 10 ]]> <![CDATA[2.02×10 3 ]]> <![CDATA[1.51×10 7 ]]> 3 - P <![CDATA[2.54×10 11 ]]> <![CDATA[4.51×10 7 ]]> <![CDATA[5.63×10 3 ]]> 3 - N <![CDATA[3.18×10 10 ]]> <![CDATA[2.40×10 3 ]]> <![CDATA[1.33×10 7 ]]>

[0024] Ninety-six clones were selected from the third-round -P eluent and subjected to monoclonal phage ELISA. Eighty-two positive clones were identified and their DNA was sequenced. Seventy-seven clones were successfully sequenced, revealing 13 unique sequences (SEQ ID NO: 1-13). Alignment of these 13 unique sequences yielded a common sequence (SEQ ID NO: 14), and complementarity-determining regions (CDRs) were identified, such as in... Figure 1 As shown in the image.

[0025] 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 were positively bound to human CLEC12A.

[0026] Table 2 - Monoclonal Phage ELISA

[0027]

[0028]

[0029] Figure 3 and Figure 4 Functional screening of humanized huCAM anti-CLEC12A clones using PBMCs is shown. Humanized huCAM anti-CLEC12A clone variants (SEQ ID NO: 1-13) were cloned as targeting domains into a bispecific backbone containing huCAM anti-CD16 and a linker. These bispecific compounds activate natural killer (NK) cells by forming a cytokine bridge between NK cells (via CD16 binding) and tumor cells (via CLEC12A binding). A previously tested and confirmed trispecific killer cell adaptor (SEQ ID NO: 15) targeting CLEC12A via a single-chain variable fragment (scFv) was used as a positive control. Data showed that clone 33 exhibited the highest activity. Some clones were produced in smaller amounts and were therefore tested at lower concentrations than scFv (the trispecific positive control) or the bispecific compound clone 33. Therefore, it is impossible to draw conclusions from the apparent lack of activity of the clones compared to the negative control.

[0030] To determine the functional specificity of the humanized CLEC12A Cameloid clone 33 antibody fragment, a CD16-clone 33 bispecific compound (SEQ ID NO:34) was used. This bispecific compound has the ability to activate natural killer (NK) cells by forming a cellular lysis bridge between NK cells (via CD16 binding) and tumor cells (via CLEC12A binding). Figure 5 Background activation of NK cells is shown as measured by NK cell degranulation (CD107a, left) and IFNγ induction (right). The bispecific compound containing clone 33 showed minimal background NK cell activation. Figure 6 The activation of NK cells in the presence of CLEC12A-positive HL60 cells was demonstrated. A bispecific compound containing an anti-CLEC12A clone 33 antibody fragment induced degranulation (left) and IFNγ production (right), with IFNγ induction being almost identical to that of the positive control, i.e., a trispecific compound containing anti-CLEC12A-scFv (SEQ ID NO:15). Figure 7 NK activation was demonstrated in the presence of CLEC12A-negative Raji (Burkitt lymphoma) cells. Compared to trispecific compounds containing anti-CLEC12A-scFv, bispecific compounds containing clon-33 induced less nonspecific degranulation against CLEC12A-negative targets.

[0031] Figure 8 The binding specificity of anti-CLEC12A clone 33 was further demonstrated. Bispecific compounds containing the anti-CLEC12A clone 33 antibody fragment and trispecific compounds containing the anti-CLEC12A scFv were both constructed to include a 10× HIS tag. These constructs were evaluated for binding with CLEC12A-positive HL60 cells using an anti-HIS-phycoerythrin-tagged antibody. Figure 8 A) and CLEC12A-negative Raji cells ( Figure 8 B) Binding. As a control, basal binding (grey bars) of the anti-HIS-PE antibody was measured in the absence of prior binding with the linker compound. As the data show, both the clonal 33 bispecific compound and the scFv trispecific compound induced minimal binding against CLEC12A negative cells. Compared with the trispecific compound containing anti-CLEC12AscFv, the clonal 33 bispecific compound induced greater binding with CLEC12A positive cells.

