Compositions that target CLL-1 and CD3 and methods of making and using the same

Abstract

Disclosed are bispecific single-chain constructs that bind to both C-type lectin-like molecule-1 (CLL-1, also known as CLEC12A) and CD3, pharmaceutical compositions comprising the constructs, methods of treatment using the pharmaceutical compositions, polynucleotides encoding the constructs, and methods of making the constructs. The disclosed methods include contacting cells, e.g., autologous T cells, with the disclosed constructs ex vivo and administering the contacted cells to a subject.

Description

Atty. Dkt. No. 650053.01224COMPOSITIONS THAT TARGET CLL-1 AND CD3 AND METHODS OF MAKING AND USING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit from U.S. Application Ser. No. 63 / 687,431, filed August 27. 2024. the entirety of which is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.SEQUENCE LISTING

[0003] A sequence listing (file name: 650053_01224.xml; size: (37,177 bytes; date generated: August 15, 2025) is hereby incorporated by reference in its entirety.BACKGROUND

[0004] The inventors performed an unbiased mass spectrometry-based analysis of surface glycoproteins (-70% of surface proteins) expressed on four acute myeloid leukemia (AML) cell lines from adults and children. The analysis revealed a variety of candidate tumor antigens including C-type lectin-like molecule- 1 (CLL-1). also known as C-type lectin domain family 12 member A (CLEC12A). CLL-1 is a member of a superfamily of proteins involved in phagocytosis, complement activation, cell adhesion, and pathogen recognition. CLL-1 is expressed on mature myeloid cells but not on normal hematopoietic stem / progenitor cells. Novel strategies to target CLL-1 expressed on tumor cells, e.g., AML cells, are needed in the art.SUMMARY

[0005] In an aspect of the current disclosure, bispecific single-chain polypeptide constructs are provided. In some embodiments, the bispecific single-chain polypeptide constructs comprise an anti-CLL-1 domain and an anti-CD3 domain. In some embodiments, the anti-CLL-1 domain comprises a first heavy chain and a first light chain and the anti-CD3 domain comprises a second heavy chain and a second light chain. In some embodiments, the first heavy chain comprises CDR1, CDR2, and CDR3 and the first light chain comprises CDR4, CDR5, and CDR6. wherein CDR1 comprises SEQ ID NO: 28, CDR2 comprises SEQ ID NO: 29, CDR3 comprises SEQ ID NO: 30, CDR4 comprises SEQ ID NO: 31, CDR5 comprises SEQ ID NO: 32, and CDR6 comprises SEQ ID NO: 33. In some embodiments, the anti-CLL-1 domain is SEQ ID NO: 1. In some embodiments, the anti-CD3 domain is SEQ ID NO: 2. In some embodiments, the first heavy chain comprises SEQ ID NO: 3. In some embodiments, the first light chain comprises SEQ IDAtty. Dkt. No. 650053.01224NO: 4. In some embodiments, the second heavy chain comprises SEQ ID NO: 5. In some embodiments, the second light chain comprises SEQ ID NO: 6. In some embodiments, the constructs further comprise a linker. In some embodiments, the linker is a flexible hydrophobic linker. In some embodiments, the linker does not comprise a hinge region. In some embodiments, the linker comprises SEQ ID NO: 7. In some embodiments, the construct further comprises an antibody Fc domain. In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the Fc domain is IgG2 Fc domain. In some embodiments, the Fc domain comprises SEQ ID NO: 8. In some embodiments, the construct comprises, from N to C terminus, an anti- CLL-1 domain, a linker, an antibody Fc domain, a linker, an anti-CD3 domain. In some embodiments, the construct comprises, from N to C terminus, an anti-CD3 domain, a linker, an antibody Fc domain, a linker, an anti-CLL-1 domain. In some embodiments, the construct comprises SEQ ID NO: 9.

[0006] In an aspect of the current disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise a construct comprising an anti-CLL-1 domain and an anti-CD3 domain and a pharmaceutically acceptable carrier or excipient. In some embodiments, the anti-CLL-1 domain comprises a first heavy chain and a first light chain and the anti-CD3 domain comprises a second heavy chain and a second light chain. In some embodiments, the anti-CLL-1 domain is SEQ ID NO: 1. In some embodiments, the anti-CD3 domain is SEQ ID NO: 2. In some embodiments, the first heavy chain comprises SEQ ID NO: 3. In some embodiments, the first light chain comprises SEQ ID NO: 4. In some embodiments, the second heavy chain comprises SEQ ID NO: 5. In some embodiments, the second light chain comprises SEQ ID NO: 6. In some embodiments, the constructs further comprise a linker. In some embodiments, the linker is a flexible hydrophobic linker. In some embodiments, the linker does not comprise a hinge region. In some embodiments, the linker comprises SEQ ID NO: 7. In some embodiments, the construct further comprises an antibody Fc domain. In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the Fc domain is IgG2 Fc domain. In some embodiments, the Fc domain comprises SEQ ID NO: 8. In some embodiments, the construct comprises, fromN to C terminus, an anti-CLL-1 domain, a linker, an antibody Fc domain, a linker, an anti-CD3 domain. In some embodiments, the construct comprises, from N to C terminus, an anti-CD3 domain, a linker, an antibody Fc domain, a linker, an anti-CLL-1 domain. In some embodiments, the construct comprises SEQ ID NO: 9.

[0007] In an aspect of the current disclosure, methods are provided. In some embodiments, the methods are methods of treating a disease or disorder associated with CLL-1 expression and comprise administering an effective amount of a pharmaceutical composition comprising aAtty. Dkt. No. 650053.01224 construct comprising an anti-CLL-1 domain and an anti-CD3 domain to a subject in need thereof to treat the disease or disorder. In some embodiments, the anti-CLL-1 domain comprises a first heavy chain and a first light chain and the anti-CD3 domain comprises a second heavy chain and a second light chain. In some embodiments, the anti-CLL-1 domain is SEQ ID NO: 1. In some embodiments, the anti-CD3 domain is SEQ ID NO: 2. In some embodiments, the first heavy chain comprises SEQ ID NO: 3. In some embodiments, the first light chain comprises SEQ ID NO: 4. In some embodiments, the second heavy chain comprises SEQ ID NO: 5. In some embodiments, the second light chain comprises SEQ ID NO: 6. In some embodiments, the constructs further comprise a linker. In some embodiments, the linker is a flexible hydrophobic linker. In some embodiments, the linker does not comprise a hinge region. In some embodiments, the linker comprises SEQ ID NO: 7. In some embodiments, the construct further comprises an antibody Fc domain. In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the Fc domain is IgG2 Fc domain. In some embodiments, the Fc domain comprises SEQ ID NO: 8. In some embodiments, the construct comprises, from N to C terminus, an anti-CLL-1 domain, a linker, an antibody Fc domain, a linker, an anti-CD3 domain. In some embodiments, the construct comprises, from N to C terminus, an anti-CD3 domain, a linker, an antibody Fc domain, a linker, an anti-CLL-1 domain. In some embodiments, the construct comprises SEQ ID NO: 9. In some embodiments, the disease or disorder is a cell-proliferative disease or disorder. In some embodiments, the cell proliferative disease or disorder is cancer. In some embodiments, the disease or disorder is acute myeloid leukemia (AML). In some embodiments, administering comprises contacting cells ex vivo with the pharmaceutical composition. In some embodiments, the method further comprises administering the cells to the subject after contacting. In some embodiments, the cells are T cells. In some embodiments, the T cells comprise CD4+T cells. In some embodiments, the T cells comprise CD8+T cells. In some embodiments, the cells are derived from the subject.

[0008] In an aspect of the current disclosure, polynucleotides are provided. In some embodiments, the polynucleotides encode a construct comprising an anti-CLL-1 domain and an anti-CD3 domain. In some embodiments, the anti-CLL-1 domain comprises a first heavy chain and a first light chain and the anti-CD3 domain comprises a second heavy chain and a second light chain. In some embodiments, the anti-CLL-1 domain is SEQ ID NO: 1. In some embodiments, the anti-CD3 domain is SEQ ID NO: 2. In some embodiments, the first heavy chain comprises SEQ ID NO: 3. In some embodiments, the first light chain comprises SEQ ID NO: 4. In some embodiments, the second heavy chain comprises SEQ ID NO: 5. In some embodiments, the second light chain comprises SEQ ID NO: 6. In some embodiments, the constructs further comprise a linker. In some embodiments, the linker is a flexible hydrophobic linker. In some embodiments, the linker doesAtty. Dkt. No. 650053.01224 not comprise a hinge region. In some embodiments, the linker comprises SEQ ID NO: 7. In some embodiments, the construct further comprises an antibody Fc domain. In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the Fc domain is IgG2 Fc domain. In some embodiments, the Fc domain comprises SEQ ID NO: 8. In some embodiments, the construct comprises, fromN to C terminus, an anti-CLL-1 domain, a linker, an antibody Fc domain, a linker, an anti-CD3 domain. In some embodiments, the construct comprises, from N to C terminus, an anti-CD3 domain, a linker, an antibody Fc domain, a linker, an anti-CLL-1 domain. In some embodiments, the construct comprises SEQ ID NO: 9.

