DLK1 Antibodies and Methods of Treating Cancer

Abstract

The present disclosure provides antigen-binding proteins that bind DLK1, bispecific antigen-binding proteins that bind DLK1 and a second antigen, and conjugates thereof.Related polypeptides, nucleic acids, vectors, host cells, and conjugates are further provided herein.Kits and pharmaceutical compositions that include such entities are further provided.Methods for producing antigen-binding proteins and methods for treating subjects with cancer are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 286,896, filed December 7, 2021, the entire contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates generally to antibodies specific for Delta-like non-canonical Notch ligand 1 (DLK1) and their use to treat cancer. [Background technology]

[0003] Antibodies are powerful therapeutic agents characterized by limited side effects due to their ability to specifically target distinct antigens in cells, bacteria, viruses, or toxins related to DLK1-related diseases.There is a clinical need to provide new antibodies, such as the antibodies described herein, to address the medical needs of patients related to DLK1-related diseases. Summary of the Invention

[0004] Provided herein are antigen binding proteins that bind to DLK1.

[0005] In various embodiments, the antigen binding protein binds to DLK1 endogenously expressed by human cancer cells. In various cases, the antigen binding protein of the present disclosure inhibits tumor growth in a subject, e.g., a human, without any other moiety that binds to the antigen binding protein. In various cases, the antigen binding protein that is not conjugated to a heterologous moiety (e.g., not conjugated to any chemotherapeutic agent, drug, or toxic moiety) inhibits tumor growth in a subject, e.g., a human.

[0006] In various embodiments, the antigen binding protein binds to DLK1 expressed by human cancer cells.Without being bound by any particular theory, the inhibitory effect of the antigen binding protein provided herein makes such entity useful in the method of suppressing tumor growth and treating subjects with tumor or cancer.As further discussed herein, in various embodiments, the antigen binding protein is an antibody, its antigen-binding antibody fragment, or an antibody protein preparation.

[0007] The present disclosure also provides antigen binding proteins that comprise at least three, four, five, or all of a particular group of amino acid sequences. In various embodiments, the antigen binding proteins comprise at least three, four, five, or six complementarity determining region (CDR) amino acid sequences of a DLK1 antibody disclosed herein, e.g., in Table 1 and Figures 19-30.

[0008] The present disclosure provides a bispecific antigen-binding protein that binds DLK1 and a second antigen. The bispecific antigen-binding protein may include any one of the antigen-binding proteins described herein. The second antigen may be a cell surface protein, optionally a protein whose binding modulates an immune response. In various embodiments, the second antigen is a cell surface protein expressed by a T cell, optionally a component of the T cell receptor (TCR), such as CD3. In some embodiments, the second antigen is CD3. In some embodiments, the second antigen is CD3E. The bispecific antigen-binding protein may take any structure, such as, for example, a diabody, a TandAb (tandem diabody), or a BiTE (bispecific T cell engager).

[0009] The present disclosure also provides a conjugate comprising an antigen-binding protein or a bispecific antigen-binding protein and a heterologous moiety (e.g., a cytotoxic drug). The conjugate may include a cleavable or non-cleavable linker. The conjugate may have a variable number of heterologous moieties (drugs) conjugated to the antigen-binding protein or bispecific antigen-binding protein described herein, preferably 1-8 drugs per protein or 3-8 drugs per protein. The conjugate may be a site-specific conjugate. The conjugate may be a homogeneous or heterogeneous conjugate.

[0010] Related polypeptides, nucleic acids, vectors, host cells, and conjugates are further provided herein. Kits and pharmaceutical compositions containing such entities are further contemplated.

[0011] Methods of producing the antigen binding proteins are also provided, in various embodiments, the methods comprise culturing a host cell comprising a nucleic acid encoding an antigen binding protein or polypeptide described herein to express the antigen binding protein or polypeptide.

[0012] Further provided herein is a method for treating a subject having cancer. In various embodiments, the method comprises administering to the subject an amount of the pharmaceutical composition of the present disclosure that is effective to treat the cancer in the subject.

