Antibodies that bind DACH1 and methods of use

Abstract

Provided herein are DACH1 antibodies or antibody fragments thereof and methods of use.

Description

ANTIBODIES THAT BIND DACH1 AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 555,717, filed on February 20, 2024, the entire disclosure of which is incorporated by reference herein in its entirety for all purposes.BACKGROUND

[0002] Human Dachshund homolog 1 (DACH1) is a nuclear protein that can be recruited to chromatin DNA and associates with DNA-binding transcription factors. DACH1 is a negative regulator of oncogenes and has been shown to be involved in breast cancer metastasis and lung adenocarcinoma tumor growth. DACH1 downregulation is indicative of poor prognosis and tumor progression and some variants and mutations are associated with pro-tumor functions. There are multiple isoforms of DACH1 including DACHla, DACHlb, and DACHlc. The DACHla isoform is known to be a tumor suppressor, inhibits cell proliferation, and DNA synthesis whereas DACHlb and DACHlc do not. Improved therapies are needed for specifically targeting isoforms of DACH1 that are involved in cancer and other disease states.SUMMARY OF THE DISCLOSURE

[0003] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1 (SEQ ID NO: 1), comprising: a) a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs) and has the amino acid sequence as set forth in any one of SEQ ID NOs: 2-7; and b) a light chain variable region (VL) comprising three CDRs and has the amino acid sequence as set forth in any one of SEQ ID NOs: 8-13. In some embodiments, the VH CDRs and the VL CDRs are defined according to AbM; the VH CDRs and the VL CDRs are defined according to Kabat; the VH CDRs and the VL CDRs are defined according to Chothia; or the VH CDRs and the VL CDRs are defined according to IMGT. In some embodiments, the VH has the amino acid sequence according to SEQ ID NO: 2, and the VL has the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the VH has the amino acid sequence according to SEQ ID NO: 3, and the VL has the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the VH has the amino acid sequence according to SEQ ID NO: 4, and the VL has the amino acid sequence according to SEQ ID NO: 10. In some embodiments, the VH has the amino acid sequence according to SEQ ID NO: 5, and the VL has the amino acid sequence according to SEQ ID NO: 11. In some embodiments, the VH has the amino acid sequence according to SEQ ID NO: 6, andthe VL has the amino acid sequence according to SEQ ID NO: 12. In some embodiments, the VH has the amino acid sequence according to SEQ ID NO: 7, and the VL has the amino acid sequence according to SEQ ID NO: 13.

[0004] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to any one of SEQ ID NOs: 14-19, (ii) a CDR2 having an amino acid sequence according to any one of SEQ ID NOs: 20-25, and (iii) a CDR3 having an amino acid sequence according to any one of SEQ ID NOs: 26-31 ; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to any one of SEQ ID NOs: 32-37, (ii) a CDR2 having an amino acid sequence according to any one of SEQ ID NOs: 38-43, and (iii) a CDR3 having an amino acid sequence according to any one of SEQ ID NOs: 44-49. In some embodiments, the VH has an amino acid sequence having at least 80% sequence identity to the sequence of any one of SEQ ID NOs: 2-7, and the VL has an amino acid sequence having at least 80% sequence identity to the sequence of any one of SEQ ID NOs: 8-13. In some embodiments, the VH has an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 2-7, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 8-13. In some embodiments, the VH has an amino acid sequence having 100% sequence identity to the sequence of any one of SEQ ID NOs: 2-7, and the VL has an amino acid sequence having 100% sequence identity to the sequence of any one of SEQ ID NOs: 8-13.

[0005] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 14, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 20, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 26; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 32, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 38, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 44. In some embodiments, the VH has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 2, and the VL has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments, the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 2, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments, the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ IDNO: 2, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 8.

[0006] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 15, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 21, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 27; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 33, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 39, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 45. In some embodiments, the VH has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 3, and the VL has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments, the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 3, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments, the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 3, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 9.

[0007] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 16, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 22, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 28; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 34, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 40, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 46. In some embodiments, the VH has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 4, and the VL has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments, the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 4, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments, the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 4, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 10.

[0008] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 17, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 23, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 29; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 35, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 41, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 47. In some embodiments, the VH has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 5, and the VL has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments, the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 5, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments, the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 5, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 11.

[0009] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 18, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 24, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 30; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 36, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 42, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 48. In some embodiments, the VH has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 6, and the VL has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments, the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 6, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments, the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 6, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 12.

[0010] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 19, (ii) aCDR2 having an amino acid sequence according to SEQ ID NO: 25, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 31 ; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 37, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 43, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 49. In some embodiments, the VH has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 7, and the VL has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments, the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 7, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments, the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 7, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 13.

[0011] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 2-7; and b) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 8-13. In some embodiments, the VH has an amino acid sequence having at least 90% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 2-7, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 8-13. In some embodiments, the VH has an amino acid sequence having 100% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 2-7, and the VL has an amino acid sequence having 100% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 8-13.

[0012] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 2; and b) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments, the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 2, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 8. In some embodiments, the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 2, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 8.

[0013] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 3; and b) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments, the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 3, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 9. In some embodiments, the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 3, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 9.

[0014] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 4; and b) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments, the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 4, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 10. In some embodiments, the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 4, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 10.

[0015] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 5; and b) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 1 1 . In some embodiments, the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 5, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 11. In some embodiments, the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 5, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 11.

[0016] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 6; and b) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments,the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 6, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 12. In some embodiments, the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 6, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 12.

[0017] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 7; and b) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments, the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 7, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 13. In some embodiments, the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 7, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 13.

[0018] Described herein, in certain embodiments, are methods of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the isolated antibody or antigen binding fragment thereof described herein.

[0019] Described herein, in certain embodiments, are methods of inhibiting DACH1 activity in a subject in need thereof, comprising administering to the subject an effective amount of the isolated antibody or antigen binding fragment thereof described herein.

[0020] Described herein, in certain embodiments, are uses of the isolated antibody or antigen binding fragment thereof described herein for treating cancer.

[0021] Described herein, in certain embodiments, are uses of the isolated antibody or antigen binding fragment thereof described herein for inhibiting DACH1 activity.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1A depicts a sequence alignment of human and mouse isoforms of DACH1 sequences. The red box indicates the peptide sequence used for generation of DACH1 monoclonal antibodies. The anti-DACHl Rabbit Monoclonal Antibody Peptide Sequence is: ERVPDSPSPAPSLEC (NP_542937.3, dachshund homolog 1 isoform a [Homo sapiens], aa434- 447) (An extra "C" is added to the C terminus to facilitate conjugation).

[0023] FIG. IB depicts a schematic representation of the DACH1 gene and its isoforms and current commercial antibodies. DACHld (V4), Dachla (VI), DACHlb (V2) and DACHlc (V3) are shown. The amino acid residues numbering is shown. DS domain is the Dach N-BOX(the dac and ski / sno DS domain). * at DACHla Ser439indicates the epitope used to derive the monoclonal antibodies 1, 3, and 2 (18A2, 15G11, and 17E12, respectively) and DACHlSer439Pthe epitope for the polyclonal antibody to DACH1 Ser439P (ZP1 ). Commercial antibodies #7 and #8 are indicated.

[0024] FIG. 1C depicts relative levels of gene expression of DACH1 isoforms in prostate cancer from The Cancer Genome Atlas (TCGA) shown as Log2 TPM (transcripts per million).

[0025] FIG. 2 depicts a schematic of antibody generation and purification from rabbits immunized with a DACH1 peptide.

[0026] FIG. 3A depicts Western blots of lysates of HEK293T cells transiently transfected with FLAG-tagged DACHla (wild type; WT). Antibodies used to probe the blot are indicated. Bands are observed at the expected molecular weight of DACH1 (-100 kDa).

[0027] FIG. 3B depicts Western blots of lysates of HEK293T cells transiently transfected with FLAG-tagged DACHlc isoform. Antibodies used to probe the blot are indicated. With the exception of the commercial control antibody and anti-FLAG antibody, bands are not observed at the expected molecular weight of DACH1 (-100 kDa).

[0028] FIG. 4A and 4B depict DACHla isoform nuclear subcellular distribution in cultured cells. FIG. 4A shows a schematic representation of the experimental protocol. FIG. 4B shows immunofluorescent and corresponding phase contrast images of LNCaP cells transfected with DACH1 mutant GFP fusion expression vectors. The arrows indicate the nuclear GFP signal in DACHla transfected cells and cytoplasmic GFP signal for DACHlb, DACHlc and DACHlaS439Amutant.

[0029] FIG. 5 depicts FLAG immunohistochemistry (IHC) of FLAG tagged DACHla wild type, DACH1 isoforms or DACHlaS439Amutant. HEK293T cells or DU-145 cells (lower panels) were transiently transfected with, vector control, FLAG tagged DACHla, other DACH1 isoforms or DACHl aS439Amutant. THC to FLAG tag shows positive nuclear staining (brown) in transfected cells.