[0009] In an aspect of the current disclosure, cells are provided. In some embodiments, the cells comprise a polynucleotide encoding a construct comprising an anti-CLL-1 domain and an anti- CD3 domain. In some embodiments, the anti-CLL-1 domain comprises a first heavy chain and a first light chain and the anti-CD3 domain comprises a second heavy chain and a second light chain. In some embodiments, the anti-CLL-1 domain is SEQ ID NO: 1. In some embodiments, the anti- CD3 domain is SEQ ID NO: 2. In some embodiments, the first heavy chain comprises SEQ ID NO: 3. In some embodiments, the first light chain comprises SEQ ID NO: 4. In some embodiments, the second heavy chain comprises SEQ ID NO: 5. In some embodiments, the second light chain comprises SEQ ID NO: 6. In some embodiments, the constructs further comprise a linker. In some embodiments, the linker is a flexible hydrophobic linker. In some embodiments, the linker does not comprise a hinge region. In some embodiments, the linker comprises SEQ ID NO: 7. In some embodiments, the construct further comprises an antibody Fc domain. In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the Fc domain is IgG2 Fc domain. In some embodiments, the Fc domain comprises SEQ ID NO: 8. In some embodiments, the construct comprises, fromN to C terminus, an anti-CLL-1 domain, a linker, an antibody Fc domain, a linker, an anti-CD3 domain. In some embodiments, the construct comprises, from N to C terminus, an anti-CD3 domain, a linker, an antibody Fc domain, a linker, an anti-CLL-1 domain. In some embodiments, the construct comprises SEQ ID NO: 9.

[0010] In some embodiments, the cells comprise a construct comprising an anti-CLL-1 domain and an anti-CD3 domain. In some embodiments, the anti-CLL-1 domain comprises a first heavy chain and a first light chain and the anti-CD3 domain comprises a second heavy chain and a second light chain. In some embodiments, the anti-CLL-1 domain is SEQ ID NO: 1. In some embodiments, the anti-CD3 domain is SEQ ID NO: 2. In some embodiments, the first heavy chain comprises SEQ ID NO: 3. In some embodiments, the first light chain comprises SEQ ID NO: 4. In some embodiments, the second heavy chain comprises SEQ ID NO: 5. In some embodiments, the second light chain comprises SEQ ID NO: 6. In some embodiments, the constructs furtherAtty. Dkt. No. 650053.01224 comprise a linker. In some embodiments, the linker is a flexible hydrophobic linker. In some embodiments, the linker does not comprise a hinge region. In some embodiments, the linker comprises SEQ ID NO: 7. In some embodiments, the construct further comprises an antibody Fc domain. In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the Fc domain is IgG2 Fc domain. In some embodiments, the Fc domain comprises SEQ ID NO: 8. In some embodiments, the construct comprises, from N to C terminus, an anti-CLL-1 domain, a linker, an antibody Fc domain, a linker, an anti-CD3 domain. In some embodiments, the construct comprises, from N to C terminus, an anti-CD3 domain, a linker, an antibody Fc domain, a linker, an anti-CLL-1 domain. In some embodiments, the construct comprises SEQ ID NO: 9. In some embodiments, the cell is selected from a bacterial cell, an archaeal cell, a fungal cell, or an animal cell. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a T cell.

[0011] In an aspect of the current disclosure, methods of making a bispecific single-chain polypeptide construct are provided. In some embodiments, the methods comprise culturing a cell comprising a bispecific single-chain polypeptide construct comprising an anti-CLL-1 domain and an anti-CD3 domain and isolating the bispecific single-chain polypeptide construct. In some embodiments, the anti-CLL-1 domain comprises a first heavy chain and a first light chain and the anti-CD3 domain comprises a second heavy chain and a second light chain. In some embodiments, the anti-CLL-1 domain is SEQ ID NO: 1. In some embodiments, the anti-CD3 domain is SEQ ID NO: 2. In some embodiments, the first heavy chain comprises SEQ ID NO: 3. In some embodiments, the first light chain comprises SEQ ID NO: 4. In some embodiments, the second heavy chain comprises SEQ ID NO: 5. In some embodiments, the second light chain comprises SEQ ID NO: 6. In some embodiments, the constructs further comprise a linker. In some embodiments, the linker is a flexible hydrophobic linker. In some embodiments, the linker does not comprise a hinge region. In some embodiments, the linker comprises SEQ ID NO: 7. In some embodiments, the construct further comprises an antibody Fc domain. In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the Fc domain is IgG2 Fc domain. In some embodiments, the Fc domain comprises SEQ ID NO: 8. In some embodiments, the construct comprises. fromN to C terminus, an anti-CLL-1 domain, a linker, an antibody Fc domain, a linker, an anti-CD3 domain. In some embodiments, the construct comprises, from N to C terminus, an anti-CD3 domain, a linker, an antibody Fc domain, a linker, an anti-CLL-1 domain. In some embodiments, the construct comprises SEQ ID NO: 9. In some embodiments, the cell is selected from a bacterial cell, an archaeal cell, a fungal cell, or an animal cell. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a T cell.Atty. Dkt. No. 650053.01224

[0012] In an aspect of the current disclosure, polypeptides are provided. In some embodiments, the polypeptides comprise any one of SEQ ID NOs: 1, 3, or 4. In some embodiments, the polypeptides comprise SEQ ID NO: 1. In some embodiments, the polypeptides comprise SEQ ID NO: 3. In some embodiments, the polypeptides comprise SEQ ID NO: 4. In some embodiments, the polypeptides further comprise an antibody constant region, a linker, a detectable marker, a chimeric antigen receptor (CAR), a cellular targeting region, a toxin, or a chemotherapeutic drug. In some embodiments, the cellular targeting region targets T cells, NK cells, or B cells. The polypeptide of claim 42, wherein the toxin comprises brentuximab

[0013] In some embodiments, the polynucleotides comprising a sequence encoding any one ofSEQ ID NOs: 1, 3, or 4. In some embodiments, the polynucleotides comprising a sequence encoding SEQ ID NO: 1. In some embodiments, the polynucleotides comprising a sequence encoding SEQ ID NO: 3. In some embodiments, the polynucleotides comprising a sequence encoding SEQ ID NO: 4.

[0014] In some embodiments, the cells comprise a polynucleotide comprising a sequence encoding any one of SEQ ID NOs: 1, 3, or 4. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a human embryonic kidney (HEK) cell. In some embodiments, the cell is a T cell. In some embodiments, the T cell is derived from stem cells. In some embodiments, the T cell is derived from induced pluripotent stem cells (iPSCs).BRIEF DESCRIPTION OF THE FIGURES

[0015] FIGs. 1A-1B show7the structure of the CLL-1 (aka CLEC12A) gene and protein. (FIG. 1A) The human CLL-1 gene maps to 12pl3. (FIG. 1B)CLL-1 consists of a single extracellular carbohydrate recognition domain with 6N-glycosylation sites, a transmembrane region, and an intracellular domain with 2 motifs of I / VXYXXL and YXXM at the NH2 terminus.

[0016] FIGs. 2A-2B show membrane Proteome Array analysis. (FIG. 2A) Testing the inventors’ lead anti-CD30 antibody candidate (8D10F10) for binding to over 6000 human surface proteins. (FIG. 2B) Validation studies confirm binding to TNFRSF8 (CD30) only.

[0017] FIG. 3 shows a schematic of the disclosed anti-CLL-l / anti-CD3 'Grappler'.

[0018] FIG. 4 shows a Western blot showing secretion of the full-length 'CLL-1 Grappler' into the supernatant of transfected Expi293F cells.