[0013] Also provided is a method for treating a subject with DLK1-expressing cancer, comprising administering to the subject the pharmaceutical composition described herein.In various embodiments, DLK1-expressing cancer expresses DLK1.Further contemplated is a method for inhibiting tumor growth in a subject, comprising administering to the subject the pharmaceutical composition described herein.

[0014] A method of reducing tumor size in a subject or preventing the recurrence of cancer in a subject, the method comprising administering to the subject a pharmaceutical composition described herein.

[0015] Also provided herein is a method for treating cancer in a subject diagnosed with low over-expression of DLK1, the method comprising administering to the subject a pharmaceutical composition described herein.

[0016] In various embodiments, the administering induces apoptosis in tumor cells, e.g., cells expressing DLK1. In various embodiments, the administering induces antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), tumor necrosis and cell death or depletion, and / or disruption of tumor cell adhesion, which results in tumor regression or slowing of tumor growth, respectively. [Brief description of the drawings]

[0017] [Figure 1] 1 shows the efficacy of a panel of anti-DLK1 chimeric mAbs in DLK1+HEPG2 human hepatocellular carcinoma cell line xenografts. The figure shows a graph of tumor volume (mm3) as a function of time (days) (A) or a graph of the mean change in tumor volume (mm3) in tumors on day 23 (B) in mice bearing DLK1-positive human liver tumors (HepG2) after treatment with a control human IgG1 antibody or four different anti-DLK1 chimeric antibodies (DLK1-547 (also called 04-547m), DLK1-548 (also called 04-548m), DLK1-557 (also called 04-557m) and DLK1-559 (also called 04-559m)) at 10 mg / kg once a week for four repeated doses, where the chimeric antibodies were generated by replacing the light and heavy chain variable regions of human IgG1 with the light and heavy chain variable regions of a mouse monoclonal anti-DLK1 antibody. [Figure 2A]The figure shows efficacy of a panel of humanized DLK1 mAbs in DLK1+CORL279 human small cell lung cancer (SCLC) cell line xenografts. The figure shows efficacy of a panel of humanized DLK1 mAbs in DLK1+CORL279 human small cell lung cancer (SCLC) cell line xenografts ... 1 represents a graph of tumor volume (mm3) as a function of time (days) in mice bearing DLK1 positive small cell lung cancer cells (COR-L279) following treatment with humanized antibodies (note that "DLK1-mab-" or "ab-" may be substituted or not listed altogether, which refer to the same humanized antibody, e.g., DLK1-547-h2 is the same as 04-547-h2, 04-0547-h2, mab-547-h2, DLK1-mab-547-h2 and 562-h2). [Figure 2B]The figure shows efficacy of a panel of humanized DLK1 mAbs in DLK1+CORL279 human small cell lung cancer (SCLC) cell line xenografts. The figure shows efficacy of a panel of humanized DLK1 mAbs in DLK1+CORL279 human small cell lung cancer (SCLC) cell line xenografts ... 1 represents a graph of the mean change in tumor volume (mm3) in tumors at day 21 in mice bearing DLK1 positive small cell lung cancer cells (COR-L279) after treatment with humanized antibodies (note that "DLK1-mab-" or "DLK1-mab-" may be substituted with "DLK1-mab-" or not listed at all, which refer to the same humanized antibody, e.g., DLK1-547-h2 is the same as 04-547-h2, 04-0547-h2, mab-547-h2, DLK1-mab-547-h2 and 562-h2). [Figure 2C]The figure shows efficacy of a panel of humanized DLK1 mAbs in DLK1+CORL279 human small cell lung cancer (SCLC) cell line xenografts. The figure shows efficacy of a panel of humanized DLK1 mAbs in DLK1+CORL279 human small cell lung cancer (SCLC) cell line xenografts ... 