[0030] FIG. 6 depicts DACH1 immunohistochemistry (IHC) of FLAG tagged DACHla wild type, DACH1 isoforms or DACHlaS439Amutant with DACHla monoclonal antibody 18A2. HEK293T cells or DU- 145 cells (lower panels) were transiently transfected with, vector control, FLAG tagged DACHla, other DACH1 isoforms or DACHlaS439Amutant. IHC to DACH1 shows positive nuclear staining (brown) in DACHla, d, and in some aS493A transfected cells.

[0031] FIG. 7 depicts DACH1 immunohistochemistry (IHC) of FLAG tagged DACHla wild type, DACH1 isoforms or DACHlaS439Amutant DACHla monoclonal antibody 15G11.HEK293T cells or DU- 145 cells (lower panels) were transiently transfected with, vector control,FLAG tagged DACHla, other DACH1 isoforms or DACHlaS439Amutant. IHC to DACH1 shows positive nuclear staining (brown) in DACHla and d transfected cells.

[0032] FIG. 8 depicts DACH1 immunohistochemistry (IHC) of FLAG tagged DACHla wild type, DACH1 isoforms or DACHlaS439Amutant DACHla monoclonal antibody 17E12. HEK293T cells or DU- 145 cells (lower panels) were transiently transfected with, vector control, FLAG tagged DACHla, other DACH1 isoforms or DACHlaS439Amutant. IHC to DACH1 shows positive nuclear staining (brown) in DACHla d, and in aS493A transfected cells.

[0033] FIG. 9 depicts DACH1 immunohistochemistry (IHC) of FLAG tagged DACHla wild type, DACH1 isoforms or DACHlaS439Amutant DACHla polyclonal antibody ZP1.HEK293T cells or DU- 145 cells (lower panels) were transiently transfected with, vector control, FLAG tagged DACHla, other DACH1 isoforms or DACHlaS439Amutant. IHC to DACH1 shows positive nuclear staining (brown) in DACHla and d transfected cells.

[0034] FIG. 10 depicts DACH1 Immunohistochemistry (IHC) of murine kidney with monoclonal antibodies (18A2, 17E12, 15G11), with DACHla polyclonal antibody (ZP1) and commercial antibody #7. Brown nuclear staining of the kidney glomerular cells is seen with commercial antibody #7, 18A2, 17E12, and ZP1.

[0035] FIG. 11 depicts DACH1 Immunohistochemistry (IHC) of human prostate cancer cells with monoclonal antibodies (18A2, 17E12, 15G11), with DACHla polyclonal antibody (ZP1) and commercial antibody #7. Brown nuclear staining of the kidney glomerular cells is seen with commercial antibody #7, 18A2, 15G11, and ZP1.

[0036] FIG. 12 depicts a summary chart of DACH1 detection by commercial antibodies, ZP1 antibody, and the isolated antibodies or antigen binding fragments thereof that bind to DACH1 described herein.

[0037] FIGs. 13A-13C depict sequences of rabbit monoclonal DACH1 antibodies 18A2-1 (FIG. 13A), 17E12-1 (FIG. 13B), and 15G1 1 -2 (FIG. 13C).

[0038] FIG. 14 depicts alignments of human DACH1 protein and mouse DACH1 protein.The anti-DACHl Rabbit Monoclonal Antibody Peptide Sequence for generation of antibodies is ERVPDSPSPAPSLEC (NP_542937.3, dachshund homolog 1 isoform a [Homo sapiens], aa434- 447) (indicated by the red box and including an extra "C" that is added to the C terminus to facilitate conjugation).

[0039] FIG. 15 depicts Western blots of DACHla, DACHlb, DACHlc, DACHld and vector control using rabbit monoclonal DACH1 (Clone 18A2-1), a rabbit polyclonal DACH1 commercial antibody, and GAPDH rabbit polyclonal antibody as a loading control.

[0040] FIG. 16 depicts Western blots of vector control, DACH1 WT, DACHla, DACHlb, DACHlc, DACHld, DACH1 L22R and DACH1 L276R using rabbit monoclonal DACH1(Clone 18A2-1), rabbit polyclonal DACH1 commercial antibody, and GAPDH rabbit polyclonal antibody as a loading control.

[0041] FIGs. 17A-17C depict staining of U2OS cells expressing FLAG-DACH1 or pLRT vector control. FIG. 17A depicts the experimental schema for expression of DACH1 induced by doxycycline and H2O2 treatment. FIG. 17B and FIG. 17C depict detection of DACH1 in control cells that are not treated with H2O2 by rabbit monoclonal DACH1 (Clone 18A2-1) antibody in pLRT vector control- and FLAG-DACH1 -expressing cells, respectively,. The nuclei are stained with DAPI.

[0042] FIGs. 18A-18C depict staining of U2OS cells expressing FLAG-DACH1 or pLRT vector control. FIG. 18A depicts the experimental schema for expression of DACH1 induced by doxycycline and H2O2 treatment. FIG. 18B and FIG. 18C depict detection of DACH1 in cells that have been treated with H2O2by rabbit monoclonal DACH1 (Clone 18A2-1) antibody in pLRT vector control and FLAG-DACH1 expressing cells, respectively. The nuclei are stained with DAPI.DETAILED DESCRIPTION

[0043] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0044] As used herein, unless otherwise indicated, the term “antibody” is understood to mean an intact antibody (e.g., an intact monoclonal antibody), or a fragment thereof, such as a Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody), or an antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), including an intact antibody, antigen-binding fragment, or Fc fragment that has been modified, engineered, or chemically conjugated. In general, antibodies are multimeric proteins that contain four polypeptide chains. Two of the polypeptide chains are called immunoglobulin heavy chains (H chains), and two of the polypeptide chains are called immunoglobulin light chains (L chains). The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by interchain disulfide bonds. A light chain consists of one variable region (VL) and one constant region (CL). The heavy chain consists of one variable region (VH) and at least three constant regions (CHI, CH2 and CH3). The variable regions determine the binding specificity of the antibody. Each variable region contains three hypervariable regions known as complementarity determining regions (CDRs) flanked by four relatively conserved regions known as framework regions (FRs). The extent of the FRs and CDRs has been defined (Kabat, E.A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). The three CDRs, referred to as CDR1, CDR2, and CDR3, contribute to the antibody binding specificity. Naturally occurring antibodies have been used as starting material for engineered antibodies, such as chimeric antibodies and humanized antibodies. Examples of antibody-based antigen-binding fragments include Fab, Fab’, (Fab’)2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies. Examples of antibodies that have been modified or engineered include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.

[0045] The terms “variable domain” and “variable region” are used interchangeably and refer to the portions of the antibody or immunoglobulin domains that exhibit variability in their sequence and that are involved in determining the specificity and binding affinity of a particular antibody. Variability is not evenly distributed throughout the variable domains of antibodies; it is concentrated in sub-domains of each of the heavy and light chain variable regions. These subdomains are called “hypervariable regions” or “complementarity determining regions” (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domains are called the “framework” regions (FRM or FR) and provide a scaffold for the six CDRs in three-dimensional space to form an antigen-binding surface.

[0046] The terms “recipient”, “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and in some embodiments, refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc. In some embodiments, the mammal is human. None of these terms require the supervision of medical personnel.

[0047] As used herein, the term “effective amount” refers to the amount of a compound (e.g., a compound of the present disclosure) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. As used herein, the term “treating” includes any effect, e.g., lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof.

[0048] As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0049] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0050] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0051] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000-fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5, fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth.

[0052] “About” a number, as used herein, refers to range including the number and ranging from 10% below that number to 10% above that number. “About” a range refers to 10% below the lower limit of the range, spanning to 10% above the upper limit of the range.

[0053] ‘Percent (%) identity” refers to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y. Generally, computer programs are employed for such calculations. Exemplary programs that compare and align pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990) and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0054] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.

[0055] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.DACH1 Antibodies or Antigen Binding Fragments Thereof

[0056] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that bind to DACH1 (SEQ ID NO: 1), comprising: a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs) and has the amino acid sequence as set forth in any one of SEQ ID NOs: 2-7; and a light chain variable region (VL) comprising three CDRs and has the amino acid sequence as set forth in any one of SEQ ID NOs: 8-13.