[0019] FIGs. 5A-5B show flow cytometry results for staining of OCIAML cells for CLL-1 expression. (FIG. 5 A) Unstained; (FIG. 5B) CLL-1 stained.Atty. Dkt. No. 650053.01224

[0020] FIGs. 6A-6B shows flow cytometry plots of labeled OCIAML2 tumor cells and labeled human T cells. T cells were coated with 'media' (FIG. 6A) and anti-CLL-l / anti-CD3 Grappler (FIG. 6B).DETAILED DESCRIPTIONBispecific single-chain constructs

[0021] The inventors performed an unbiased mass spectrometry-based analysis of surface glycoproteins (-70% of surface proteins) expressed on four AML cell lines from adults and children which revealed 700+ proteins, of which, the inventors prioritized 10 candidate tumor antigens. C-type lectin-like molecule-1 (CLL-1), also known as C-type lectin domain family 12 member A (CLEC12A), is a member of a superfamily of proteins involved in phagocytosis, complement activation, cell adhesion, and pathogen recognition. The sequence of human CLL-1 is provided as SEQ ID NO: 27. CLL-1 is expressed on mature myeloid cells but not on normal hematopoietic stem / progenitor cells.

[0022] In some embodiments, autologous T cells are coated ex vivo with the disclosed novel cojoined biAb that binds to both CLL-1 and CD3, simultaneously. Ex vivo coating uses less biAb than direct injection does (by at least 1000-fold) and helps ensure that one of the two target receptors (CD3 on the T cells) is already engaged prior to reinfusion of the engineered immune effector cells.

[0023] To synthesize this novel immunotherapy reagent, the inventors first overexpressed and purified the extracellular domain of human CLL-1. The inventors used this enriched polypeptide to screen the inventors’ in-house humanized single-chain antibody I i bran that is constructed in bacteriophage. After panning with increased stringency, the inventors were able to isolate a unique phage clone that binds CLL-1 specifically. Next, the inventors found an scFv for human CD3 that was known in the art. The inventors then joined these sequences together with the CH2 and CH3 regions of the human IGHG2 gene behind a tissue independent secretory sequence that leads to high expression (HMM38). The inventors recently demonstrated that this novel single, linked polypeptide could be expressed in the supernatant of transfected producer cells and migrates at the expected size on PAGE. It also binds to both T cells and tumor cells at the same time and pulls them together; as the inventors expected it would.

[0024] In some embodiments, the disclosed constructs comprise scFvs to target CD3, e.g., SEQ ID NO: 2 and CLL-1, e.g., SEQ ID NO: 1, an IgG2 Fc region, e.g., SEQ ID NO: 8, and flexible, hydrophilic linkers connecting the scFvs to the Fc region of the antibody, e.g.. SEQ ID NO: 7. Rather than using a traditional (GGGS)nlinker, the disclosed compounds may comprise a flexible,Atty. Dkt. No. 650053.01224 hydrophilic linker, which is designed to improve the bioavailability and decrease the volume of distribution of the Grapplers, while still allowing for the flexibility traditional (GGGS)n linkers impart. In some embodiments, the constructs comprise an Fc region which may be derived from an IgG2 antibody. This subclass of IgG is designed to result in lower systemic responses to the Grappler. In addition, an Fc region may be designed to allow the Grappler to use the FcRn recycling pathway to increase the half-life of the Grappler protein beyond what would be expected for a protein of its size.

[0025] Accordingly, in an aspect of the current disclosure, bispecific single-chain constructs are provided. In some embodiments, the constructs comprise an anti-CLL-1 domain and an anti-CD3 domain. In some embodiments, the anti-CLL-1 domain and the anti-CD3 domain are antibody single chain variable fragments (scFvs) which are made up of an antibody heavy chain and light chain linked together. Thus, in some embodiments, the anti-CLL-1 domain comprises a first heavy chain and a first light chain, and the anti-CD3 domain comprises a second heavy chain and a second light chain. The CLL-1 targeting scFv may be defined by complementarity determining regions (CDRs). The first heavy chain may comprise CDR1, CDR2, and CDR3 and the first light chain may comprise CDR4. CDR5. and CDR6, wherein CDR1 comprises SEQ ID NO: 28. CDR2 comprises SEQ ID NO: 29, CDR3 comprises SEQ ID NO: 30, CDR4 comprises SEQ ID NO: 31, CDR5 comprises SEQ ID NO: 32, and CDR6 comprises SEQ ID NO: 33.

[0026] In some embodiments, the first heavy chain (anti-CLL-1) comprises SEQ ID NO: 3. In some embodiments, the first light chain (anti-CLL-1) comprises SEQ ID NO: 4. In some embodiments, the second heavy chain (anti-CD3) comprises SEQ ID NO: 5. In some embodiments, the second light chain (anti-CD3) comprises SEQ ID NO: 6. In some embodiments, the anti-CLL-1 domain has the sequence SEQ ID NO: 1, or a sequence with at least about 50% identity, at least about 55% identity, at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to SEQ ID NO: 1.

[0027] In some embodiments, the anti-CD3 domain is SEQ ID NO: 2, or a sequence with at least about 50% identity, at least about 55% identity, at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity,Atty. Dkt. No. 650053.01224 at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to SEQ ID NO: 2. SEQ ID NO: 2 is derived from a humanized anti-CD3s monoclonal antibody (0KT3), see, e.g., U.S. Pat. No. 6,491,916.

[0028] In some embodiments, the constructs comprise a linker. The inventors believe that a flexible hydrophobic linker, which does not comprise a hinge region, may be beneficial for properties of the disclosed constructs. Therefore, in some embodiments, the linker is a flexible hydrophobic linker which, in some embodiments, does not comprise a hinge region, e.g., the linker with the amino acid sequence SEQ ID NO: 7.

[0029] The inventors believe that the addition of an antibody Fc domain also improves the properties of the disclosed constructs by, for example, increasing the half-life of the construct in a subject. Accordingly, in some embodiments, the disclosed constructs comprise an antibody Fc domain. In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the Fc domain is IgG2 Fc domain. In some embodiments, the Fc domain comprises SEQ ID NO: 8, or a sequence with at least about 90% identity to SEQ ID NO: 8. In some embodiments the Fc domain is SEQ ID NO: 8.

[0030] In some embodiments, the directionality of the construct may be defined. Accordingly, in some embodiments, the construct comprises, from N to C terminus, an anti-CLL-1 domain, a linker, an antibody Fc domain, a linker, an anti-CD3 domain. In other embodiments, the construct comprises, from N to C terminus, an anti-CD3 domain, a linker, an antibody Fc domain, a linker, an anti-CLL-1 domain. In some embodiments, the construct comprises SEQ ID NO: 9.

[0031] Disclosed herein are novel CLL-1 binding peptides. Accordingly, disclosed herein is a polypeptide comprising any one of SEQ ID NOs: 1, 3, 4, or 28-33. the polypeptides may further comprise an antibody constant region, a linker, a detectable marker, a chimeric antigen receptor (CAR), a toxin, a cellular targeting region, or a chemotherapeutic drug. Exemplary toxins include, but are not limited to, brentuximab, though any toxin may be linked to the polypeptides by methods known in the art, e.g., bio-orthogonal click chemistry. Exemplary cellular targeting regions include, but are not limited to, anti-CD3 targeting regions (T cell targeting regions), e g., anti-CD3 scFvs, targeting regions specific for NK. cells, targeting regions specific for B cells, etc. Methods of making CARs are known in the art and the skilled artisan could use the disclosed novel anti- CLL-1 targeting regions as antigen-binding regions in a CAR construct. Similarly, polynucleotides encoding the CARs and cells comprising the polynucleotides are also disclosed herein as well as making CAR T cells using the polynucleotides.Atty. Dkt. No. 650053.01224

[0032] The disclosed constructs may comprise or be encoded wholly or in part by one of SEQ ID NOs: 1-26 or 28-33, depending on whether the sequence is an amino acid sequence or anucleotide sequence, or may comprise a sequence with at least about 50% identity, at least about 55% identity, at least about 60% identity, at least about 65% identity, at least about 70% identity', at least about 75% identity, at least about 80% identity', at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity, or more to one of SEQ ID NOs: 1-26 or 28-33.Polynucleotides

[0033] Also contemplated in the current disclosure are polynucleotides that encode the disclosed constructs. Accordingly, in another aspect of the current disclosure, polynucleotides are provided. In some embodiments, the polynucleotides encode a bispecific single-chain construct comprising an anti-CLL-1 domain and an anti-CD3 domain. Thus, in some embodiments, the first heavy chain is encoded by a sequence comprising SEQ ID NO: 13. One of skill in the art will understand that certain modifications to the DNA sequence encoding the disclosed constructs, or portions thereof, could be modified to generate constructs with essentially the same amino acid structure, for example, through codon optimization or conservative codon usage. Thus, in some embodiments, the first heavy chain (anti-CLL-1 ) may be encoded by a sequence with at least about 50% identity, at least about 55% identity’, at least about 60% identity, at least about 65% identity', at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity’, at least about 98% identity, or at least about 99% identity to SEQ ID NO: 13.