1 depicts a graph of the percent change in body weight in mice bearing DLK1 positive small cell lung cancer cells (COR-L279) following treatment with humanized antibodies (note that "DLK1-547-h2" may be replaced with "04-547-h2," "mab-," or "DLK1-mab-" or not listed at all, which refer to the same humanized antibody, e.g., DLK1-547-h2 is the same as 04-547-h2, 04-0547-h2, mab-547-h2, DLK1-mab-547-h2 and 562-h2). [Diagram 3] 1 shows selective tumor volume (mm3) reduction of DLK1-positive (A-C) over DLK1-negative (D-F) human tumor xenografts following treatment with DLK1-ADCs including DLK1-561-f1, a humanized anti-DLK1 antibody conjugated to the cytotoxic monomethyl auristatin E (MMAE). Hu-IgG1 control is an unconjugated non-targeting human IgG1 control antibody. DLK1-positive human tumor cell lines: small cell lung cancer cell lines COR-L279 (A) and H524 (C), and rhabdomyosarcoma cell line JR-1 (also referred to as JR) (B); DLK1-negative human tumor cell lines: colorectal cancer cell lines SNU-C1 (also referred to as SNUC1) (D) and LS513 (E), and melanoma cell line M202 (F). [Figure 4A]Figure 1 shows the efficacy of humanized DLK1-ADC in DLK1-positive CORL279 human SCLC cell line xenografts. This figure shows a graph of tumor volume (mm3) as a function of time in mice bearing the human DLK1-positive human small cell lung cancer cell line COR-L279 after treatment with antibody drug conjugates (ADCs) of control IgG antibody or humanized anti-DLK1 antibody conjugated to MMAE (DLK1-ADC-547-h2, DLK1-ADC-562-h10 and DLK1-ADC-561-f1) at 5 mg / kg once a week for three repeated doses. IgG is an unconjugated non-targeting control antibody. [Figure 4B] Figure 1 shows the efficacy of humanized DLK1-ADC in DLK1-positive CORL279 human SCLC cell line xenografts. This figure shows a graph of the mean change in tumor volume (mm3) on day 13 in mice bearing the human DLK1-positive human small cell lung cancer cell line COR-L279 after treatment with antibody drug conjugates (ADCs) of control IgG antibody or humanized anti-DLK1 antibody conjugated to MMAE (DLK1-ADC-547-h2, DLK1-ADC-562-h10 and DLK1-ADC-561-f1) at 5 mg / kg once a week for three repeated doses. IgG is an unconjugated non-targeting control antibody. [Figure 4C] Figure 1 shows the efficacy of humanized DLK1-ADC in DLK1-positive CORL279 human SCLC cell line xenografts. This figure shows a graph of the percent change in body weight in mice bearing the human DLK1-positive human small cell lung cancer cell line COR-L279 after treatment with a control IgG antibody or an antibody drug conjugate (ADC) of a humanized anti-DLK1 antibody conjugated to MMAE (DLK1-ADC-547-h2, DLK1-ADC-562-h10 and DLK1-ADC-561-f1) at 5 mg / kg once a week for three repeated doses. IgG is an unconjugated non-targeting control antibody. [Diagram 5]Figure 1 shows that humanized DLK1-ADC has no antitumor activity in DLK1-negative M202 human melanoma cell line xenografts. This figure shows a graph of tumor volume (mm3) as a function of time (A) and a graph of the mean change in tumor volume (mm3) at day 31 (B) in mice bearing the human DLK1-negative human melanoma cell line M202 after treatment with antibody drug conjugates (ADCs) of a control IgG antibody or a humanized anti-DLK1 antibody conjugated to MMAE (DLK1-ADC-547-h2, DLK1-ADC-562-h10 and DLK1-ADC-561-f1) at 5 mg / kg once a week for three repeated doses. IgG is an unconjugated non-targeting control antibody. [Figure 6-1] The biochemical, biophysical and cell biological properties of the three lead humanized anti-DLK1 antibodies are summarized. [Figure 6-2] The biochemical, biophysical and cell biological properties of the three lead humanized anti-DLK1 antibodies are summarized. [Figure 7] 1 shows internalization of three humanized anti-DLK1 antibody-MMAE conjugates and one humanized anti-DLK1 antibody into a human DLK1-positive small cell lung cancer cell line (COR-L279). [Figure 8]Summarizing the effect of long-term storage at different temperatures on the aggregation state and stability of humanized anti-DLK1 antibodies (04-0561-F1 and 04-0562-h10) analyzed by size exclusion chromatography and non-reducing SDS-PAGE. The bottom set of panels shows non-reducing SDS-PAGE analysis after 5 weeks of storage at the indicated temperatures and concentrations. Top panel: Long-term storage (5 weeks) and temperature stress (RT or 37° C.) did not cause significant antibody aggregation and degradation at any concentration of 10, 20, 50, or 75 mg / ml (SEC). Specifically, after 5 weeks of storage at 37° C., less than 3.5% degradation (all four concentrations) and approximately 4% aggregation (only at 75 mg / ml) was observed for 04-0561-F1, and less than 8% degradation was observed for 04-562-h10. Bottom panel: Long-term storage (5 weeks) and temperature stress (RT or 37° C.) did not significantly affect antibody integrity at any concentration of 10, 20, 50, or 75 mg / ml (non-reducing SDS-PAGE). However, some antibody degradation was detected after 5 weeks of storage at 37° C. at all concentrations (10, 20, 50, and 75 mg / ml); 04-0561-F1 had lower degradation levels compared to 04-0562-h10. [Figure 9] Figure 1 shows the effect of different temperatures and long-term storage on the stability of DLK1 hAb 04-0547-h2 (10 mg / ml) (antibody integrity assessed by non-reducing SDS PAGE and antibody aggregation by SEC (-80°C vs. 4°C, RT, and 37°C for 7 days). The figure shows SDS-PAGE analysis of the stability of humanized anti-DLK1 antibody 04-0547-h2 stored at -80°C, 4°C, RT, or 37°C for 7 days, with the left side of each gel showing stored antibody subjected to reducing conditions immediately prior to electrophoresis and the right side showing stored antibody subjected to non-reducing conditions immediately prior to electrophoresis (left panel). The aggregation and fragmentation state of the stored antibodies was analyzed by size exclusion chromatography as shown in the chromatograms and summarized below (right panel). [Figure 10-1]1 shows a sequence alignment of human DLK1 (UniProtKB Accession No. P80370-1), cynomolgus DLK1 (UniProtKB Accession No. A0A2K5TMQ6), mouse DLK1 (UniProtKB Accession No. Q09163) and rat DLK1 (UniProtKB Accession No. O70534) protein sequences, with the yellow highlight indicating the extracellular domain of the human DLK1 transmembrane protein. The percent identity of the above DLK1 proteins to the human DLK1 amino acid sequence as well as the UniProtKB and NCBI accession numbers are shown in the table below. [Figure 10-2] 1 shows a sequence alignment of human DLK1 (UniProtKB Accession No. P80370-1), cynomolgus DLK1 (UniProtKB Accession No. A0A2K5TMQ6), mouse DLK1 (UniProtKB Accession No. Q09163) and rat DLK1 (UniProtKB Accession No. O70534) protein sequences, with the yellow highlight indicating the extracellular domain of the human DLK1 transmembrane protein. The percent identity of the above DLK1 proteins to the human DLK1 amino acid sequence as well as the UniProtKB and NCBI accession numbers are shown in the table below. [Figure 11-1] Flow cytometry (upper half) and KinExA (lower half) assessment of the binding activity of three humanized anti-DLK1 antibodies (04-561-F1, 04-562-h10 and 04-547-h2) to HEK293T cells overexpressing human, monkey, mouse or rat DLK1 as a fusion protein with mGFP fluorescent protein. [Figure 11-2] Flow cytometry (upper half) and KinExA (lower half) assessment of the binding activity of three humanized anti-DLK1 antibodies (04-561-F1, 04-562-h10 and 04-547-h2) to HEK293T cells overexpressing human, monkey, mouse or rat DLK1 as a fusion protein with mGFP fluorescent protein. [Figure 12] 1 shows the time course of internalization of anti-DLK1 antibodies into cultured COR-L279 DLK1-positive human small cell lung cancer cells. [Figure 13]Figure 1 shows the cellular internalization of anti-DLK1 antibody into cultured HEPG2 hepatoma cells. Negative controls - hIgG and no antibody. The number of seconds in each image indicates the exposure time. [Figure 14] 1 shows the time course of internalization of humanized anti-DLK1 antibodies into cultured COR-L279 DLK1-positive human small cell lung cancer cells. [Figure 15] 1 shows the time course of internalization of humanized anti-DLK1 antibodies into cultured COR-L279 DLK1-positive human small cell lung cancer cells. [Figure 16] Figure 1 shows the time course of internalization of humanized anti-DLK1 antibodies into cultured COR-L279 DLK1 positive human small cell lung cancer cells. Negative controls - non-targeting human IgG1 (hIgG1) and no antibody. [Figure 17] Fluorescence (red) associated with anti-DLK1 antibodies 48 hours after the start of a cellular internalization study of anti-DLK1 antibodies into cultured COR-L279 DLK1-positive human small cell lung cancer cells. Negative controls - hIgG and no antibody. [Figure 18] Figure 1 shows the time course of cellular internalization of three antibody-drug conjugates of humanized anti-DLK1 antibodies with MMAE and one non-ADC-conjugated humanized anti-DLK1 antibody into cultured COR-L279 DLK1-positive human small cell lung cancer cells. The figure shows that all three anti-DLK1 ADCs bind well to DLK1 and are completely internalized within 90 minutes of binding. [Figure 