[0032] Therefore, this disclosure describes peptides that target CLEC12A on myeloid malignancies. Because these peptides target CLEC12A, they induce less targeting in normal myeloid cells compared to peptides that target other antigens associated with myeloid malignancies (e.g., CD33). This disclosure explicitly describes 12 unique humanized single-domain antibody (sdAb) sequences targeting CLEC12A, which are common sequences derived from alignments of 12 unique anti-CLEC12A sdAb sequences, and provides guidance for other variants by identifying highly conserved and variable regions in a total of 13 sequences.

[0033] The anti-CLEC12A peptide described herein can be incorporated into biological constructs that can be used in the context of, for example, therapeutic, diagnostic, and / or detection methods. For example, the anti-CLEC12A peptide described herein can be incorporated into bispecific killer cell adaptor molecules (BiKE, e.g., SEQ ID NO:34), trispecific killer cell adaptor molecules (TriKE, e.g., SEQ ID NO:35), tetraspecific killer cell adaptor molecules (TetraKE), pentaspecific killer cell adaptor molecules (PentaKE), bispecific T cell adaptor molecules (BiTE), trispecific T cell adaptor molecules (TriTE), tetraspecific T cell adaptor molecules (TetraTE), pentaspecific T cell adaptor molecules (PentaTE), and chimeric antigen receptors (CARs, for use in, for example, CAR T cells, CAR... Applications include: expression in NK cells, CAR macrophages, etc.; complete antibody constructs (e.g., to induce antibody-dependent cytotoxicity (ADCC)); antibody-drug conjugate (ADC) molecules (e.g., for toxin delivery); labeled constructs (e.g., for commercial evaluation of antigen expression); radiolabeled forms (e.g., for positron emission tomography (PET) imaging or directed radiation delivery); applications delivering cytokines and / or chemokines; or other general immunotherapy regimens.

[0034] While other agents (e.g., CD33-binding antibodies and / or antibody fragments) target CD33-expressing myeloid malignancies, they also target normal myeloid cells expressing lower levels of the target. In contrast, the anti-CLEC12A peptides described herein result in a much lower likelihood of on-target, off-tumor toxicity by targeting an antigen more specifically expressed on myeloid malignancies (e.g., acute myeloid leukemia (AML) cells or myelodysplastic syndrome (MDS) cells). CLEC12A is also expressed in leukemia stem cells. Therefore, targeting CLEC12A can limit the likelihood and / or severity of relapse. Since sdAbs are more stable than scFvs, the anti-CLEC12A peptides described herein also offer advantages over scFv-forming constructs (e.g., BiKE, TriKE, BiTE, CAR, etc.). Finally, the anti-CLEC12A sequences described herein are derived from humanized libraries and are therefore unlikely to be rejected in human patients.

[0035] This disclosure describes anti-CLEC12A peptides. Exemplary anti-CLEC12A peptides include peptides comprising the amino acid sequence of any one of SEQ ID NO:1-14 or SEQ ID NO:29-31, or structurally similar to, or functional variants thereof. SEQ ID NO:1-13 are single-domain antibody sequences selected by phage display because they specifically bind to CLEC12A. SEQ ID NO:14 is derived from an alignment analysis of SEQ ID NO:1-13 (…). Figure 1 The shared sequence of SEQ ID NO: 29 is the shared sequence of CDR1, as seen from... Figure 1 The alignment analysis shown indicates that SEQ ID NO: 30 is a common sequence of CDR2, as determined by the comparison analysis. Figure 1 The alignment analysis shown indicates that SEQ ID NO: 31 is a common sequence of CDR3, as determined by... Figure 1 The comparison analysis shown indicates that...

[0036] As used herein, an anti-CLEC12A peptide is considered "structurally similar" to a reference peptide or a "functional variant" of the reference peptide if the amino acid sequence of the anti-CLEC12A peptide has a specified amount of identity with the reference peptide. If a "functional fragment" amino acid sequence contains fewer amino acids than the full-length amino acid sequence of the reference amino acid sequence, then the amino acid sequence is a "functional fragment" of the reference amino acid sequence. A "functional fragment" may further have a specified amount of sequence identity or sequence specificity compared to the reference amino acid sequence.