[0034] In some embodiments, the first light chain (anti-CLL-1) is encoded by a sequence comprising SEQ ID NO: 15, or a sequence with at least about 50% identity, at least about 55% identity, at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity’, at least about 80% identity, at least about 85% identity, at least about 90% identity’, at least about 91% identity', at least about 92% identity’, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to SEQ ID NO: 15.Atty. Dkt. No. 650053.01224

[0035] In some embodiments, the second heavy chain (anti-CD3) is encoded by a sequence comprising SEQ ID NO: 21, or a sequence with at least about 50% identity, at least about 55% identity, at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity', at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to SEQ ID NO: 21.

[0036] In some embodiments, the second light chain (anti-CD3) is encoded by a sequence comprising SEQ ID NO: 23, or a sequence with at least about 50% identity, at least about 55% identity, at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity', at least about 95% identity, at least about 96% identity', at least about 97% identity, at least about 98% identity, or at least about 99% identity to SEQ ID NO: 23.

[0037] In some embodiments, the construct is encoded by a sequence comprising SEQ ID NO: 10. or a sequence with at least about 50% identity, at least about 55% identity, at least about 60% identity', at least about 65% identity', at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity', at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to SEQ ID NO: 10.Pharmaceutical compositions

[0038] The disclosed bispecific constructs are contemplated to be used as a pharmaceutical intervention. Thus, in another aspect of the current disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise a bispecific singlechain construct comprising an anti-CLL-1 domain and an anti-CD3 domain. As will be understood by one of skill in the art. the disclosed pharmaceutical compositions may be formulated such that they include appropriate pharmaceutical carriers or excipients to maximize the desired pharmaceutical composition’s function. Such pharmaceutical formulations are considered routine.

[0039] In some embodiments, the pharmaceutical compositions are formulated for parenteral administration, i.e., they may be formulated for administration by subcutaneous (SC / SQ), intraperitoneal (IP), intravenous (IV), intradermal (ID), and intramuscular (IM) route. Suitably, the pharmaceutical compositions are formulated for intravenous administration. In someAtty. Dkt. No. 650053.01224 embodiments, the pharmaceutical compositions are formulated such that they consist only of GMP approved compounds, i.e., they have defined characteristics and may be used in conjunction with GMP prepared cells, e.g., the compositions may be used in conjunction with cells intended for adoptive transfer therapy.

[0040] The disclosed compositions may further comprise a therapeutic, e.g., a chemotherapeutic, or an immunosuppressant.Methods of treatment

[0041] In another aspect of the current disclosure, methods of treating a disease or disorder associated with CLL-1 expression are provided. In some embodiments, the methods comprise administering an effective amount of a pharmaceutical composition comprising a bispecific singlechain construct comprising an anti-CLL-1 domain and an anti-CD3 domain to a subject in need thereof to treat the disease or disorder. As discussed above, CLL-1 is expressed on a variety' of transformed cells, e.g., transformed cells present in a subject with a cell-proliferative disease or disorder. In some embodiments, the cell proliferative disease or disorder is acute myeloid leukemia (AML); however, it is to be understood that the diseases and disorders associated with CLL-1 expression are not limited to the foregoing examples. Accordingly, cancers that are characterized by CLL-1 expression on the surface of cancerous cells are also contemplated to be targets for treatment by administration of the disclosed compositions, pharmaceutical compositions and by use of the disclosed methods.

[0042] As used herein, "‘administration” may comprise any acceptable administrative route, e.g., intravenous administration or intrathecal administration. Alternatively, administration may comprise administration by percutaneous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The route of administration may be varied in any way, limited by the physical properties of the compounds being employed and the convenience of the subject and the caregiver, etc.

[0043] In some embodiments, the disclosed compositions, e.g., bispecific antibodies, can be directly administered to a subject in need thereof (e.g., a subject diagnosed with or suffering from a CLL-1 associated disease or condition, such as AML). By way of example, but not by way of limitation, administration of the bispecific antibody may include intravenous, intrathecal, intracranial, or intratumoral administration.

[0044] CLL-1 can also be expressed on granulocy tes, e.g., neutrophils, which are associated with certain autoimmune diseases or disorders, e.g., systemic lupus erythematosus (SLE). The subjectAtty. Dkt. No. 650053.01224 may have been diagnosed with a disease or disorder associated with CLL-1 and / or with granulocytes expressing CLL-1, e.g., an autoimmune disease or disorder, e.g., SLE.

[0045] The disclosed compositions may be administered simultaneously, or in addition to, either before or after, another therapeutic, e.g., a chemotherapeutic, an immunosuppressant, an antihistamine, etc.

[0046] The inventors envision that in the disclosed methods, cells, e.g., T cells, may be isolated from the subject by leukapheresis. e.g., a subject with cancer, e.g.. AML, and contacted with the disclosed bispecific single-chain constructs or pharmaceutical compositions comprising the same, ex vivo / in vitro. Thus, the cells, e.g., T cells, are pre-loaded with the construct. Without wishing to be limited by any theory or mechanism, the inventors believe that this strategy increases positive interactions between immune cells, e.g., T cells, and the disclosed constructs as compared to administration to a subject, or administration to a subject after infusion of T cells. In other words, the disclosed constructs are more likely to bind to the T cells ex vivo due to higher relative concentration of the construct to the T cells in comparison with administration of the disclosed constructs to the subject, e.g., intravenously, which requires that the disclosed constructs bind to the immune cells, e.g., T cells, and target cells in situ, i.e.. in the context of the bloodstream, lymphatic system, or in tissues. Further, this strategy requires significantly less of the construct for each administration event as the target cells are pre-bound with the construct. In some embodiments, the T cells are stimulated ex vivo with, e.g., anti-CD3 antibodies and / or anti-CD28 antibodies and expanded by culturing the cells with interleukin 2 (IL-2). Then, the inventors envision that, in one embodiment, the cells are re-infused into the subject. Accordingly, the pre- loaded cells are brought into close contact with target cells, i.e., cells expressing CLL-1. Thus, the inventors believe that the immune cells, e.g., T cells, become stimulated near the target cells and initiate a cytotoxic response and / or a cytokine-based inflammatory response targeting the CLL-1+cells. The inventors envision that the immune cells may be, e.g., CD8+T cells, CD4+T cells, or a combination of CD8+T cells and CD4+T cells.

[0047] Thus, in one embodiment, T cells from a patient with CLL-1 positive cancerous tissue may be harvested via blood collection. Then the T cells are armed with the CLL-1 Grappler. The armed T cells will be infused back into the patient. When the armed T cells come near CLL-1 positive cells, the CLL-1 Grappler would bind to the CLL-1 proteins on the cancerous cells. The T cells would then be able to release both lytic granzymes and inflammatory cytokines. The granzymes would induce lysis of the cancerous bound cell. The cytokines would recruit other immune cells to the site of the cancerous tissue allowing for greater levels of targeted cell lysis.Atty. Dkt. No. 650053.01224Cells

[0048] The instant disclosure provides polynucleotides that encode the disclosed novel bispecific single-chain constructs. Accordingly, in another aspect of the current disclosure, cells comprising the disclosed polynucleotides encoding the bispecific single-chain constructs are provided. In some embodiments, the cells are a mammalian cells, e.g., human cells. In some embodiments, the cells are HEK293 cells.

[0049] The cells may comprise T cells, e.g., human T cells. The T cells may be derived from stem cells, e.g., induced pluripotent stem cells or embr onic stem cells.

[0050] In addition to the cells provided by the instant disclosure, methods of making the disclosed bispecific single-chain constructs are provided. In some embodiments, the methods comprise introducing the disclosed polynucleotides into a cell and allowing the cell to express the polynucleotides to generate the disclosed bispecific single chain constructs in vitro. In some embodiments, the constructs are further purified from the cells by means known in the art. In some embodiments, the constructs comprise affinity' tags, e.g., streptavidin tags, histidine tags, FLAG tags, HA tags, etc., which allow their efficient purification.