19-1] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0547-h2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 19-2] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0547-h2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 19-3]The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0547-h2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 19-4] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0547-h2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 20-1] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0561-F1 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 20-2] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0561-F1 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 20-3] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0561-F1 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 20-4] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0561-F1 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 21-1] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0562-h10 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 21-2] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0562-h10 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 21-3] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0562-h10 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 21-4] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0562-h10 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 22-1] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0548-h4 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 22-2] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0548-h4 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 22-3] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0548-h4 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 22-4]The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0548-h4 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 23-1] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0557-F2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 23-2] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0557-F2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 23-3] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0557-F2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 23-4] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0557-F2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 24-1] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0557-h3 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 24-2] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0557-h3 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 24-3] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0557-h3 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 24-4] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0557-h3 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 25-1] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0559-F2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 25-2] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0559-F2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 25-3] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0559-F2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 25-4] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0559-F2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 26-1]The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0559-h4 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 26-2] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0559-h4 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 26-3] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0559-h4 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 26-4] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0559-h4 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 27-1] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0565-F2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 27-2] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0565-F2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 27-3] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0565-F2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 27-4] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of humanized anti-DLK1 antibody 04-0565-F2 are shown, along with the lengths, positions and amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the Kabat and AbM methods. [Figure 28-1] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of three humanized anti-DLK1 antibodies 04-0547-h2, 04-0561-F1, and 04-0562-h10 are shown, along with the amino acid sequences of the complementarity determining regions (CDR) and framework regions (FR) defined by the ImMunoGeneTics (IMGT) method, and the name of the expression vector that produces the antibodies. The expression vector p04-0561-F1 was deposited on December 7, 2022 at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia 20110-2209, and was assigned the accession number _____. This deposit is maintained under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. [Figure 28-2] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of three humanized anti-DLK1 antibodies 04-0547-h2, 04-0561-F1, and 04-0562-h10 are shown, along with the amino acid sequences of the complementarity determining regions (CDR) and framework regions (FR) defined by the ImMunoGeneTics (IMGT) method, and the name of the expression vector that produces the antibodies. The expression vector p04-0561-F1 was deposited on December 7, 2022 at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia 20110-2209, and was assigned the accession number _____. This deposit is maintained under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. [Figure 29-1]The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of five humanized anti-DLK1 antibodies 04-0548-h4, 04-0557-F2, 04-0559-F2, 04-0559-h4, and 04-0565-F2 are shown, along with the amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the IMGT method, and the names of the expression vectors that produce the antibodies. [Figure 29-2] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of five humanized anti-DLK1 antibodies 04-0548-h4, 04-0557-F2, 04-0559-F2, 04-0559-h4, and 04-0565-F2 are shown, along with the amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the IMGT method, and the names of the expression vectors that produce the antibodies. [Figure 29-3] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of five humanized anti-DLK1 antibodies 04-0548-h4, 04-0557-F2, 04-0559-F2, 04-0559-h4, and 04-0565-F2 are shown, along with the amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the IMGT method, and the names of the expression vectors that produce the antibodies. [Figure 29-4] The amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of five humanized anti-DLK1 antibodies 04-0548-h4, 04-0557-F2, 04-0559-F2, 04-0559-h4, and 04-0565-F2 are shown, along with the amino acid sequences of the complementarity determining regions (CDRs) and framework regions (FRs) defined by the IMGT method, and the names of the expression vectors that produce the antibodies. [Diagram 30] The amino acid sequences of the human immunoglobulin light chain kappa constant region (IGHKC; UniProt ID: P01834) and the human immunoglobulin heavy chain constant regions gamma 1 (IGHG1; UniProt ID: P01857) and gamma 2 (IGHG2; UniProt ID: P01859) in some embodiments of the humanized anti-DLK1 antibody of the present invention are shown. [Figure 31-1]1 shows the nucleic acid sequences encoding the heavy chain variable region (VH) and light chain variable region (VL) of nine humanized anti-DLK1 antibodies 04-0565-F2, 04-0562-h10, 04-0561-F1, 04-0559-h4, 04-0559-F2, 04-0557-h3, 04-0557-F2, 04-0548-h4, and 04-0547-h2. [Figure 31-2] 1 shows the nucleic acid sequences encoding the heavy chain variable region (VH) and light chain variable region (VL) of nine humanized anti-DLK1 antibodies 04-0565-F2, 04-0562-h10, 04-0561-F1, 04-0559-h4, 04-0559-F2, 04-0557-h3, 04-0557-F2, 04-0548-h4, and 04-0547-h2. [Figure 31-3] 1 shows the nucleic acid sequences encoding the heavy chain variable region (VH) and light chain variable region (VL) of nine humanized anti-DLK1 antibodies 04-0565-F2, 04-0562-h10, 04-0561-F1, 04-0559-h4, 04-0559-F2, 04-0557-h3, 04-0557-F2, 04-0548-h4, and 04-0547-h2. [Figure 31-4] 1 shows the nucleic acid sequences encoding the heavy chain variable region (VH) and light chain variable region (VL) of nine humanized anti-DLK1 antibodies 04-0565-F2, 04-0562-h10, 04-0561-F1, 04-0559-h4, 04-0559-F2, 04-0557-h3, 04-0557-F2, 04-0548-h4, and 04-0547-h2. [Diagram 32] 1 shows the nucleic acid sequences encoding the human immunoglobulin light chain