[0037] Structural similarity and / or sequence identity between two peptides can be determined by comparing the amino acid residues of two peptides (e.g., a candidate anti-CLEC12A peptide and, for example, a peptide from any one of SEQ ID NO: 1-14 or SEQ ID NO: 29-31) to optimize the number of identical amino acids along their sequence length. To optimize the number of identical amino acids, vacancies are allowed in either or both sequences during the alignment, although the amino acids in each sequence must maintain their proper order. The candidate anti-CLEC12A peptide is the peptide to be compared with a reference peptide (e.g., any one of SEQ ID NO: 1-14 or SEQ ID NO: 29-31). The candidate peptide can be isolated, for example, from animals, or generated using recombinant techniques, or synthesized chemically or enzymatically.

[0038] Pairwise contrast analysis of amino acid sequences can be performed using the BESTFIT algorithm in the GCG package (version 10.2, Madison WI). Alternatively, peptides can be contrasted using the Blastp procedure with the BLAST 2 search algorithm, as described by Tatiana et al. FEMS Microbiol Lett As described in , 174, 247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website. All BLAST 2 search parameters can be used with default values, including matrix = BLOSUM62; open void penalty = 11, extended void penalty = 1, void x_dropoff = 50, expectation = 10, word length = 3, and filter on.

[0039] When comparing two amino acid sequences, structural similarity can be expressed as a percentage of "identity" or a percentage of "similarity." "Identity" indicates the presence of the same amino acid. "Similarity" indicates not only the presence of the same amino acid but also the presence of a conserved substitution. Conserved substitutions of amino acids in the anti-CLEC12A peptide can be selected from other members of the same amino acid group. For example, it is well known in protein biochemistry that amino acids belonging to a specific size or property (such as charge, hydrophobicity, and hydrophilicity) can substitute for another amino acid without altering the protein's activity, especially in protein regions 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 substitution for Arg and vice versa, to maintain a positive charge; Glu substitution for Asp and vice versa, to maintain a negative charge; Ser substitution for Thr, thereby maintaining free -OH; and Gln substitution for Asn to maintain free -NH2. Similarly, bioactive analogs of peptides containing the deletion or addition of one or more continuous or discontinuous amino acids that do not eliminate the functional activity of the peptide are also considered.

[0040] In some embodiments, the anti-CLEC12A peptide as described herein may include a peptide 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.

[0041] In some embodiments, the anti-CLEC12A peptide as described herein may include 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 with a reference amino acid sequence.

[0042] In some embodiments, the anti-CLEC12A peptide as described herein may also be designed to provide additional sequences, such as amino acids added to the C-terminus or N-terminus of the anti-CLEC12A peptide. Such additional amino acids may include, for example, a signal sequence (e.g., SEQ ID NO:32) or a tag that facilitates purification by trapping the labeled anti-CLEC12A peptide on a column or by using an antibody. Exemplary tags include, for example, histidine-rich tags that allow purification of the peptide on a nickel column (e.g., SEQ ID NO:33).

[0043] This disclosure also describes polynucleotides encoding anti-CLEC12A peptides. Exemplary polynucleotides encoding anti-CLEC12A peptides include polynucleotides comprising the nucleotide sequence of any one of SEQ ID NO:16-28. However, SEQ ID NO:16-28 are merely exemplary. Because the genetic code is well known, this disclosure describes any polynucleotide encoding the anti-CLEC12A peptide as described herein.