[0057] In some embodiments, the DACH1 comprises a sequence at least at least 60% (e.g., at least 70%, at least 80%, at least 85%, 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%, at least 99%, or 100%) identical to SEQ ID NO: 1. In some embodiments, the DACH1 comprises a sequence at least at least 70% identical to SEQ ID NO: 1. In some embodiments, the DACH1 comprises a sequence at least at least 80% identical to SEQ ID NO: 1. In some embodiments, the DACH1 comprises a sequence at least at least 85% identical to SEQ ID NO: 1. In some embodiments, the DACH1 comprises a sequence at least at least 90% identical to SEQ ID NO: 1. In some embodiments, the DACH1 comprises a sequence at least at least 70% identical to SEQ ID NO: 1. In some embodiments, the DACH1 comprises a sequence at least at least 91% identical to SEQ ID NO: 1. In some embodiments, the DACH1 comprises a sequence at least at least 92% identical to SEQ ID NO: 1. In some embodiments, the DACH1 comprises a sequence at least at least 93% identical to SEQ ID NO: I. In some embodiments, the DACH1 comprises a sequence at least at least 94% identical to SEQ ID NO: 1 . In some embodiments, the DACH1 comprises a sequence at least at least 95% identical to SEQ ID NO: 1. In some embodiments, the DACH1 comprises a sequence at least at least 96% identical to SEQ ID NO: 1. In some embodiments, the DACH1 comprises a sequence at least at least 97% identical to SEQ ID NO: 1. In some embodiments, the DACH1 comprises a sequence at least at least 98% identical to SEQ ID NO: 1. In some embodiments, the DACH1 comprises a sequence at least at least 99% identical to SEQ ID NO: 1. In some embodiments, the DACH1 comprises a sequence identical to SEQ ID NO: 1.MAVPAALIPPTQLVPPQPPISTSASSSGTTTSTSSATSSPAPSIGPPASSGPTL FRPEP1ASAAAAAATVTSTGGGGGGGGSGGGGGSSGNGGGGGGGGGGS NCNPNLAAASNGSGGGGGGISAGGGVASSTPINASTGSSSSSSSSSSSSSSS SSSSSSSSSCGPLPGKPVYSTPSPVENTPQNNECKMVDLRGAKVASFTVEGCELICLPQAFDLFLKHLVGGLHTVYTKLKRLEITPVVCNVEQVRILRGLGA IQPGVNRCKLISRKDFETLYNDCTNASSRPGRPPKRTQSVTSPENSHIMPHS VPGLMSPGIIPPTGLTAAAAAAAAATNAAIAEAMKVKKIKLEAMSNYHAS NNQHGADSENGDMNSSVGLELPFMMMPHPLIPVSLPPASVTMAMSQMN HLSTIANMAAAAQVQSPPSRVETSVIKERVPDSPSPAPSLEEGRRPGSHPSS HRSSSVSSSPARTESSSDRIPVHQNGLSMNQMLMGLSPNVLPGPKEGDLA GHDMGHESKRMHIEKDETPLSTPTARDSLDKLSLTGHGQPLPPGFPSPFLF PDGLSSIETLLTNIQGLLKVAIDNARAQEKQVQLEKTELKMDFLRERELRE TLEKQLAMEQKNRAIVQKRLKKEKKAKRKLQEALEFETKRREQAEQTLK QAASTDSLRVLNDSLTPEIEADRSGGRTDAERTIQDGRLYLKTTVMY (SEQ ID NO: 1)

[0058] In some embodiments, the DACH1 is DACH1 isoform DACHla. In some embodiments, the isolated antibodies or antigen binding fragments thereof that bind to DACH1 bind to a region of DACH1 found in isoform DACHla and not in DACHlb or DACHlc. In some embodiments, the region of DACH1 found in isoform DACHla and not in DACHlb or DACHlc contains the amino acid sequence according to SEQ ID NO: 50 (ERVPDSPSPAPSLEC). In some embodiments, the region of DACH1 found in isoform DACHla and not in DACHlb or DACHlc is important in restraining epithelial to mesenchymal transition (EMT). In some embodiments, the isolated antibodies or antigen binding fragments thereof that bind to DACH1 do not bind to a region of the c-terminus of DACH1 common to isoforms DACHla, DACHlb, and DACHlc.

[0059] In some embodiments, the VH CDRs and the VL CDRs are defined according to AbM; the VH CDRs and the VL CDRs are defined according to Kabat; the VH CDRs and the VL CDRs are defined according to Chothia; or the VH CDRs and the VL CDRs are defined according to IM GT.

[0060] In some embodiments, the VH has the amino acid sequence according to SEQ ID NO: 2, and the VL has the amino acid sequence according to SEQ ID NO: 8.

[0061] In some embodiments, are isolated antibodies or antigen binding fragments thereof that bind to DACH1, wherein the VH has the amino acid sequence according to SEQ ID NO: 3, and the VL has the amino acid sequence according to SEQ ID NO: 9.

[0062] In some embodiments, are isolated antibodies or antigen binding fragments thereof that bind to DACH1, wherein the VH has the amino acid sequence according to SEQ ID NO: 4, and the VL has the amino acid sequence according to SEQ ID NO: 10.

[0063] In some embodiments, are isolated antibodies or antigen binding fragments thereof that bind to DACH1, wherein the VH has the amino acid sequence according to SEQ ID NO: 5, and the VL has the amino acid sequence according to SEQ ID NO: 11.

[0064] In some embodiments, are isolated antibodies or antigen binding fragments thereof that bind to DACH1, wherein the VH has the amino acid sequence according to SEQ ID NO: 6, and the VL has the amino acid sequence according to SEQ ID NO: 12.

[0065] In some embodiments, are isolated antibodies or antigen binding fragments thereof that bind to DACH1, wherein the VH has the amino acid sequence according to SEQ ID NO: 7, and the VL has the amino acid sequence according to SEQ ID NO: 13.

[0066] In some embodiments, are isolated antibodies or antigen binding fragments thereof that bind to DACH1, comprising a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to any one of SEQ ID NOs: 14-19, (ii) a CDR2 having an amino acid sequence according to any one of SEQ ID NOs: 20-25, and (iii) a CDR3 having an amino acid sequence according to any one of SEQ ID NOs: 26-31; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to any one of SEQ ID NOs: 32-37, (ii) a CDR2 having an amino acid sequence according to any one of SEQ ID NOs: 38-43, and (iii) a CDR3 having an amino acid sequence according to any one of SEQ ID NOs: 44-49.

[0067] Amino acid sequences of exemplary CDRs of DACH1 antibodies or antigen binding fragments thereof are provided in Table 1.Table 1. Sequences of CDRs of Antibody 1-6

[0068] Described herein, in certain embodiments, are isolated antibodies or antigen binding fragments thereof that bind to DACH1 comprising a heavy chain variable region comprising a CDR1, CDR2, and CDR3 as listed in Table 1. In some embodiments, the isolated antibodies or antigen binding fragments thereof that bind to DACH1 comprise a heavy chain variable region comprising (i) a CDR1 having an amino acid sequence according to any one of SEQ ID NOs: 14-19, (ii) a CDR2 having an amino acid sequence according to any one of SEQ ID NOs: 20-25, and (iii) a CDR3 having an amino acid sequence according to any one of SEQ ID NOs: 26-31.

[0069] In some embodiments, the isolated antibodies or antigen binding fragments thereof that bind to DACH1 comprise a light chain variable region comprising a CDR1, CDR2, and CDR3 as listed in Table 1. In some embodiments, the isolated antibodies or antigen binding fragments thereof that bind to DACH1 comprise a light chain variable region comprising (i) a CDR1 having an amino acid sequence according to any one of SEQ ID NOs: 32-37, (ii) a CDR2 having an amino acid sequence according to any one of SEQ ID NOs: 38-43, and (iii) a CDR3 having an amino acid sequence according to any one of SEQ ID NOs: 44-49.

[0070] In some embodiments, the isolated antibodies or antigen binding fragments thereof that bind to DACH1 comprise a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to any one of SEQ ID NOs: 14-19, (ii) a CDR2 having an amino acid sequence according to any one of SEQ ID NOs: 20-25, and (iii) a CDR3 having an amino acid sequence according to any one of SEQ ID NOs: 26-31; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to any one of SEQ ID NOs: 32-37, (ii) a CDR2 having an amino acid sequence according to any one of SEQ ID NOs: 38-43, and (iii) a CDR3 having an amino acid sequence according to any one of SEQ ID NOs: 44-49.

[0071] In some embodiments, the isolated antibodies or antigen binding fragments thereof that bind to DACH1 comprise a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 14, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 20, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 26; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 32, (ii) a CDR2 having an amino acidsequence according to SEQ ID NO: 38, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 44.

[0072] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 15, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 21, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 27; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 33, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 39, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 45.

[0073] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 16, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 22, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 28; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 34, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 40, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 46.

[0074] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 17, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 23, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 29; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 35, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 41, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 47.

[0075] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 18, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 24, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 30; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 36, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 42, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 48.

[0076] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 19, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 25, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 31 ; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 37, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 43, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 49.