[0051] Disclosed are polynucleotides comprising a sequence encoding any one of SEQ ID NOs: 1, 3, or 4. Disclosed are cells comprising the polynucleotides, e.g., a mammalian cell, a human cell, a human embry onic kidney (HEK) cell.

[0052] The present invention is described herein using several definitions, as set forth below and throughout the application.Definitions

[0053] The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0054] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0055] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary' skill in the art and will vary' to some extent on the context in which they are used. If there are uses of the term w hich are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plusAtty. Dkt. No. 650053.01224 or minus 10% of the particular term and "substantially" and ‘‘significantly” will mean more than plus or minus 10% of the particular term.

[0056] As used herein, the terms '‘include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0057] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0058] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0059] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4. or 6 members, and so forth.

[0060] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use aAtty. Dkt. No. 650053.01224 specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb "‘may” has the same meaning and connotation as the auxiliary verb “can / ’EXAMPLES

[0061] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.Example 1 - Generation and testing of bispecific single-chain constructs

[0062] There is a significant unmet need for immunotherapies in adult and pediatric AML. The inventors plan to address this by developing an immunotherapy that uses a patient's own T cells to attack their cancer. The inventors plan to take T cells out from the patient and coat them 'out of body' with a unique cojoined bispecific antibody the inventors developed. This anti-CLL-1 Grappler (as the inventors call it) will bind to the patient's T cells and to the CLL-1 tumor antigen present on their AML cells. After reinfusion, these T cells will be redirected to attack the patient's AML cells specifically in their bodies.

[0063] As disclosed herein, the inventors examined potential tumor antigen expression on AML cell lines. The inventors identified the candidate target: CLL-1. Next, the inventors pulled out a novel humanized anti-CLL-1 single-chain antibody from a 'library' the inventors had generated previously. The inventors fused this unique antibody directly to a known antibody that binds T cells. As shown herein, the bispecific antibody can be produced, and is shown to binds both T cells and tumor cells simultaneously.

[0064] The autologous therapy disclosed herein does not require genetic modification of cells. This will simplify clinical trials and reduce costs. The inventors plan on coating autologous T cells ex vivo with a novel co-joined bi Ab that the inventors conceptualized and then synthesized that binds to both CLL-1 and CD3, simultaneously. Ex vivo coating uses less biAb than direct injection does (by at least 1000-fold) and helps ensure that one of the two target receptors (CD3 on the T cells) is already engaged prior to reinfusion of the engineered immune effector cells.

[0065] To synthesize this novel immunotherapy reagent, the inventors first overexpressed and purified the extracellular domain of human CLL-1. The inventors used this enriched polypeptide to screen the inventors’ in-house humanized single-chain antibody library that is constructed in bacteriophage. After panning with increased stringency, the inventors were able to isolate a unique phage clone that binds CLL-1 specifically. Next the inventors found an scFv for human CD3 from an expired patent. The inventors then joined these sequences together with the CH2 and CHS regionsAtty. Dkt. No. 650053.01224 of the human IGHG2 gene behind a tissue independent secretory sequence that leads to high expression (HMM38). The inventors demonstrated that this novel single, linked polypeptide could be expressed in the supernatant of transfected producer cells and migrates at the expected size on PAGE. It also binds to both T cells and tumor cells at the same time and pulls them together; as the inventors expected it would.

[0066] The inventors' objectives for this proposal are to continue development and testing of this co-joined biAb towards clinical implementation utilizing ex vivo-loaded autologous T cells to treat pediatric AML at MCW / Children's Wisconsin. The inventors will perform expression / stability studies, binding affinity studies, and binding specificity studies along with in vitro and in vivo testing in xenotransplant AML models. Meanwhile an experienced clinical trialist and expert in AML, will generate a draft clinical protocol.Introduction / Significance

[0067] CAR-T cells have revolutionized immunotherapy for cancer. Yet the number of candidate tumor antigens being targeted by CAR-T cells is limited [1]. Further, CAR-T cells require genetic modification. This lengthens production time, increases costs, complicates clinical protocols, and requires long-term monitoring. CAR-T cells can also induce cytokine release syndrome (CRS). In contrast, bispecific antibodies (biAbs) allow for directed targeting of tumor antigens but are easier to implement. The inventors generated a novel anti-CD30 / anti-CD3 bi Ab for treatment of HL in adults and children [2], The inventors’ approach involves ex vivo loading of autologous T cells followed by multiple infusions into non-conditioned recipients. For that study, a response to a previous FDA 'Clinical Hold' letter has recently been communicated (NCT05544968).

[0068] For AML, the inventors undertook an unbiased, mass spectrometry -based analysis of surface glycoproteins (-70% of surface proteins) expressed on four AML cell lines from adults and children. The inventors revealed 700+ proteins; the inventors prioritized 10 candidate tumor antigens. C-type lectin-like molecule-1 (CLL-1) is a member of a superfamily of proteins involved in phagocytosis, complement activation, cell adhesion, and pathogen recognition [3], CLL-1 is expressed on mature myeloid cells but not on normal hematopoietic stem / progenitor cells. CLL-1 has been validated as a tumor antigen: CARs against CLL-1 have been developed [4] and infused into children with AML [5] and adults [6], In other studies, biAbs co-targeting CLL-1 and CD3 (effector T cells) have been developed for direct infusions [7, 8],

[0069] The inventors have a unique pipeline at Children's Wisconsin to implement novel biAbs for treatment of pediatric AML. As mentioned, the inventors’ approach involves ex vivo loading of the biAbs onto autologous T cells. This requires 1000-fold less biAb than direct injection. The inventors’ collaborator at UVa has treated many patients for other cancers and has not reached anyAtty. Dkt. No. 650053.01224DLTs. Patients received multiple doses of billions of biAb-loaded T cells (e.g., [9]) without toxicities or anti-idiotype antibodies.

[0070] The inventors developed a unique tethering system for the inventors’ single-chain antibodies that directly links them to an analogous single-chain antibody that binds to CD3. This cojoined bispecific reagent does not require chemical conjugation for linking.Research Plan

[0071] Background: Immunotherapy for the treatment of acute myeloid leukemia (AML) has proven difficult as most potential antigenic targets are present on not only cancerous cells, but also normal hematopoietic stem cells (HSCs). The inventors have chosen to focus on a relatively new cancer target that came up in the inventors’ mass spectrometry -based screen of surface glycoproteins on AML cell lines. C-type lectin-like molecule- 1 (CLL-1), a membrane glycoprotein also known as C-type lectin domain family 12 member A (FIGs. 1A-1B), is encoded by the CLEC 12A gene. It was discovered in 2004 while researchers were scanning for AML tumor targets using a panel of single-chain Fv fragments from a phage-display library

[0010] , CLL-1 expression is restricted to myeloid cells present in peripheral blood and bone marrow; expression has been found on 92% of cells from primary AML patients examined

[0010] . CLL-1 has also been found on CD34+CD38- leukemic stem cells in 87% of AML patients

[0011] , CLL-1 expression on CD34+ cells in CML and myelodysplastic syndrome was variable (10-60%), and virtually absent on cells from primary- ALL patients (7.7%)

[0010] , Strong AML upregulation on tumors and restricted expression on normal hematopoietic cells makes CLL-1 a compelling target for novel AML immunotherapies.