[0044] The anti-CLEC12A peptides described herein can be incorporated into biologics and then formulated with pharmaceutically acceptable carriers. As used herein, "anti-CLEC12A biologic" refers to a biological compound comprising an anti-CLEC12A peptide. Anti-CLEC12A biologics may include additional functional motifs depending on the basic structural platform of the biologic (e.g., BiKE, TriKE, CAR, etc.). As used herein, "carrier" includes any solvent, dispersion medium, medium, coating agent, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delay agent, buffer, carrier solution, suspension, colloid, etc. The use of such media and / or reagents for pharmaceutically active substances is well known in the art. Their use in therapeutic compositions is anticipated, except where any conventional media or reagent is incompatible with the active ingredient. Complementary active ingredients may also be incorporated into the composition. As used herein, “pharmaceutically acceptable” means that the material is not biologically or otherwise undesirable, meaning that the material can be administered to an individual in conjunction with the anti-CLEC12A biological product without causing any undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition containing the material.

[0045] Therefore, anti-CLEC12A biological products can be formulated into pharmaceutical compositions. These pharmaceutical compositions can be formulated into various forms suitable for preferred routes 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, transdermal, rectal, etc.). The pharmaceutical compositions can be applied to mucosal surfaces, such as by application to, for example, the nasal or respiratory mucosa (e.g., via a spray or aerosol). The compositions can also be administered via sustained or delayed release.

[0046] Therefore, anti-CLEC12A biological products can be provided in any suitable form, including but not limited to solutions, suspensions, emulsions, sprays, aerosols, or mixtures. The composition can be delivered in a formulation together with any pharmaceutically acceptable excipient, carrier, or medium. For example, the formulation can be delivered in conventional topical dosage forms, such as creams, ointments, aerosol formulations, non-aerosol sprays, gels, lotions, etc. The formulation may further include one or more additives, including, for example, adjuvants, skin penetration enhancers, colorants, fragrances, flavorings, humectants, thickeners, etc.

[0047] The formulation can be conveniently presented in unit dosage form and can be prepared using methods well-known in the pharmaceutical field. Methods for preparing compositions using pharmaceutically acceptable carriers include the step of binding the anti-CLEC12A biological product to a carrier constituting one or more adjuvants. Generally, formulations can be prepared by uniformly and / or tightly binding the active compound to a liquid carrier, a finely pulverized solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

[0048] Therefore, in another aspect, this disclosure describes a method for treating any condition in which targeting cells overexpressing CLEC12A has a therapeutic benefit. In many embodiments, the condition may be a myeloid malignancy. However, in other embodiments, the condition may be an autoimmune condition—for example, a condition in which myeloid cells infiltrate the central nervous system. Typically, the method includes administering to the subject an amount of an anti-CLEC12A biologic effective for treating the condition. “Treatment” or variations thereof means any reduction in symptoms or signs associated with the condition, limitation of the progression of symptoms or signs associated with the condition, improvement or remission of symptoms or signs associated with the condition. As used herein, “improvement” means any reduction in the degree, severity, frequency and / or likelihood of symptoms or clinical signs characteristic of a particular condition; “symptom” means any subjective evidence of a disease or patient condition; and “sign” or “clinical sign” means an objective physical finding related to a particular condition that could be detected by someone other than the patient.

[0049] "Treatment" can be therapeutic or preventative. "Therapeutic" and its variations refer to treatment that improves one or more existing symptoms or clinical signs associated with the condition. "Preventative" and its variations refer to treatment that limits the development and / or occurrence of symptoms or clinical signs of the condition to any extent. Typically, "therapeutic" treatment is initiated after the subject develops the condition, while "preventative" treatment is initiated before the subject develops the condition. Therefore, in some embodiments, the method may involve preventative treatment of subjects at risk of developing the condition. "At risk" means a subject who may actually have or may not actually have said risk. Thus, for example, a subject at "risk" of developing a particular condition is a subject with one or more markers indicating an increased risk of having or developing the particular condition, regardless of whether the subject exhibits any symptoms or clinical signs of having or developing the condition. Exemplary markers of the condition may include, for example, genetic predisposition, ancestry, age, sex, geographic location, lifestyle, or medical history. Treatment may also continue after symptoms have subsided, for example, to prevent or delay their recurrence.