[0077] The amino acid sequence boundaries of a CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including those described by Kabat et al., supra (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); Martin (Enhanced Chothia or AbM) Abhinandan and Martin, Mol Immunol. 2008 Aug;45(14):3832-9; MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegger and Pliickthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme); each of which is incorporated by reference in its entirety.

[0078] Table 2 provides the positions of CDR-L1 , CDR-L2, CDR-L3, CDR-H1 , CDR-H2, and CDR-H3 as identified by the Kabat, Chothia, AbM, Contact, and IMGT schemes. For CDR- Hl, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0079] CDRs may be assigned, for example, using antibody numbering software, such as Abnum, available at bioinf.org.uk / abs / abnum / , and described in Abhinandan and Martin, Immunology, 2008, 45:3832-3839, incorporated by reference in its entirety. Descriptions of the various antibody numbering schemes are available at bioinf.org.uk / abs / info.html.Table 2. Residues in CDRs according to the indicated numbering schemes.* The C-terminus of CDR-H1, when numbered using the Kabat numbering convention, varies between H32 and H34, depending on the length of the CDR.

[0080] The “EU numbering scheme” is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al., supra). Unless stated otherwise, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

[0081] Amino acid sequences of exemplary heavy chain variable regions (VH) and light chain variable regions (VL) of DACH1 antibodies or antigen binding fragments thereof are provided in Table 3. Table 3. Sequences of heavy chain variable regions (VH) and light chain variable regions(VL) of DACH1 antibodies or antigen binding fragments thereof

[0082] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: SEQ ID NOs: 2-7. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region (VH) comprising an amino acid sequence according to any one of SEQ ID NOs: 2-7.

[0083] In some embodiments, the N-terminal glutamine (Q) is replaced with an N-terminal glutamic acid (E) residue. This N-terminal “Q” residue can be in the VH or the VL of the antibody. In some embodiments, the N-terminal glutamine (Q) of the VH comprising an amino acid sequence according to any one of SEQ ID NOs: 2-7 is replaced with an N-terminal glutamic acid (E) residue. In some embodiments, the VH and VL sequences of the antibodies provided here can be expressed with a signal sequence, which can be used to facilitate the expression of the antibody. The signal sequence can be cleaved during the processing of the antibody sequences by the cell from which the antibody is produced. These sequences are common and any appropriate sequence can be used to facilitate the expression.

[0084] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a light chain variable region (VL) comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to D ACH 1 comprises a light chain variable region (VL) comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH 1 comprises a light chain variable region (VL) comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a light chain variable region (VL) comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a light chain variable region (VL) comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a light chain variable region (VL) comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a light chain variable region (VL) comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen bindingfragment thereof that binds to DACH1 comprises a light chain variable region (VL) comprising an amino acid sequence according to any one of SEQ ID NOs: 8-13.

[0085] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region (VH) that comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, 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%, at least 99%, or 100%) identical to the heavy chain variable region (VH) of an DACH1 antibody or antigen binding fragment thereof disclosed in Table 3, and a light chain variable region (VL) that comprises an amino acid sequence at least 60% (e.g., at least 70%, at least 80%, at least 85%, 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%, at least 99%, or 100%) identical to the light chain variable region (VL) of the same the isolated antibody or antigen binding fragment thereof that binds to DACH1 disclosed in Table 3.

[0086] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7; and a light chain variable region comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH 1 comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7; and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7 ; and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7; and a light chain variable region comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds toDACH1 comprises a heavy chain variable region comprising an amino acid sequence having atleast 96% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7 ; and a light chain variable region comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7; and a light chain variable region comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7; and a light chain variable region comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to any one of SEQ ID NOs: 2-7 ; and a light chain variable region comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according any one of SEQ ID NOs: 8-13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence according to any one of SEQ ID NOs: 2-7; and a light chain variable region comprising an amino acid sequence according to any one of SEQ ID NOs: 8-13.

[0087] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 2; and a light chain variable region comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 8. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 2; and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 8. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH 1 comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 2; and a light chain variable regioncomprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 8. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 2; and a light chain variable region comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 8. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 2; and a light chain variable region comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 8. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 2; and a light chain variable region comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 8. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 2; and a light chain variable region comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 8. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to SEQ ID NO: 2; and a light chain variable region comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according SEQ ID NO: 8. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 2; and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 8.

[0088] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 3; and a light chain variable region comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 3; and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 3; and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 3; and a light chain variable region comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 3; and a light chain variable region comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 3; and a light chain variable region comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 3; and a light chain variable region comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to SEQ ID NO: 3; and a light chain variable region comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according SEQ ID NO: 9. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acidsequence according to SEQ ID NO: 3; and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 9.

[0089] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 4; and a light chain variable region comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 10. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 4; and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 10. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 4; and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 10. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 4; and a light chain variable region comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 10. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 4; and a light chain variable region comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 10. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 4; and a light chain variable region comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 10. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 4; and a light chain variable region comprising an amino acid sequence having at least 98% sequence identity with an amino acidsequence according to SEQ ID NO: 10. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to SEQ ID NO: 4; and a light chain variable region comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according SEQ ID NO: 10. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 4; and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 10.

[0090] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 5; and a light chain variable region comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 11. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 5; and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 11. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH 1 comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 5; and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 11. In some embodiments, the isolated antibody or antigen binding fragment thereof that hinds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 5; and a light chain variable region comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 11. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 5; and a light chain variable region comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 11. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 97%sequence identity with an amino acid sequence according to SEQ ID NO: 5; and a light chain variable region comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 11. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 5; and a light chain variable region comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 11. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to SEQ ID NO: 5; and a light chain variable region comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according SEQ ID NO: 11. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 5; and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 11.

[0091] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 6; and a light chain variable region comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 12. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 6; and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 12. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 6; and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 12. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 6; and a light chain variable region comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according toSEQ ID NO: 12. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 6; and a light chain variable region comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 12. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 6; and a light chain variable region comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 12. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH 1 comprises a heavy chain variable region comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 6; and a light chain variable region comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 12. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to SEQ ID NO: 6; and a light chain variable region comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according SEQ ID NO: 12. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 6; and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 12.

[0092] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 13. In some embodimentsthe isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence having at least 85% sequence identity with an amino acid sequence according to SEQ ID NO: 13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity withan amino acid sequence according to SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence according to SEQ ID NO: 13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence having at least 95% sequence identity with an amino acid sequence according to SEQ ID NO: 13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence having at least 96% sequence identity with an amino acid sequence according to SEQ ID NO: 13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence having at least 97% sequence identity with an amino acid sequence according to SEQ ID NO: 13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence having at least 98% sequence identity with an amino acid sequence according to SEQ ID NO: 13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according to SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence having at least 99% sequence identity with an amino acid sequence according SEQ ID NO: 13. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a heavy chain variable region comprising an amino acid sequence according to SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence according to SEQ ID NO: 13.

[0093] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 with a KD lower than or equal to 10 nanomolar (nM), 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1with a KD within the range of about 10 pM - about 1 nM, about 10 pM - about 0.9 nM, about 10 pM - about 0.8 nM, about 10 pM - about 0.7 nM, about 10 pM - about 0.6 nM, about 10 pM - about 0.5 nM, about 10 pM - about 0.4 nM, about 10 pM - about 0.3 nM, about 10 pM - about 0.2 nM, about 10 pM - about 0.1 nM, about 10 pM - about 50 pM, 0.1 nM - about 10 nM, about 0.1 nM - about 9 nM, about 0.1 nM - about 8 nM, about 0. 1 nM - about 7 nM, about 0.1 nM - about 6 nM, about 0.1 nM - about 5 nM, about 0. 1 nM - about 4 nM, about 0. 1 nM - about 3 nM, about 0.1 nM - about 2 nM, about 0.1 nM - about 1 nM, or about 0.1 nM - about 0.5 nM. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 with a Ko less than about 1 nanomolar (nM). In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 with a KD less than about 0.9 nanomolar (nM). In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 with a Ko less than about 0.8 nanomolar (nM). In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 with a Ko less than about 0.7 nanomolar (nM). In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 with a KD less than about 0.6 nanomolar (nM). In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 with a Ko less than about 0.5 nanomolar (nM). In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 with a KD less than about 0.4 nanomolar (nM). In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 binds DACH1 with a Ko less than about 0.3 nanomolar (nM). In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 with a Ko less than about 0.2 nanomolar (nM). In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 with a KD less than about 0.1 nanomolar (nM). In some embodiments, the KD is measured by surface plasmon resonance (SPR). In some embodiments, the KD is measured by Biolayer Interferometry (BLI).