[0072] Monoclonal antibodies targeting CLL-1 have been developed by others and shown to induce dose-dependent cy totoxicity against AML cell lines, primary AML patient cells, and xenograft mice implanted with HL-60 AML cells

[0012] . Also, nanomicelles delivering daunorubicin were surface-loaded with novel anti-CLLl -targeting peptides and found to bind CD34+CLL-1 + primary AML cells to deliver their payload - but were found to not bind to normal hematopoietic stem cells

[0013] , Chimeric Antigen Receptor (CAR) transduced -T cell products have been found to demonstrate potent activity against CLL-1 expressed on several AML cell lines and on primary patient-derived AML blasts in in vitro analyses while sparing healthy HSCs [4, 14] . Furthermore, in NOD scid gamma (NSG) xenograft models of disseminated AML generated using CLL1- positive U937 or HL60 cell lines, anti-CLL-1 CAR-T cells demonstrated anti -tumor activity [4, 14, 15] and prolonged survival

[0015] , Eight children with relapsed or refractory AML have been treated clinically with an anti-CLL-1 CAR T strategy [5], Patients received one dose of autologous anti-CLLl CAR-T cells after lymphodepletion conditioning. Four of the eight treated patientsAtty. Dkt. No. 650053.01224 achieved a morphologic leukemia-free state (MFLS) with minimal residual disease (MRD) negativity, one patient achieved MLFS with MRD positivity, one patient achieved complete remission with incomplete hematologic recovery but MRD positivity, one patient achieved a partial remission, and one patient demonstrated stable disease and CLL1 -positive AML blast clearance [5],

[0073] CLL-1 has also been investigated in a bi Ab setting, with a high-affinity monkey cross- reactive anti-CLL-1 antibody tested alongside several anti-CD3 antibodies of varying affinity. High-affinity CD3’s were up to 100-fold more potent in vitro, but in a monkey model only the low-affinity CD3 / CLL-1 bispecific was found to be well tolerated and functional. This suggests that a well -engineered CLL-1 -CD3 bispecific may be effective for the treatment of AML [7],

[0074] The inventors’ planned approach to treat pediatric AML is to engineer a novel anti-CLL- l / anti-CD3 biAb and use it to load autologous patient T cells in an ex vivo fashion. These cells will then be re-infused (multiple times, if needed) into patients. Ex vivo loading requires 1000-fold less biAb than direct injections and ensures that one end (CD3) of the compound is already bound to its cognate target. This saves that rate- and dose-limiting process from having to occur in vivo in patients. BiAb-loading of cells also does not require genetic modification of the immunotherapy effector population, thus saving money and time and increasing safety. Lastly, the inventors’ biAb loaded cells can be infused multiple times into nonconditioned recipients in out-patient procedures; a distinct advantage over CAR-T cells, which require conditioning that can contribute to CRS.

[0075] Preliminary Data: Constructing and implementing a therapeutic pipeline: As mentioned, the inventors have previously engineered novel biAbs against the anti-cancer target antigen CD30 for the eventual treatment of HL in adults and children [2], The inventors did this by generating murine antibodies against the extracellular domain of CD30 that the inventors had overexpressed, purified, and used as an immunogen. Following screening, hybridoma formation, and antibody purification, the inventors tested binding affinities, specificities, and epitope recognition patterns of the inventors’ lead monoclonal antibody. When the inventors chemically conjugate the inventors’ lead anti-CD30 antibody candidate (8D10) with OK3, a well-known murine antibody developed against CD3, and preload human T cells with this bi Ab combination, the inventors can directly pull tumor cells and T cells together. This leads to cytokine secretion and specific killing of the tumor cells [2], Further, as the inventors are developing this biAb combination for clinical implementation, and this is a new antibody against CD30, the inventors wanted to test binding to other cell surface antigens instead of just the intended target. The inventors tested binding to over 6000 human surface proteins by Membrane Proteome Analysis (FIGs. 2A-2B). Only TNFRSF8 (i. e. , CD30) bound. This was further validated in confirmatory studies using plasmid transfectionsAtty. Dkt. No. 650053.01224 to overexpress candidate surface proteins (FIG. 2B). Again, only CD30 bound. The inventors established a Master Cell Bank from a clonally isolated single-cell clone with a collaborator who purified the inventors’ antibody 8D10F10 under GMP conditions to support our IND application to the FDA to open a trial in HL. The inventors have developed a novel humanized anti-CLL-1 single-chain antibody. To launch this project, the inventors first performed an unbiased, mass spectrometry-based analysis of surface glycoproteins (-70% of surface proteins) expressed on four AML cell lines originating from both adults and children (Kasumi 1, KG-1. OCIAML, and U937). This mirrors a previous discovery study the inventors published in 2020 for Multiple Myeloma

[0016] , The inventors’ AML study revealed 700+ glycosylated surface proteins on the AML cell lines; the inventors prioritized 10 candidate tumor antigens by in silico expression analyses. One candidate tumor antigen was the C-type lectin-like molecule- 1 (CLL-1 aka CLEC12A).

[0076] The inventors overexpressed the extracellular domain (ECD) of human CLL-1 and purified this polypeptide. DNA encoding tPA signal sequence-Hisx6 purification tag-GSG linker- CLL-1 ECD amino acids 65-265 was synthesized. Expi293 cells were transfected, media was harvested after 5 days, and the Hisx6-CLL-1 ECD purified to near homogeneity with Ni-NTA resin. Identity was confirmed by Western blot with a polyclonal anti-CLL-1 antibody. The expressed protein appeared somewhat larger than expected. The ECD of CLL-1 is reported to be highly glycosylated; reanalysis after deglycosylation confirmed this as the protein was reduced to the expected size of ~25kDa (not shown). Next, the single chain fragment variable (scFv) sequence specific for the ECD of human CLL-1 was obtained by us from the inventors’ proprietary human scFv phage display library. Purified CLL-1 ECD (made above) was used to pan the inventors’ scFv phage display library 3 successive times to obtain anti-CLL-1 scFv sequence(s). Briefly, the library was panned, eluted phage was precipitated and panned a 2ndtime, etc. With each panning the stringency of the wash was increased. After ELISA screening, DNA was prepared from 3 phage clones and sequenced. All 3 clones contained an identical scFv sequence. This likely indicates a high-affinity interaction. Specific Hisx6-CLL-1 ECD scFv binding was demonstrated via ELISA, which also included a Hisx6-non-CLL-l protein as a control (not shown).

[0077] The inventors generated a novel cojoined bi Ab consisting of dual single-chains that bind both CLL-1 and CD3 linked by Fc (Cm and Cm) regions. A schematic is shown in FIG. 3. This anti-CLL-1 / anti-CD3 'Grappler' is an IgG-like molecule containing the Cm and CH3 domains of the IgG Fc region along with domains that will bind to the CLL-1 protein and to the CD3 molecule on the surface of T cells. The anti-CD3 scFv sequence was obtained from expired patent: U.S. 6,491,916 BL The inventors acquired the amino acids for the VH and VL and used Benchling (an on-line software program) to back translate and humanize the sequences. The inventors also addedAtty. Dkt. No. 650053.01224 a CD138 scFv linker to connect the VH and VL regions. A major advantage of this novel construct is the fact that the biAb will now be produced as one single polypeptide from transfected cells. This will get around issues that the FDA has with the use of murine hybridomas to produce clinical- grade immunotherapy reagents (and the additional manufacturing testing that goes along with this). This approach will also produce already co-joined scFvs, thus eliminating the need for chemical conjugations as the inventors have done for the inventors’ anti-CD30 biAbs [2], Those previous chemical conjugations are about 50% efficient in the inventors’ hands and could also be generating many spatial permutations of cross-linked biAbs; some of which may have reduced or altered function or even decreased stability.

[0078] To produce additional preliminary data for this present application, a codon-optimized nucleic acid sequence, CLL-1 OKT3 scFv2-Fc (or 'CLL-1 Grappler'), containing a secretion signal, was synthesized into the pcDNA3.4-TOPO plasmid backbone. This plasmid construct was transiently transfected into Expi293F cells. A culture of transfected cells was allowed to express the 'CLL- 1 Grappler' for 5 days. After that period, cells were removed, pelleted, and lysed. Proteins in the supernatant and cell lysates were separated by SDS-PAGE alongside pre-transfection supernatant and recombinant human IgG-Fc as controls. The gel was transferred to PVDF membrane and probed with anti-human IgG-Fc-HRP antibody. FIG. 4 shows the results of that Western blot. This is important milestone data: it demonstrates that the inventors’ novel 'CLL-1 Grappler' can be synthesized, is stable when secreted, and migrates at the expected MW in PAGE.

[0079] Lastly, the inventors recently measured binding of the inventors’ anti-CLL-1 Grappler to both effector T cells and tumor cells; does this new polypeptide have the capacity to actively bring tumor cells and T cells together? This very preliminary assay was performed as the inventors have done before [2]. Briefly: OCIAML2 cells (which are CLL-1 positive from the inventors’ original mass spec scan and from flow cytometry analyses: FIGs. 5A-5B) were labeled with an unconjugated anti-human CD 123 antibody and then an AF488-cojugated anti-mouse IgG secondary antibody. Human T cells were obtained from de-identified and discarded leukocyte reduction system cones or apheresis products or purchased from Stem Cell Technologies. These cells were labeled with AF700 conjugated to anti-CD8 antibody and loaded with the inventors’ anti-CLL-1 Grappler (straight from the supernatant of transfected cells). Unarmed (media control) human T cells served as a control. T cells and tumor cells were combined at a 1 : 1 ratio and analyzed on a BD LSR Fortessa X-20 flow cytometer. FIGs. 6A-6B shows that a new combinatorial flow peak was obtained in the upper right quadrant when the specific anti-CLL-1 / anti-CD3 Grappler supernatant was utilized.Atty. Dkt. No. 650053.01224

[0080] Characterize the cojoined anti-CLL-l / anti-CD3 biAb. Overexpress and purify large quantities from the supernatant of transfected cells. Further test binding of the purified polypeptide (and dilutions) to both T cells and tumor cells by flow cytometry. Test binding affinity to soluble CLL-1 by surface plasmon resonance. Test binding to other human cell surface proteins for both anti-CLL- 1 and anti-CD3 by Membrane Proteome Array.