[0050] In some implementations, "preventative" treatment also includes treatment in cases of relapse. In other words, a subject may have previously exhibited symptoms or clinical signs of the disease but received successful treatment, thus being considered to be in remission. Such a subject may no longer exhibit symptoms or clinical signs of the disease during remission and is instead at "risk" of relapse. In this case, treatment including anti-CLEC12A biologics may be considered "preventative" to reduce the likelihood and / or severity of relapse.

[0051] Therefore, the anti-CLEC12A biologic can be administered to subjects before, during, or after the first appearance of symptoms or clinical signs of the condition. Treatment initiated before the first appearance of symptoms or clinical signs associated with the condition may result in a reduced likelihood of clinical evidence of the condition, a reduced severity of symptoms and / or clinical signs, and / or complete remission of the condition compared to subjects who have not received the anti-CLEC12A biologic. Treatment initiated before the first appearance of symptoms or clinical signs associated with a recurrence of the condition may result in a reduced likelihood of clinical evidence of a recurrence, a reduced severity of symptoms and / or clinical signs, and / or complete remission of the condition compared to subjects who have not received the anti-CLEC12A biologic. Treatment initiated concurrently with the appearance of symptoms or clinical signs associated with the condition may result in a reduced severity of symptoms and / or clinical signs, and / or complete remission of the condition compared to subjects who have not received the anti-CLEC12A biologic.

[0052] The amount of anti-CLEC12A biologic administered can vary depending on a variety of factors, including, but not limited to, the specific anti-CLEC12A biologic administered, the subject's weight, physical condition and / or age, and / or route of administration. Therefore, the absolute weight of the anti-CLEC12A biologic contained in a given unit dosage form can vary widely and depends on factors such as the subject's species, age, weight and physical condition, and / or method of administration. Therefore, it is impractical to generally list the amounts of anti-CLEC12A biologics that constitute an effective amount for all possible applications. However, those skilled in the art can readily determine appropriate amounts by properly taking such factors into account.

[0053] In some embodiments, the method may include administering sufficient anti-CLEC12A biologic to provide the subject with a dose, for example, from about 100 ng / kg / day to about 50 mg / kg / day, although in some embodiments, the method may be performed by administering anti-CLEC12A biologic at a dose outside this range.

[0054] In some embodiments, the method may include administering sufficient anti-CLEC12A biologic to provide a minimum dose of at least 100 ng / kg / day, such as 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.

[0055] In some embodiments, the method includes administering sufficient anti-CLEC12A biologic to provide a maximum dose not exceeding 10 mg / kg / day, such as not exceeding 5 mg / kg / day, not exceeding 4 mg / kg / day, not exceeding 3 mg / kg / day, not exceeding 2 mg / kg / day, not exceeding 1 mg / kg / day, not exceeding 900 μg / kg / day, not exceeding 800 μg / kg / day, not exceeding 700 μg / kg / day, not exceeding 600 μg / kg / day, not exceeding 500 μg / kg / day, not exceeding 400 μg / kg / day, not exceeding 300 μg / kg / day, not exceeding 200 μg / kg / day, not exceeding 100 μg / kg / day, not exceeding 90 μg / kg / day, not exceeding 80 μg / kg / day, not exceeding 70 μg / kg / day, not exceeding 60 μg / kg / day, not exceeding 50 μg / kg / day, not exceeding 40 μg / kg / day, not exceeding 30 μg / kg / day, not exceeding 20 mg ... μg / kg / day or not exceeding 10 μg / kg / day. When the anti-CLEC12A biologic is not absent but present in an amount that includes the specified amount, the anti-CLEC12A biologic is provided at a dose “not greater than” the specified amount.

[0056] In some embodiments, the method includes administering sufficient anti-CLEC12A biologic to provide a dose characterized by a range having endpoints defined by any minimum dose determined above and any maximum dose greater than the selected minimum dose. For example, in some embodiments, the method may include administering sufficient anti-CLEC12A biologic to provide a subject with a dose from about 10 µg / kg / day to about 10 mg / kg / day, a dose from about 100 µg / kg / day to about 1 mg / kg / day, a dose from 5 µg / kg / day to 100 µg / kg / day, etc.