[0094] In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a binding affinity to DACH1 at least 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, or 20 fold more than a binding affinity of a comparator antibody to DACH1. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a binding affinity to DACH1 at least 1.5 fold more than a binding affinity of a comparator antibody to DACH1. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a binding affinity to DACH1 at least 2 fold more than a binding affinity of a comparator antibody to DACH1. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a binding affinity to DACH1 at least 3 fold more than a bindingaffinity of a comparator antibody to DACH1. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a binding affinity to DACH1 at least 4 fold more than a binding affinity of a comparator antibody to DACH1. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a binding affinity to DACH1 at least 5 fold more than a binding affinity of a comparator antibody to DACH1. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a binding affinity to DACH1 at least 6 fold more than a binding affinity of a comparator antibody to DACH1 . In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a binding affinity to DACH1 at least 7 fold more than a binding affinity of a comparator antibody to DACH1. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a binding affinity to DACH1 at least 8 fold more than a binding affinity of a comparator antibody to DACH1. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a binding affinity to DACH1 at least 9 fold more than a binding affinity of a comparator antibody to DACH1. In some embodiments, the isolated antibody or antigen binding fragment thereof that binds to DACH1 comprises a binding affinity to DACH1 at least 10 fold more than a binding affinity of a comparator antibody to DACH1.

[0095] Further described herein, in certain embodiments, are methods of treating a cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of an isolated antibody or antigen binding fragment thereof that binds to DACH1 comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to any one of SEQ ID NOs: 14-19, (ii) a CDR2 having an amino acid sequence according to any one of SEQ ID NOs: 20-25, and (iii) a CDR3 having an amino acid sequence according to any one of SEQ ID NOs: 26-31 and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to any one of SEQ ID NOs: 32-37, (ii) a CDR2 having an amino acid sequence according to any one of SEQ ID NOs: 38-43, and (iii) a CDR3 having an amino acid sequence according to any one of SEQ ID NOs: 44-49.

[0096] The antibodies provided herein can also be used in methods and kits for diagnosing and treating cancers. Examples of such methods can be found, for example, in PCT Publication No. W02022087023, which is hereby incorporated by reference in its entirety.

[0097] In some embodiments, methods for aiding a subject suffering from cancer are provided. In some embodiments, the methods comprise determining a sensitivity of one or more cancer cells by administering an immunohistochemical stain configured to determine an abundance of DACH1 gene. In some embodiments, determining the sensitivity of the one ormore cancer cells comprises the step of determining a deletion of the DACH1 gene in the one or more cancer cells. In some embodiments, the immunohistochemical stain is an antibody configured to target DACH1 gene, a mRNA thereof, or a DACH1 protein. In some embodiments, the antibody is configured to target the DACH1 gene or the DACH1 protein.

[0098] In some embodiments, the immunohistochemical stain comprises a marker selected from nestin, p3-tubulin, vimentin, rhodopsin, Ki-67, PKC-a marker, GDNF, GATA6, GFAP, and a combination of two or more thereof. In some embodiments, the immunohistochemical stain comprises a DACH1 polyclonal antibody, a DACH1 monoclonal antibody, or a combination thereof. In some embodiments, the immunohistochemical stain comprises propidium iodide and an anti-BrdU antibody.

[0099] In some embodiments, the methods further comprise providing a DNA-PK inhibitor, a WEE1 inhibitor, a prodrug thereof, a salt thereof, or a combination of two or more thereof to the subject. In some embodiments, the method further comprises administering a therapeutically effective amount of a composition comprising DNA-PK inhibitor, a WEE1 inhibitor, a prodrug thereof, a salt thereof, or a combination of two or more thereof or administering a composition comprising a PARP inhibitor, a prodrug thereof, a salt thereof, or a combination of two or more thereof based on the determined sensitivity of the one or more cancer cells.

[0100] In some embodiments, the WEE1 inhibitor is selected from AZD1775 (MK1775), 2- allyl-l-[6-(l-hydroxy-l-methylethyl)pyridin-2-yl]-6- { [4-(4- methylpiperazin-l-yl)phenyl]aminol- l,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one, 3-(2,6- dichlorophenyl)-4-imino-7-[(2'-methyl- 2',3'-dihydro-rH-spiro[cyclopropane-l,4'-isoquinolin]- 7'-yl)amino]-3,4-dihydropyrimido[4,5- d]pyrimidin-2(lH)-one, and a combination of two or more thereof. The structures of such compounds can be found, for example, in the referenced PCT publication above. In some embodiments, the DNA-PK inhibitor is AZD7648, which is also described in PCT Publication No. W02022087023, which is hereby incorporated by reference in its entire. In some embodiments, the method does not include administering a PARP inhibitor.

[0101] In some embodiments, the cancer cell is a human prostate cancer cell, a human lung cancer cell, or a human breast cancer cell. In some embodiments, the cancer cell is an ovarian cancer cell, brain cancer cell, kidney cancer cell, esophogeal cancer cell, or any other type of cancer cell. In some embodiments, the cancer cell is a human prostate cancer cell.

[0102] In some embodiments, methods for aiding a human suffering from cancer are provided, wherein the methods comprise: (a) administering, to a human having one or more cancer cells, an immunohistochemical stain configured target to a DACH1 gene or a DACH1 protein; (b) determining a deletion of the DACH1 gene in the one or more cancer cells; and (c) determining a sensitivity of the one or more cancer cells based on the determination of thedeletion of the DACH1 gene from the one or more cancer cells. In some embodiments, the method further comprises (d) administering a composition comprising a DNA-PK inhibitor, a WEE1 inhibitor, a prodrug thereof, a salt thereof, or a combination thereof. In some embodiments, the composition administered in such methods is free of or substantially free of a PARP inhibitor. In some embodiments, the composition further comprises a DNA- targeted agent. In some embodiments, the DNA-targeted agent comprises a DNA alkylating agents, a topoisomerase inhibitor, or a combination of two or more thereof. In some embodiments, the DNA-targeted agent is selected from cisplatin, capecitabine, carboplatin, cyclophosphamide, cytarabine, dauoribicin, docetaxel, doxorubicin, 5- fluorouracil, gemcitabine, methotrexate, paclitaxel, premetrexed, irinotecan temozolomide, topotecan, radiation, and a combination of two or more thereof.

[0103] In some embodiments, the sensitivity is determined by the presence of absence of DACH1, which can be measured by utilizing the antibodies provided for herein. In some embodiments, the antibodies are directly labeled so that the antibody is detected directly, or the antibody binding to DACH1 is detected by an “indirect” method of detection, such as an ELISA or other immunoabsorbant assay. Methods of labeling antibodies are known in the art and are routine.

[0104] In some embodiments, methods of detecting DACH1 in a biological sample are provided. In some embodiments, the methods comprise contacting the sample with the isolated antibody or antigen binding fragment thereof as provided for herein. In some embodiments, as provided for herein, the antibody is a labeled antibody, such as with a fluorophore. In some embodiments, the biological sample is a tumor cell sample, such as those exemplified herein.

[0105] In some embodiments, methods of determining the sensitivity of a subject with cancer to a therapeutic treatment are provided. The sensitivity, can for example, be determined by the presence of DACH1 . Thus, in some embodiments, the methods comprise contacting a biological sample obtained from the subject and detecting the presence of DACH1 by contacting the sample with the isolated antibody or antigen binding fragment thereof, as provided for herein. In some embodiments, if DACH1 is detected, the subject is determined to have a cancer that is sensitive to a therapeutic treatment. In some embodiments, the methods further comprise administering the therapeutic treatment to the subject who is determined to be sensitive to the therapeutic treatment. In some embodiments, the antibody, or antigen binding fragment thereof, is labeled with a fluorophore.

[0106] The antibodies provided for herein that can be used with the presently provided methods or other methods of detection of DACH1 can be labeled with any means or molecule that can be used to detect the presence of the antibody bound to DACH1. Fluorophores are justone example. Other examples include, biotin, streptavidin, radioactive tags, and the like. The moiety used to detect the antibody can be any method known in the art, which can also use a secondary reagent to detect the antibody, such as what is done in an ELISA.

[0107] In some embodiments, kits are provided to perform such methods. The kits can contain reagents and / or instructions to perform such methods. The reagants can include, for example, the antibodies provided for herein, which can be already labeled. Alternatively, the kit can include reagents or instructions on how to label the antibody so that it can utilized in the methods provided for herein. In some embodiments, the antibodies provided herein that bind to DACH1 are not labeled.

[0108] Accordingly, in some embodiments, a kit is provided, wherein the kit comprises (a) an immunohistochemical stain configured target to a DACH1 gene or a DACH1 protein; and (b) instructions for determining a deletion of the DACH1 gene in the one or more cancer cells, and determining a sensitivity of the one or more cancer cells based on the determination of the deletion of the DACH1 gene from the one or more cancer cells. In some embodiments, the kit may comprises an additional therapeutic agent, such as, but not limited to, a DNA-PK inhibitor, a WEE1 inhibitor, a prodrug thereof, a salt thereof, or a combination thereof, and / or a DNA targeted agent, such as those provided for herein.