[0081] Test in vitro stimulation / killing when the inventors’ cojoined biAb is loaded on human T cells. Optimize loading and dosing. Test stability, cytokine production, and tumor cell killing.

[0082] Test in vivo effects in xeno transplanted immune-deficient mice. Animals will be bearing human AML cells and then infused with the inventors’ cojoined antibody-loaded human T cells.

[0083] Approach / Methodology: The inventors’ first goal is to prepare large amounts of purified anti-CLL- 1 Grappler. The inventors will transfect Expi293F cells as part of the Expi293 Expression System following the manufacturer’s directions. IgGs bind to Protein A or G-coated Sepharose beads via their Fc region

[0017] . Protein A is used for purification of most IgG proteins, but some are better purified using Protein G

[0018] . Proteins will be eluted from the Sepharose resin using a low pH elution buffers. The inventors will empirically determine to which protein (A or G) the anti-CLL- 1 Grappler binds to best, and which elution buffer efficiently dislodges it from the resin. Once purified protein is obtained the inventors will repeat the flow cytometry-based cell binding assays.

[0084] Next the inventors will test surface plasmon resonance (SPR) binding affinity analyses will be performed to examine the interaction of the cojoined the inventors’ anti-CLL- 1 Grappler with ligand. Methods here are analogous to those used in experiments the inventors have performed before with the inventors’ anti-CD30 / anti-CD3 biAb [2], Specifically, recombinant CLL-1 ECD (from above) will be immobilized on a CM5 sensor chip and serial dilutions of the inventors’ anti- CLL- 1 Grappler cojoined biAb injected. SPR signal will be detected with a Biacore S200 instrument with concentration curves fitted to a 1 : 1 Langmuir binding model and kinetic constants and equilibrium constants calculated. The reverse will also be carried out with cojoined biAb immobilization.

[0085] The inventors will test the specificity of binding of the inventors’ anti-CLL- 1 Grappler by a Membrane Proteome Array analysis by Integral Molecular (see FIGs. 2A-2B). Their library contains over 6000 human membrane proteins including 94% of all single-pass, multi-pass, and GPI-anchored proteins. This is key data to predict 'on-target off-cancer' effects. Arguably, there is no better way to do this as animals express species-specific variants of proteins that can differ significantly in structure and epitope access. Here the inventors predict that they will see bindingAtty. Dkt. No. 650053.01224 to both human CLL-1 and human CD3 for this particular co-joined biAb. Identified targets will be validated in secondary screens using plasmid transfections as the inventors have done before [2],

[0086] Once the inventors have determined that the inventors’ anti-CLL-1 Grappler binds specifically and brings tumor cells and effector T cells together, the inventors will determine if that interaction leads to T cell stimulation and target cell killing. The inventors will optimize concentrations. T cell stimulation will be measured by a Luminex assay as the inventors have done before [2]. The inventors will use unarmed human T cells as a control. The inventors will measure supernatant expression of CD40L, EGF, Eotaxin, FGF-P, Flt-3 ligand, Fractalkine, G-CSF, GM- CSF, Granzyme B, GRO-a, GRO-0, IFN-a, IFN-p, IFN-y, IL-10, IL-12 p70, IL-13, IL-15, IL- I7A, IL-17E, IL-la, IL-1 p, IL-2, IL-3, IL-33, IL-4, IL-5, IL-6, IL-7, IL-8, IL-lra, IP-10, MCP-1, MIP-la, MIP-ip, MIP-3a, MIP-3p, PD-L1, PDGF-AA, PDGF-AB / BB, RANTES, TGF-a, TGF -P,TNF-a, TRAIL, and VEGF (Magnetic Luminex Performance Assay XL Kit and additional assays) once tumor cells and T cells are brought together. The inventors expect changes in IFN-y, TNF-a, IL-2, and GM-CSF as those are commonly upregulated with T cell engagement [2].

[0087] For in vitro cytotoxicity studies, the inventors will use two methods. One is a standard 4-hour chromium release assay as the inventors have done before [2]. For that assay, the inventors will first determine an optimal loading dose of the inventors’ anti-CLL-1 Grappler onto human T cells. Next, the inventors will incubate CLL-1 positive AML cells lines (and controls) with51Cr for 2 hours. Then loaded or control T cells will be plated with target cells for 4 hours at various effector-to-target ratios. Supernatants will be collected and read using a Perkin Elmer MicroBeta 2 Microplate counter. Spontaneous51Cr release will be measured by incubation of target cells with media alone. The inventors’ second set of cytotoxicity experiments will use the Incucyte system to allow us to track cell killing in real time. Target cells labeled with Incucyte CytoLight Rapid Red Reagent will be mixed with effector cells at an E / T ratio of 1 : 1 in the presence of Incucyte Caspase 3 / 7 Green Apoptosis Reagent. Images of each well will be captured in real-time every 2 hours for 48 hours using an Incucyte Live-Cell Imaging System (Sartorius) which assesses the number of apoptotic cells (green) and target cells (red). Cytotoxicity is expressed as the percentage of cells with both green and red signals as the fraction of total red cells.10088] After establishing specific in vitro killing of the inventors’ tumor target cell lines versus controls, the inventors will perform in vivo assays in immune-deficient mice. For these studies the inventors will use the OCIAML (and control) cells labeled with eGFP / Luc. To make these reporter cells, the inventors will infect tumor cells with a recombinant lentiviral vector that the inventors’ lab has produced that engineers dual expression of eGFP and luciferase. Cells will be sorted forAtty. Dkt. No. 650053.01224 eGFP expression by flow cytometry and placed into cell culture at limiting dilutions. Individual clonal populations will be isolated and eGFP+luciferase expression confirmed. Dually labeled cells will be injected into the lateral tail veins of 6-8-week-old NSG mice. This generates a disseminated xenograft mouse model of AML

[0019] , Seven days later mice will be injected intravenously with anti-CLL-1 Grappler-loaded T cells. Various doses of effector cells will be tested. Tumor growth will be measured by bioluminescence imaging using an IVIS Spectrum In Vivo Imaging System. Mice will be monitored and euthanized according to MCW IACUC standards.

[0089] Statistics: All statistical analyses will be performed using Graph-Pad Prism version 10 for Mac. Results from conjugation assays will be compared by one-way ANOVA with Tukey’s multiple comparison test. Luminex cytokine data and T cell cytokine release data will be compared by two-way ANOVA with Dunnett’s multiple comparisons test. Cytotoxicity data will be compared by two-way ANOVA with Tukey’s multiple comparison test. In vivo data will be compared by a log-rank (Mantel-Cox) test.

[0090] The inventors expect that a validated novel humanized cojoined biAb can be used as a key element of a novel immunotherapy submission as an IND to the FDA.Innovation and Impact:

[0091] AML is a devastating disorder. New therapies are needed. The inventors’ approach is 'old- school' in one way (employing monoclonal antibodies against an antigen on the surface of most AML cells) but 'new-school' in other ways (bispecific orientation and ex vivo autologous effector cell loading. No genetic modification used). Indeed, the inventors are bringing the best of these immunotherapy-based technologies together. The inventors are capitalizing on the cellular potency of CAR-T cells (driven by the single-chain antibody on the end of the artificial receptor that binds the tumor antigen) with the great repeatability of the bispecific antibody strategy all while avoiding associated costs, manufacturing delays, and potential toxicities.