[0057] In some embodiments, the method includes administering sufficient anti-CLEC12A biologic to provide a dose equal to any of the minimum or maximum doses listed above. Thus, for example, in some embodiments, the method may include administering sufficient anti-CLEC12A biologic to provide doses such as 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.

[0058] In some embodiments, the anti-CLEC12A biologic can be administered, for example, from a single dose to multiple doses weekly, although in some embodiments, the method can be performed by administering the anti-CLEC12A biologic at frequencies exceeding this range. In some embodiments, the anti-CLEC12A biologic can be administered from approximately once a month to approximately five times a week. In some embodiments, the above-indicated doses are administered in a 7-day cycle of 4 days of treatment and 3 days of rest, described as the amount of anti-CLEC12A biologic administered over a 24-hour period.

[0059] In some embodiments, the anti-CLEC12A biologic can be administered, for example, from a single dose to multiple treatment cycles, although in some embodiments, the method can be performed by administering the anti-CLEC12A biologic for a duration exceeding this range. In some embodiments, the anti-CLEC12A biologic can be administered for three weeks. In such embodiments, one week can be a treatment cycle, such as the exemplary treatment cycle described in the preceding paragraph. In other embodiments, the anti-CLEC12A biologic can be administered for a larger number of treatment cycles without intervals between a set of treatment cycles and subsequent sets of treatment cycles. The interval between a set of treatment cycles and subsequent sets of treatment cycles can be a week or more, a month or more, or a year or more.

[0060] In some embodiments, the method further includes administering one or more additional therapeutic agents. 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 administered co-administered. As used herein, “co-administered” means the administration of two or more components in a combination such that the combined therapeutic or preventative effect is greater than the therapeutic or preventative effect of either component administered alone. The two components may be administered simultaneously or sequentially. Components administered simultaneously may be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes situations where the application of components allows each component to be present simultaneously at the treatment site. Alternatively, sequential co-administration of two components may include situations where at least one component has been cleared from the treatment site, but at least one cellular effect of the administered 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. Therefore, in some cases, the co-administered combination may include components that would never coexist in a chemical mixture. In other embodiments, the anti-CLEC12A biologic and additional therapeutic agents may be administered as part of a mixture or blend. In some respects, administration of the anti-CLEC12A biologic may allow for the effectiveness of lower doses of other therapeutic modalities compared to administration of one or more other therapeutic agents alone, thereby reducing the likelihood, severity, and / or extent of toxicity observed when higher doses of other one or more therapeutic agents are administered.

[0061] Other exemplary therapeutic agents include hexamethicone, acridine, L-asparaginase, asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, and fluorouracil. Fludarabine, formustin, ganciclovir, gemcitabine, hydroxyurea, idarubicin, isophosphoramide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, thioguanine, thiotepa, topotecan, vincristine, vinblastine, vindesine, and vinorelbine.

[0062] In the foregoing description and the appended claims, the term “and / or” means one or all of the listed elements or any combination of two or more of the listed elements; the terms “comprising,” “including,” and variations thereof shall be interpreted as open-ended—that is, additional elements or steps are optional and may or may not be present; unless otherwise indicated, “a,” “an,” “the,” and “at least one” are used interchangeably and refer to one or more than one; and the enumeration of numerical ranges by endpoints includes all numbers included in that range (e.g., 1-5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0063] In the foregoing description, for clarity, specific embodiments may be described individually. Unless otherwise expressly stated, features of a particular embodiment are incompatible with features of another embodiment, some embodiments may include combinations of compatible features described herein in conjunction with one or more embodiments.

[0064] For any method disclosed herein that includes discrete steps, the steps can be performed in any feasible order. Furthermore, depending on the circumstances, any combination of two or more steps can be performed simultaneously. Example

[0065] Construction of the CD16-huCAMCLEC12A bispecific compound (SEQ ID NO:34)

[0066] As previously described, the construct encoding the CD16-huCAMCLEC12A bispecific compound was synthesized using PCR and HiFi cloning techniques (Vallera et al., 2016). Clin Cancer Res 22:3440-3450). Each fully assembled fragment includes the EcoRI restriction site, the ATG start codon, and the coding sequence of the humanized camelid antibody-CD16 sdAb (Vincke et al., 2007). Protein Eng Des Sel The sequences encoding huCAMCLEC12A sdAb (SEQ ID NO: 16-28) and the sequence encoding a 10X His tag were obtained. 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 validated to confirm the sequence and location of the gene insertion (Biomedical Genomics Center, University of Minnesota, Minneapolis, MN).