[0109] The immunohistochemical stains employed with the methods are typically an antibody configured to target DACH1 gene, a mRNA thereof, or a DACH1 protein. The immunohistochemical stains can those provided for herein.PHARMACEUTICAL COMPOSITIONS

[0110] The present disclosure also features pharmaceutical compositions that contain a therapeutically effective amount of the isolated antibody or antigen binding fragment thereof that binds to DACH1 described herein. The composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533, 1990).

[0111] In some embodiments, a pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta- cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; saltforming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see, Remington’ s Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990)).

[0112] In some embodiments, a pharmaceutical composition may contain nanoparticles, e.g., polymeric nanoparticles, liposomes, or micelles.

[0113] In some embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly (2- hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art.

[0114] Pharmaceutical compositions containing an isolated antibody or antigen binding fragment thereof that binds to DACH1 disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes ofadministration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration.

[0115] Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see Remington ’s Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. In some embodiments, the formulation for parenteral administration is citrate-free.

[0116] These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as-is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.

[0117] A polyol, which acts as a tonicifier and may stabilize the isolated antibody or antigen binding fragment thereof that binds to DACH1, may also be included in the formulation. The polyol is added to the formulation in an amount which may vary with respect to the desired isotonicity of the formulation. In some embodiments, the aqueous formulation is isotonic. The amount of polyol added may also be altered with respect to the molecular weight of the polyol. For example, a lower amount of a monosaccharide (e.g., mannitol) is added, compared to a disaccharide (such as trehalose). In some embodiments, the polyol which is used in the formulation as a tonicity agent is mannitol.

[0118] A detergent or surfactant may also be added to the formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g., polysorbates 20, 80 etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated antibody and / or minimizes the formation of particulates in the formulation and / or reduces adsorption. In some embodiments, the formulation may include a surfactant which is a polysorbate. In some embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitanmonooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edi., 1996).

[0119] In embodiments, the protein product of the present disclosure is formulated as a liquid formulation. In some embodiments, the liquid formulation is prepared in combination with a sugar at stabilizing levels. In some embodiments, the liquid formulation is prepared in an aqueous carrier. In some embodiments, a stabilizer is added in an amount no greater than thatwhich may result in a viscosity undesirable or unsuitable for intravenous administration. In some embodiments, the sugar is disaccharides, e.g., sucrose. In some embodiments, the liquid formulation may also include one or more of a buffering agent, a surfactant, and a preservative.

[0120] In some embodiments, the pH of the liquid formulation is set by addition of a pharmaceutically acceptable acid and / or base. In some embodiments, the pharmaceutically acceptable acid is hydrochloric acid. In some embodiments, the base is sodium hydroxide.

[0121] The aqueous carrier of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.

[0122] A preservative may be optionally added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.

[0123] The isolated antibody or antigen binding fragment thereof that binds to DACH1 may be lyophilized to produce a lyophilized formulation including the proteins and a lyoprotectant. The lyoprotectant may be sugar, e.g., disaccharides. In some embodiments, the lyoprotectant is sucrose or maltose. The lyophilized formulation may also include one or more of a buffering agent, a surfactant, a bulking agent, and / or a preservative.

[0124] The amount of sucrose or maltose useful for stabilization of the lyophilized drug product may be in a weight ratio of at least 1 :2 protein to sucrose or maltose. In some embodiments, the protein to sucrose or maltose weight ratio is of from 1 :2 to 1 :5. In some embodiments, the pH of the formulation, prior to lyophilization, is set by addition of a pharmaceutically acceptable acid and / or base. In some embodiments, the pharmaceutically acceptable acid is hydrochloric acid. Tn some embodiments, the pharmaceutically acceptable base is sodium hydroxide.

[0125] A patient’s dose can be tailored to the approximate body weight or surface area of the patient. Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex, and medical condition of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those skilled in the art, especially in light of the dosage information and assays disclosed herein. The dosage can also be determined through the use of known assays for determining dosages used in conjunction with appropriate dose-response data. An individual patient's dosage can be adjusted as the progress of the disease is monitored. Blood levels of the targetable construct or complex in a patient can bemeasured to see if the dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics may be used to determine which targetable constructs and / or complexes, and dosages thereof, are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308: 43-53, 2001; Steimer et al., Clinica Chimica Acta 308: 33-41, 2001).METHODS OF PREPARATION

[0126] The isolated antibody or antigen binding fragment thereof that binds to DACH1 described above can be made using recombinant DNA technology well known to a skilled person in the art. For example, one or more isolated polynucleotides encoding the isolated antibody or antigen binding fragment thereof that binds to DACH 1 can be ligated to other appropriate nucleotide sequences, including, for example, constant region coding sequences, and expression control sequences, to produce conventional gene expression constructs (i.e., expression vectors) encoding the desired isolated antibody or antigen binding fragment thereof that binds to DACH1. Production of defined gene constructs is within routine skill in the art.

[0127] Nucleic acids encoding the desired isolated antibody or antigen binding fragment thereof that binds to DACH1 can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques.Exemplary host cells are E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that do not otherwise produce IgG protein. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode DACH1 antibodies or antibody fragments thereof.

[0128] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence. The expressed protein may be secreted. The expressed protein may accumulate in retractile or inclusion bodies, which can be harvested after disruption of the cells by French press or sonication. The retractile bodies then are solubilized, and the protein may be refolded and / or cleaved by methods known in the art.

[0129] If the engineered gene is to be expressed in eukaryotic host cells, e.g. , CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon. Optionally, the vector or gene construct maycontain enhancers and introns. In embodiments involving fusion proteins comprising an DACH1 antibodies or antibody fragments thereof, the expression vector optionally contains sequences encoding all or part of a constant region, enabling an entire, or a part of, a heavy or light chain to be expressed. The gene construct can be introduced into eukaryotic host cells using conventional techniques.

[0130] In some embodiments, in order to express an isolated antibody or antigen binding fragment thereof that binds to DACH1, an N-terminal signal sequence is included in the protein construct. Exemplary N-terminal signal sequences include signal sequences from interleukin-2, CD-5, IgG kappa light chain, trypsinogen, serum albumin, and prolactin.

[0131] After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limited dilution, ELISA, FACS, microscopy, or Clonepix. Clones can be cultured under conditions suitable for bio-reactor scale-up and maintained expression of the isolated antibody or antigen binding fragment thereof that binds to DACH1.

[0132] The isolated antibody or antigen binding fragment thereof that binds to DACH1 can be isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography .EXAMPLES

[0133] The disclosure now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and is not intended to limit the disclosure.Example 1. Generation and Validation of Antibodies with Affinity to DACH1

[0134] This Example describes the generation and validation of isolated antibodies or antigen binding fragments thereof that binds to DACH1 described herein.

[0135] A peptide sequence was generated from the DACH1 A isoform in a region conserved between humans and mice (FIG. 1A). An additional cystine residue at the C-terminus to facilitate conjugation to KLH. The peptide comprises the amino acid sequence of SEQ ID NO: 50 (ERVPDSPSPAPSLEC). As shown in FIG. IB, the human DACH1 isoforms all contain a DS domain (Dach N-Box (dac and ski / sno DS domain)) also known as the Dachshund homology domain and predicted to form a highly organized structure of a-helices and -strands. Onlyisoforms DACHla and DACHld contain the peptide region (indicated by arrow) used to generate antibodies 1-3 (18A2, 17E12, and 15G11) at residue Ser439(DACHla numbering). The heavy chain and light chain DNA and amino acid sequences for antibodies 1-3 (18A2, 17E12, and 15G11) are depicted in FIGs. 13A-13C. The DACHla isoform shows the highest level of expression in prostate cancer as indicated by data from the Cancer Genome Atlas (TCGA) See FIG. 1C. Monoclonal antibodies were generated by methods immunizing New Zealand rabbits, subsequently isolating PBMCs, enriching for memory B cells, and stimulating the B cells. See FIG. 2. B cell clones were screened by ELISA. As a control, a polyclonal antibody was also generated against a phosphorylated form (Ser439P(antibody ZP1)) of the DACH1 peptide. Commercial polyclonal rabbit comparator antibodies #7, and #8 were generated against amino acids 361-706 of DACHla (SEQ ID NO. 1), and the peptide ERTIQDGRLYLKTTVMY (amino acids 690-706; SEQ ID NO. 51) of DACHla (SEQ ID NO. 1), respectively. ELISA plates were coated with DACH1, incubated with undiluted B cell clone lysates, and binding was detected through use of an anti-rabbit conjugated secondary antibody. Results are summarized in Table 4. Table 4.