[0092] The inventors have engineered a novel 'co-joined' polypeptide that is synthesized effectively, is stable when overexpressed from producer cells, and facilitates the physical association between immune effector T cells and tumor cells. The inventors plan to optimize the binding / dosing of this novel reagent. Then the inventors plan to show that this linkage is stable and leads to effective killing of a variety of tumor cells both in vitro and in vivo. Meanwhile, should the inventors’ additional data warrant, the inventors will generate a clinical protocol in parallel. The inventors believe that their clinical protocol will be simple enough that the 'loaded' effector cells can be infused autologously away from major medical centers in out-patient procedures. The inventors also expect that their approach will reduce costs and arm-to-arm times dramatically. These advances offer the opportunity to improve treatment for AML.Atty. Dkt. No. 650053.01224Example 2 - Treatment of cancer with the disclosed bispecific single-chain constructs

[0093] In one example, a subject suffering from cancer, e.g., acute myeloid leukemia (AML), is administered a therapeutically effective amount of a pharmaceutical composition comprising the disclosed bispecific single-chain constructs. The pharmaceutical composition may suitably be administered by any route that is indicated by the particular treatment needs of the subject, e.g., intravenously. Signs and symptoms of the cancer, e.g., AML, may be reduced by the administration of the pharmaceutical composition. Treatment may be administered daily, every other day, every third day, once a week, once a month, or on a schedule as determined by the patient's progress, pursuant to a physician's decision. It is anticipated that the subject may experience a reduction in signs or symptoms of the cancer as compared to an untreated subject. Methods of measuring reductions in signs and symptoms of cancer are known in the art, e.g., reduction in tumor burden, activation or differentiation of tumor-specific immune cells, e.g., tumor infiltrating lymphocytes (TILs).Example 3 - Treatment of cancer by administration of immune cells pre-treated with the disclosed bispecific single-chain constructs

[0094] In one example, a subject suffering from cancer, e.g., acute myeloid leukemia (AML), is administered a therapeutically effective amount of a pharmaceutical composition comprising cells that have been contacted, or “pre-treated” or “armed,” with the disclosed bispecific single-chain constructs in vitro or ex vivo. The cells may suitably be administered by any route that is indicated by the particular treatment needs of the subject, e.g., intravenously, intratumorally. The cells may be any suitable CD3 -expressing cell, e.g., T cells. The cells may be autologous cells, e.g., autologous T cells. Signs and symptoms of the cancer may be reduced by the administration of the cells. Treatment may be administered daily, every other day, every third day, once a week, once a month, or on a schedule as determined by the patient's progress, pursuant to a physician's decision. It is anticipated that the subject may experience a reduction in signs or symptoms of the cancer as compared to an untreated subject. Methods of measuring reductions in signs and symptoms of cancer are known in the art, e.g., reduction in tumor burden, activation or differentiation of tumorspecific immune cells, e.g., tumor infiltrating lymphocytes (TILs).

[0095] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitablyAtty. Dkt. No. 650053.01224 may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0096] Citations to a number of patent and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.SequencesAtty. Dkt. No. 650053.01224Atty. Dkt. No. 650053.01224Atty. Dkt. No. 650053.01224Atty. Dkt. No. 650053.01224Atty. Dkt. No. 650053.01224Atty. Dkt. No. 650053.01224

Claims

Atty. Dkt. No. 650053.01224CLAIMS1. A bispecific single-chain polypeptide construct comprising an anti-CLL-1 domain and an anti-CD3 domain.

2. The construct of claim I, wherein the anti-CLL-1 domain comprises a first heavy chain and a first light chain and the anti-CD3 domain comprises a second heavy chain and a second light chain.

3. The construct of claim 2. wherein the first heavy chain comprises CDR1, CDR2, and CDR3 and the first light chain comprises CDR4, CDR5, and CDR6, wherein CDR1 comprises SEQ ID NO: 28, CDR2 comprises SEQ ID NO: 29, CDR3 comprises SEQ ID NO: 30, CDR4 comprises SEQ ID NO: 31, CDR5 comprises SEQ ID NO: 32, and CDR6 comprises SEQ ID NO: 33.

4. The construct of claim 1. wherein the anti-CLL-1 domain is SEQ ID NO: 1.

5. The construct of claim 1, wherein the anti-CD3 domain is SEQ ID NO: 2.

6. The construct of claim 2, wherein the first heavy' chain comprises SEQ ID NO: 3.

7. The construct of claim 2. wherein the first light chain comprises SEQ ID NO: 4.

8. The construct of claim 2, wherein the second heavy chain comprises SEQ ID NO: 5.

9. The construct of claim 2, wherein the second light chain comprises SEQ ID NO: 6.

10. The construct of claim 1. further comprising a linker.

11. The construct of claim 10, wherein the linker is a flexible hydrophobic linker.

12. The construct of claim 10, wherein the linker does not comprise a hinge region.

13. The construct of claim 10, wherein the linker comprises SEQ ID NO: 7.Atty. Dkt. No. 650053.0122414. The construct of claim 1, comprising an antibody Fc domain.

15. The construct of claim 14, wherein the Fc domain is an IgG Fc domain.

16. The construct of claim 14, wherein the Fc domain is IgG2 Fc domain.

17. The construct of claim 14, wherein the Fc domain comprises SEQ ID NO: 8.

18. The construct of claim 1. wherein the construct comprises, from N to C terminus, an anti-CLL-1 domain, a linker, an antibody Fc domain, a linker, an anti-CD3 domain.

19. The construct of claim 1, wherein the construct comprises, from N to C terminus, an anti-CD3 domain, a linker, an antibody Fc domain, a linker, an anti-CLL-1 domain.

20. The construct of claim 1. wherein the construct comprises SEQ ID NO: 9.

21. A pharmaceutical composition comprising the construct of claim 1 and a pharmaceutically acceptable carrier or excipient.

22. A method of treating a disease or disorder associated with CLL-1 expression comprising administering an effective amount of the pharmaceutical composition of claim 21 to a subject in need thereof to treat the disease or disorder.

23. The method of claim 22, wherein the disease or disorder is a cell-proliferative disease or disorder.

24. The method of claim 23, wherein the cell proliferative disease or disorder is cancer.

25. The method of claim 23, wherein the disease or disorder is acute myeloid leukemia (AML).

26. The method of claim 22, wherein administering comprises contacting cells ex vivo with the pharmaceutical composition.Atty. Dkt. No. 650053.0122427. The method of claim 26, wherein the method further comprises administering the cells to the subject after contacting.

28. The method of claim 26 or 27, wherein the cells are T cells.

29. The method of claim 28, wherein the T cells comprise CD4+T cells.

30. The method of claim 28, wherein the T cells comprise CD8+T cells.

31. The method of any one of claims 26-30. wherein the cells are derived from the subject.

32. A polynucleotide encoding the construct of claim 1.

33. A cell comprising the polynucleotide of claim 32.

34. A cell comprising the construct of claim 1.

35. The cell of claim 33 or 34, wherein the cell is selected from a bacterial cell, an archaeal cell, a fungal cell, or an animal cell.

36. The cell of claim 35. wherein the cell is a human cell.

37. The cell of claim 36, wherein the human cell is a T cell.

38. A method of making a bispecific single-chain polypeptide construct comprising an anti-CLL-1 domain and an anti-CD3 domain, the method comprising culturing the cell of any one of claims 34-36 and isolating the bispecific single-chain polypeptide construct.

39. A polypeptide comprising any one of SEQ ID NOs: 1, 3, 4, or 28-33.

40. A polypeptide comprising SEQ ID NO: 1.

41. A polypeptide comprising SEQ ID NO: 3.Atty. Dkt. No. 650053.0122442. A polypeptide comprising SEQ ID NO: 4.

43. The polypeptide of any one of claims 39-42. further comprising an antibody constant region, a linker, a detectable marker, a chimeric antigen receptor (CAR), a cellular targeting region, a toxin, or a chemotherapeutic drug.

44. A polynucleotide comprising a sequence encoding any one of SEQ ID NOs: 1, 3, 4, or 28-33.

45. A polynucleotide comprising a sequence encoding SEQ ID NO: 1 .

46. A polynucleotide comprising a sequence encoding SEQ ID NO: 3.

47. A polynucleotide comprising a sequence encoding SEQ ID NO: 4.

48. A cell comprising the polynucleotide of any one of claims 44-47.

49. The cell of claim 48, wherein the cell is a mammalian cell.

50. The cell of claim 49, wherein the cell is a human cell.

51. The cell of claim 50. wherein the cell is a human embryonic kidney (HEK) cell.

52. The cell of claim 49, wherein the cell is a T cell.

53. The cell of claim 52, wherein the T cell is derived from stem cells.

54. The cell of claim 53, wherein the T cell is derived from induced pluripotent stem cells(iPSCs).

55. The polypeptide of claim 43, wherein the cellular targeting region targets T cells, NK cells, or B cells.

56. The polypeptide of claim 43. wherein the toxin comprises brentuximab.

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