[0067] Construction of CD16-huCAMCLEC12A trispecific compound (SEQ ID NO:35)

[0068] As previously described, constructs encoding the CD16-huCAMCLEC12A trispecific compound were synthesized using PCR and HiFi cloning techniques (Vallera et al., 2016). Clin Cancer Res 22:3440-3450). Each fully assembled fragment includes the EcoRI restriction site, the ATG start codon, and the coding sequence of the humanized camelid antibody-CD16 sdAb (Vincke et al., 2007). Protein Eng Des Sel The assembled fragments included the coding sequences for 21:1-10), the adapter, the human IL-15 fragment (amino acids 162-175 of SEQ ID NO:35), the whitlow amino acid adapter (GSTSGSGKPGSGEGSTKG; SEQ ID NO:36), the huCAMCLEC12A sdAb (one of SEQ ID NO:16-28), and the sequence encoding the 10X His 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 validated to confirm the sequence and location of the inserted gene (BiomedicalGenomics Center, University of Minnesota, Minneapolis, MN).

[0069] Production and isolation of CD16-huCAMCLEC12A bispecific and trispecific compounds

[0070] Following the manufacturer's protocol, all cloned plasmids were transfected into Expi293 cells (Thermo Fisher Scientific, Inc., Waltham, MA). The supernatant was collected and the protein was purified using HisPur cobalt resin (Thermo Fisher Scientific, Inc., Waltham, MA) and PIERCE centrifuge columns (Thermo Fisher Scientific, Inc., Waltham, MA). The protein was eluted with 250 mM imidazole solution and desalted using pre-packaged single-use PD-10 columns (GE Healthcare Systems, Chicago, IL). Purity and size were determined by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis on a Simply Blue Life Stain (Invitrogen, Carlsbad, CA).

[0071] Cancer cell lines (HL60 and Raji)

[0072] HL60 promyelocytes (ATCC CCL-240, American Center for Type Culture Collection, Manassas, VA) were obtained from ATCC and used as the CLEC12A expression line. Raji Burkitt lymphoma lymphoblasts (ATCC CCL-86, American Center for Type Culture Collection, Manassas, VA) were also obtained from ATCC and used as the CLEC12A-negative line.

[0073] Functional evaluation of different clones in CLEC12A-positive HL60 cells using PBMCs

[0074] Healthy donor blood was obtained from the Memorial Blood Bank (Minneapolis, MN) and processed using a density gradient Ficoll-Paque (GE Healthcare Systems, Chicago, IL) to obtain peripheral blood mononuclear cells (PBMCs). As previously described, NK cell function was assessed by flow cytometry (Vallera et al., 2016, Clin Cancer Res22:3440-3450). PBMCs, HL60 cells, and treatment agents (30 nM) were co-cultured and stained with FITC-conjugated anti-CD107a (H4A3, BioLegend, San Diego, CA). One hour after the addition of anti-CD107a, cells were administered Golgi Stop and Golgi Plug (BD Biosciences, San Jose, CA) and incubated for three hours. At the end of incubation, cells were stained with 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 infiltrated. Infiltrating cells were stained with BV650-conjugated IFNγ (4S.B3, BioLegend, San Diego, CA) and expression was evaluated by flow cytometry.

[0075] NK cells were used to evaluate the functional specificity of clone 33 in blank, CLEC12A-positive HL60 cells, or CLEC12A-negative Raji cells.