[0136] 293T cells were transiently transfected with FLAG-tagged DACH1 and supernatants were separated by gel electrophoresis for Western blot analysis. DACH1 was probed for using commercial rabbit polyclonal antibody #7, a rabbit polyclonal anti-phospho-DACHl antibody control generated by the same antibody manufacturer that produced the anti-DACHl monoclonal antibodies, and the anti-DACHl monoclonal antibodies purified from recombinant supernatants from B cell clones (Antibodies 1-3 (18A2, 17E12, 15G11, respectively)), and a commercial mouse monoclonal anti-FLAG (M2) antibody. Antibody 3 (clone 15G1 1) was tested at two concentrations as indicated in FIG. 3A. Clones that were immunoreactive with DACH1 displayed bands at -100 KDa, the molecular weight of DACH1 (FIG. 3A). Supernatants from 293T cells transfected with FLAG-tagged DACHlc were also analyzed by Western blot (FIG. 3B). The commercial antibody #7 directed further towards the carboxyl terminus of DACH1 was equally reactive with D ACH 1 c. None of the D ACH 1 a isoform-specific anti-sera reached with DACHlc.Example 2. Characterization of Antibodies with Affinity to DACH1

[0137] This Example describes the characterization of the localization of DACH1 and use of isolated antibodies or antigen binding fragments thereof that binds to DACH1 described herein to detect cellular DACH1.

[0138] To first determine DACHla subcellular distribution, LNCapP cells were transfected with control vector or EGFP-tagged DACH1 isoforms and a DACHlaS493Amutant and analyzed by imaging as depicted in the FIG. 4A schematic. Shown in FIG. 4B are immunofluorescent images indicating nuclear DACHla and cytoplasmic expression of DACHlb, c, d, and the aS439A mutant with red arrows.

[0139] Next, HEK293T and DU145 cells were transfected with vector control, FLAG-tagged DACHla, and DACHlb, c, d, and the aS439A mutant. Immunohistochemical (IHC) staining using an anti-FLAG (M2) antibody showed positive nuclear staining for transfected cells in both cell types (FIG. 5). Antibodies 1 (18A2), 3 (15G11), 2 (17E12), and antibody ZP1 were used insimilar IHC experiments in transfected HEK293T and DU145 cells (FIGs. 6, 7, 8, and 9). Antibodies 1, 2, 3, and ZP1 were able to detect nuclear DACHla and d isoforms but not b or c isoforms. Antibodies 1 and 3 also detected nuclear aS439A mutant DACH1. The ZP1 antibody generated to Ser439Pwas unable to detect the aS439A mutant DACH1.

[0140] Additional IHC experiments were performed to analyze mouse kidney cells. IHC was conducted in mouse kidney sections (FIG. 10). The commercial antibody #7, antibodies 1 (18A2), 2 (17E12), and ZP1 antibody stained the kidney glomerular cells (FIG. 10). Antibody 3 (15G11) appeared similar to unstained control cells. Similarly, IHC experiments were performed on human prostate cancer cells (FIG. 11). Positive cell staining was observed in the human prostate cancer sections with the commercial antibody #7, antibodies 1 (18A2) and ZP1 antibody. Antibodies 2 (17E12) and 3 (15G11) appeared similar to unstained control cells.

[0141] Characterization data is summarized in the chart depicted in FIG. 12.Example 3. Detection of DACH1 by antibody 18A2

[0142] This Example describes the detection of DACH1 using antibody 18A2.

[0143] Briefly, 293T cells were transiently transfected by calcium phosphate precipitation transfection with tetracycline inducible FLAG-DACH1 WT correct (DACHla), FLAG- DACHlb, FLAG-DACHlc, FLAG-DACHld, or pLRT vector control. On the next day, cells were changed to medium with 1 mg / mL doxycycline. One day later after doxycycline treatment, total cellular lysate (2x Laemmli loading buffer with DTT) was subjected to Western blot analysis (7% SDS-PAGE). The following antibodies were used for the Western blot analysis: rabbit monoclonal DACH1 (Clone 18A2-1), rabbit polyclonal DACH1 antibody (commercial antibody), and the loading control GAPDH rabbit polyclonal antibody. The 18A2 antibody showed strong detection of DACHla and DACHld as seen in FIG. 15.

[0144] Next, 293T cells were transiently transfected using calcium phosphate precipitation with pLRT-DACHl wild-type correct or mutants (L222R and L276R), CMVIO-DACHla, b, c, d or pLRT vector control. On the next day, cells were changed to medium with 1 mg / mL doxycycline. One day later after doxycycline treatment total protein lysates (2x Laemmli loading buffer with DTT) were subjected to Western blot analysis. The following antibodies were used for Western blot analysis: rabbit monoclonal DACH1 antibody (clone 18A2), rabbit polyclonal DACH1 antibody (commercial antibody), and rabbit polyclonal GAPDH antibody as a loading control. The 18A2 antibody showed strong detection of DACHla, DACHld, and the L222R and L276R mutants without detecting DACHlb and DACHlc as seen in FIG. 16.

[0145] FIG. 17A shows an experimental immunofluorescence assay schema. Briefly, 5xl04U2OS cells stably transfected with tetracycline inducible FLAG-DACH1 or pLRT vector controlwere plated in 8-well chamber slide. The next day cells were treated with doxycycline at 2 mg / mL. One day later, cells were treated with 2 mM H2O2 for 10 minutes. Cells were washed once with PBS post H2O2 treatment before being fixed in ice-cold methanol: acetone (1 :1 , v / v) at -20 °C for 15 minutes. Fixative was removed and cells were washed twice with PBS then blocked in blocking buffer (3% BSA in PBS with 0.2% Tween 20). Cells were incubated in primary antibody rabbit monoclonal anti-DACHl (Clone 18A2-11:500) diluted in blocking buffer overnight at 4 °C. Primary antibody was removed from wells and the cells were washed three times in PBS with 0.1% Triton X-100. Cells were then incubated with a secondary antibody diluted 1 :500 in blocking buffer for 1.5 hours followed by incubation with 1 pg / mL DAPI in blocking buffer for 30 minutes at room temperature. Cells were washed five times in PBS with 0.1% Triton X-100 prior to imaging. Cells were imaged on confocal microscope at 40x oil. Data for control cells is seen in FIGs. 17B-17C.

[0146] FIG. 18A shows an experimental immunofluorescence assay schema. Briefly, 5xl04U2OS cells stably transfected with tetracycline inducible FLAG-DACH1 or pLRT vector control were plated in 8-well chamber slide. The next day cells were treated with doxycycline at 2 mg / mL. One day later cells were treated with 2 mM H2O2 for 10 minutes. Cells were washed once with PBS post H2O2 treatment before being fixed in ice-cold methanol: acetone (1 :1, v / v) at -20 °C for 15 minutes. Fixative was removed and cells were washed twice with PBS then blocked in blocking buffer (3% BSA in PBS with 0.2% Tween 20). Cells were incubated in primary antibody rabbit monoclonal anti-DACHl) diluted in blocking buffer overnight at 4 °C. Primary antibody was removed from wells and the cells were washed three times in PBS with 0.1% Triton X-100. Cells were then incubated in secondary antibody diluted 1:500 in blocking buffer for 1.5 hours followed by incubation withl pg / mL DAPI in blocking buffer for 30 minutes at room temperature. Cells were washed five times in PBS with 0.1% Triton X-100 prior to imaging. Cells were imaged on confocal microscope at 40x oil. Data for H202-treated cells is seen in FIGs. 18B-18C.

[0147] The data and embodiments provided herein demonstrate that the present antibodies can be used to detect DACH1 in biological samples, such as the types of samples provided for herein.EQUIVALENTS

[0148] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the disclosure described herein. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.SEQUENCE LISTING

Claims

CLAIMS1. An isolated antibody or antigen binding fragment thereof that binds to DACH1 (SEQ ID NO: 1), comprising: a) a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs) and has the amino acid sequence as set forth in any one of SEQ ID NOs: 2-7; and b) a light chain variable region (VL) comprising three CDRs and has the amino acid sequence as set forth in any one of SEQ ID NOs: 8-13.

2. The isolated antibody or antigen binding fragment thereof of claim 1 , wherein a) the VH CDRs and the VL CDRs are defined according to AbM; b) the VH CDRs and the VL CDRs are defined according to Kabat; c) the VH CDRs and the VL CDRs are defined according to Chothia; or d) the VH CDRs and the VL CDRs are defined according to IM GT.

3. The isolated antibody or antigen binding fragment thereof of any one of claims 1-2, wherein the VH has the amino acid sequence according to SEQ ID NO: 2, and the VL has the amino acid sequence according to SEQ ID NO: 8.

4. The isolated antibody or antigen binding fragment thereof of any one of claims 1-2, wherein the VH has the amino acid sequence according to SEQ ID NO: 3, and the VL has the amino acid sequence according to SEQ ID NO: 9.

5. The isolated antibody or antigen binding fragment thereof of any one of claims 1-2, wherein the VH has the amino acid sequence according to SEQ ID NO: 4, and the VL has the amino acid sequence according to SEQ ID NO: 10.

6. The isolated antibody or antigen binding fragment thereof of any one of claims 1-2, wherein the VH has the amino acid sequence according to SEQ ID NO: 5, and the VL has the amino acid sequence according to SEQ ID NO: 11.

7. The isolated antibody or antigen binding fragment thereof of any one of claims 1-2, wherein the VH has the amino acid sequence according to SEQ ID NO: 6, and the VL has the amino acid sequence according to SEQ ID NO: 12.

8. The isolated antibody or antigen binding fragment thereof of any one of claims 1-2, wherein the VH has the amino acid sequence according to SEQ ID NO: 7, and the VL has the amino acid sequence according to SEQ ID NO: 13.

9. An isolated antibody or antigen binding fragment thereof that binds to DACH1, comprising:a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to any one of SEQ ID NOs: 14-19, (ii) a CDR2 having an amino acid sequence according to any one of SEQ ID NOs: 20-25, and (iii) a CDR3 having an amino acid sequence according to any one of SEQ ID NOs: 26-31; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to any one of SEQ ID NOs: 32-37, (ii) a CDR2 having an amino acid sequence according to any one of SEQ ID NOs: 38-43, and (iii) a CDR3 having an amino acid sequence according to any one of SEQ ID NOs: 44-49.

10. The isolated antibody or antigen binding fragment thereof of claim 9, wherein the VH has an amino acid sequence having at least 80% sequence identity to the sequence of any one of SEQ ID NOs: 2-7, and the VL has an amino acid sequence having at least 80% sequence identity to the sequence of any one of SEQ ID NOs: 8-13.

11. The isolated antibody or antigen binding fragment thereof of any one of claims 9-10, wherein the VH has an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 2-7, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 8-13.

12. The isolated antibody or antigen binding fragment thereof of any one of claims 9-11, wherein the VH has an amino acid sequence having 100% sequence identity to the sequence of any one of SEQ ID NOs: 2-7, and the VL has an amino acid sequence having 100% sequence identity to the sequence of any one of SEQ ID NOs: 8-13.

13. An isolated antibody or antigen binding fragment thereof that binds to DACH1 , comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 14, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 20, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 26; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 32, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 38, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 44.

14. The isolated antibody or antigen binding fragment thereof of claim 13, wherein the VH has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 2, and the VL has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 8.

15. The isolated antibody or antigen binding fragment thereof of any one of claims 13-14, wherein the VH has an amino acid sequence having at least 90% sequence identity to thesequence of SEQ ID NO: 2, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 8.

16. The isolated antibody or antigen binding fragment thereof of any one of claims 13-15, wherein the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 2, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 8.

17. An isolated antibody or antigen binding fragment thereof that binds to DACH1 , comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 15, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 21, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 27; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 33, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 39, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 45.

18. The isolated antibody or antigen binding fragment thereof of claim 17, wherein the VH has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 3, and the VL has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 9.

19. The isolated antibody or antigen binding fragment thereof of any one of claims 17-18, wherein the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 3, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 9.

20. The isolated antibody or antigen binding fragment thereof of any one of claims 17-19, wherein the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 3, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 9.

21. An isolated antibody or antigen binding fragment thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 16, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 22, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 28; andb) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 34, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 40, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 46.

22. The isolated antibody or antigen binding fragment thereof of claim 21, wherein the VH has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 4, and the VL has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 10.

23. The isolated antibody or antigen binding fragment thereof of any one of claims 21-22, wherein the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 4, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 10.

24. The isolated antibody or antigen binding fragment thereof of any one of claims 21-23, wherein the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 4, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 10.

25. An isolated antibody or antigen binding fragment thereof that binds to DACH1 , comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 17, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 23, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 29; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 35, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 41, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 47.

26. The isolated antibody or antigen binding fragment thereof of claim 25, wherein the VH has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 5, and the VL has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 11.

27. The isolated antibody or antigen binding fragment thereof of any one of claims 25-26, wherein the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 5, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 11.

28. The isolated antibody or antigen binding fragment thereof of any one of claims 25-27, wherein the VH has an amino acid sequence having 100% sequence identity to the sequence ofSEQ ID NO: 5, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 11.

29. An isolated antibody or antigen binding fragment thereof that binds to DACH1 , comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 18, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 24, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 30; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 36, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 42, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 48.

30. The isolated antibody or antigen binding fragment thereof of claim 29, wherein the VH has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 6, and the VL has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 12.

31. The isolated antibody or antigen binding fragment thereof of any one of claims 29-30, wherein the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 6, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 12.

32. The isolated antibody or antigen binding fragment thereof of any one of claims 29-31 , wherein the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 6, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 12.

33. An isolated antibody or antigen binding fragment thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 19, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 25, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 31; and b) a light chain variable region (VL) comprising (i) a CDR1 having an amino acid sequence according to SEQ ID NO: 37, (ii) a CDR2 having an amino acid sequence according to SEQ ID NO: 43, and (iii) a CDR3 having an amino acid sequence according to SEQ ID NO: 49.

34. The isolated antibody or antigen binding fragment thereof of claim 33, wherein the VH has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ IDNO: 7, and the VL has an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 13.

35. The isolated antibody or antigen binding fragment thereof of any one of claims 33-34, wherein the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 7, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 13.

36. The isolated antibody or antigen binding fragment thereof of any one of claims 33-35, wherein the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 7, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 13.

37. An isolated antibody or antigen binding fragment thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 2-7; and b) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 8-13.

38. The isolated antibody or antigen binding fragment thereof of claim 37, wherein the VH has an amino acid sequence having at least 90% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 2-7, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 8-13.

39. The isolated antibody or antigen binding fragment thereof of any one of claims 37-38, wherein the VH has an amino acid sequence having 100% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 2-7, and the VL has an amino acid sequence having 100% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 8-13.

40. An isolated antibody or antigen binding fragment thereof that binds to DACH1 , comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 2; and b) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 8.

41. The isolated antibody or antigen binding fragment thereof of claim 40, wherein the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 2, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 8.

42. The isolated antibody or antigen binding fragment thereof of any one of claims 40-41, wherein the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 2, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 8.

43. An isolated antibody or antigen binding fragment thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 3; and b) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 9.

44. The isolated antibody or antigen binding fragment thereof of claim 43, wherein the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 3, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 9.

45. The isolated antibody or antigen binding fragment thereof of any one of claims 43-44, wherein the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 3, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 9.

46. An isolated antibody or antigen binding fragment thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 4; and b) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 10.

47. The isolated antibody or antigen binding fragment thereof of claim 46, wherein the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 4, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 10.

48. The isolated antibody or antigen binding fragment thereof of any one of claims 46-47, wherein the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 4, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 10.

49. An isolated antibody or antigen binding fragment thereof that binds to DACH1, comprising:a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 5; and b) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 11.

50. The isolated antibody or antigen binding fragment thereof of claim 49, wherein the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 5, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 11 .

51. The isolated antibody or antigen binding fragment thereof of any one of claims 49-50, wherein the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 5, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 11 .

52. An isolated antibody or antigen binding fragment thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 6; and b) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 12.

53. The isolated antibody or antigen binding fragment thereof of claim 52, wherein the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 6, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 12.

54. The isolated antibody or antigen binding fragment thereof of any one of claims 52-53, wherein the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 6, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 12.

55. An isolated antibody or antigen binding fragment thereof that binds to DACH1, comprising: a) a heavy chain variable region (VH) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 7; and b) a light chain variable region (VL) comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 13.

56. The isolated antibody or antigen binding fragment thereof of claim 55, wherein the VH has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ IDNO: 7, and the VL has an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 13.

57. The isolated antibody or antigen binding fragment thereof of any one of claims 55-56, wherein the VH has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 7, and the VL has an amino acid sequence having 100% sequence identity to the sequence of SEQ ID NO: 13.

58. A method of detecting DACH1 in a biological sample, the method comprising contacting the sample with the isolated antibody or antigen binding fragment thereof of any one of claims 1-57.

59. The method of claim 58, wherein the antibody is a labeled antibody, such as with a fluorophore.

60. A method of determining the sensitivity of a subject with cancer to a therapeutic treatment, the method comprising contacting a biological sample obtained from the subject and detecting the presence of DACH1 by contacting the sample with the isolated antibody or antigen binding fragment thereof of any one of claims 1-57, wherein if DACH1 is detected, the subject is determined to have a cancer that is sensitive to a therapeutic treatment.

61. The method of claim 60, further comprising administering the therapeutic treatment to the subject who is determined to be sensitive to the therapeutic treatment.

62. The method of claims 60 or 61, wherein the antibody, or antigen binding fragment thereof, is labeled with a fluorophore.

63. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the isolated antibody or antigen binding fragment thereof of any one of claims 1-57.

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