[0076] Healthy donor blood was obtained from the Memorial Blood Bank (Minneapolis, MN) and processed using a density gradient Ficoll-Paque (GE Healthcare Systems, Chicago, IL) to obtain peripheral blood mononuclear cells (PBMCs). PBMCs were used for magnetic enrichment of 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, 2017). Clin Cancer Res22:3440-3450). Enriched NK cells were incubated individually, co-incubated with HL60 cells, or co-incubated with Raji cells and the specified treatment agent (30 nM). Cells and treatment agent were co-cultured and stained with FITC-conjugated anti-CD107a (H4A3, BioLegend, San Diego, CA). One hour after adding anti-CD107a, cells were administered Golgi Stop and Golgi Plug (BDBiosciences, San Jose, CA) and incubated for three hours. At the end of incubation, cells were stained with 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 infiltrated. Infiltrating cells were stained with BV650-conjugated IFNγ (BioLegend, San Diego, CA) and expression was evaluated by flow cytometry.

[0077] Combined with specific determination

[0078] To determine binding specificity, 30 nM of the clone 33 bispecific compound or a trispecific compound containing anti-CLEC12A scFv was incubated with CLEC12A-positive HL60 cells or CLEC12A-negative Raji cells at 37°C for 30 min. Cells were centrifuged and washed twice. Anti-HIS, phycoerythrin (PE)-labeled antibody (OriGene Technologies, Inc., Rockville, MD) was added and incubated with the cells at 4°C for 20 min. Cells were washed twice, fixed with 2% paraformaldehyde, and flow cytometry was run to evaluate the percentage of cells binding to the specified construct.

[0079] All disclosures of patents, patent applications, and publications cited herein, as well as electronically available material (including, for example, nucleotide sequences submitted in GenBank and RefSeq, and amino acid sequences submitted in SwissProt, PIR, PRF, and PDB, and translations from annotation coding regions in GenBank and RefSeq), are incorporated herein by reference in their entirety. In the event of any inconsistency between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application shall prevail. The foregoing detailed description and examples are given for clarity only. Unnecessary limitations should not be construed from them. The invention is not limited to the exact details shown and described, as variations obvious to those skilled in the art will be included within the scope of the invention as defined by the claims.

[0080] Unless otherwise stated, all figures representing component amounts, molecular weights, etc., used in the specification and claims should in all cases be understood to be modified by the term "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters listed in the specification and claims are approximate values ​​that may vary according to the desired performance sought to be obtained according to the invention. At least, and not in an attempt to limit the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant digits recorded and by applying ordinary rounding techniques.

[0081] Although the numerical ranges and parameters described in this invention are approximate, the values ​​given in the specific embodiments are reported as precisely as possible. However, all values ​​inherently include a range, which necessarily derives from the standard deviation found in their respective experimental measurements.

[0082] All headings are for the reader's convenience and should not be used to limit the meaning of the text following them unless otherwise stated.

[0083] Sequence List Free Text

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Claims

1. An anti-CLEC12A single-domain antibody sdAb, comprising the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 as shown in SEQ ID NO:

9. The amino acid sequence of CDR1 is YDDMG, the amino acid sequence of CDR2 is GIQNTDGSTYYADSVKGRFT, and the amino acid sequence of CDR3 is LYDRMVGKEEQLAS.

2. The anti-CLEC12A sdAb according to claim 1, wherein the sdAb comprises the amino acid sequence of SEQ ID NO:

9.

3. A biological therapeutic compound comprising the anti-CLEC12AsdAb according to claim 1 or 2, The aforementioned biotherapeutic compound is a bispecific killer cell adaptor molecule (BiKE) or a trispecific killer cell adaptor molecule (TriKE).

4. The biotherapeutic compound according to claim 3, wherein the biotherapeutic compound comprises the amino acid sequence of SEQ ID NO:34 or SEQ ID NO:

35.

5. A pharmaceutical composition comprising: The biotherapeutic compound according to claim 3 or 4; and Pharmaceutically acceptable carrier.

6. Use of the biotherapeutic compound according to claim 3 or 4 in the preparation of a medicament for treating a subject with myeloid malignancies.

7. The use according to claim 6, wherein the malignant tumor is acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).

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