kappa constant region (IGHKC) and the human immunoglobulin heavy chain constant region gamma 1 (IGHG1) used in some embodiments of the present invention to generate full-length humanized anti-DLK1 antibodies. [Diagram 33] The nucleic acid sequences encoding the heavy chain variable region (VH) and light chain variable region (VL) of the chimeric anti-DLK1 antibody 04-0561-m, as well as the humanized VH and VL in its humanized version, 04-0561-F1, are shown. [Diagram 34]Control HEK293T cells or engineered HEK293T cells expressing mGFP fusions with the long form of the human DLK1 protein as full-length protein (HEK293T DLK1 mGFP G06 (long form)) or human DLK1 protein in region 1 (834 HEK293T DLK1HM1-mGFP), region 2 (835 HEK293T DLK1HM2-mGFP), region 3 (836 HEK293T DLK1HM3-mGFP), region 4 (837 HEK293T DLK1HM4-mGFP), region 5 (850 HEK293T DLK1HM5-mGFP), region 6 (851 HEK293T DLK1HM6-mGFP), region 7 (848 HEK293T DLK1HM7-mGFP), or region 8 (849 HEK293T Epitope mapping data for DLK1-561-F1, a humanized anti-DLK1 antibody directly labeled with Alexa Fluor647 (AF647) dye, which binds to engineered HEK293T cells expressing an mGFP fusion with a human-mouse chimeric DLK1 protein in which protein segments containing one or more EGF-like domains from DLK1 (HM8-mGFP) have been replaced with the corresponding regions from the mouse DLK1 protein. The DLK1-561-F1 antibody binds to a region for regions 6 and 7 (HM6 and HM7) of the human DLK1 protein, which corresponds to amino acid residues 55-113. Top table: AF647 fluorescence of control HEK293T cells and engineered HEK293T cells is reported for the DLK1-561-F1-AF647 antibody as well as hIgG1-AF647 antibody control and no antibody control. Bottom table: GFP fluorescence. [Diagram 35] The amino acid sequences of the long form of the human DLK1 protein and the regions (light blue; the top two arrows below each set of aligned sequences) that were used to exchange segments of human DLK1 with the corresponding segments of the mouse DLK1 protein are shown. The amino acid sequence differences between the mouse, rat, and cynomolgus DLK1 proteins and the human DLK1 protein are also shown. [Diagram 36]The 04-561-F1 antibody binding site in the predicted structure of human DLK1 protein long form (UniProtKB: P80370) and the topology of long and short forms of human DLK1 protein is shown. This figure shows (A) the predicted structure and topology of long form human DLK1 protein (UniProtKB: P80370), and (B) long and short forms of human DLK1 protein. (A) For the long form of human DLK1 protein (DLK-FL), the predicted structure, topology, and the positions of amino acid numbers 24 and 169 are shown, along with those of the signal peptide and transmembrane domain followed by the cytoplasmic domain. Six EGF-like domains are found at amino acid numbers 24 to 245. Based on the epitope mapping experiment results in FIG. 34, DLK1-561-F1 antibody binds to epitopes in the EGF-like 2 and EGF-like 3 domains corresponding to amino acid numbers 55 to 113. (B) Diagram of the two known human isoforms of DLK1: full-length cleavable isoform 1 (DLK1-FL) and membrane-bound isoform 2 (DLK1-MB). The large bolt identifies the ADAM17-mediated juxtamembrane cleavage site of the full-length ligand. The smaller bolts identify other protease cleavage sites. [Figure 37] Flow cytometric assessment of binding activity of DLK1-561-F1 (also called 561-F1), LegoChem 18A5, and human IgG1 (hIgG1) control antibodies to parental HEK293T cells (HEK293T parent), HEK293 cells expressing a long form of the human DLK1-mGFP fusion protein (HEK293T DLK1 mGFP G06 (long form)), or HEK293T cells expressing a short form of the human DLK1-mGFP fusion protein (HEK293T DLK1 Iso2-mGFP large population (short form)). Upper panel: (direct) flow cytometric analysis of cells bound to anti-DLK1 antibodies conjugated to the AF-647 fluorophore. Lower panel: (indirect) flow cytometric analysis of cells bound to anti-DLK1 antibody detected with a secondary anti-His epitope tag antibody conjugated to the AF-647 fluorophore. [Figure 38]The DLK1-561-F1 antibody binds to both isoforms of the DLK1 protein, whereas the LegoChem 18A5 antibody binds only to the long chain form. This figure shows the binding curves and EC50 values ​​of the DLK1-561-F1 and LegoChem 18A5 antibodies. [Figure 39] Figure 1 shows that DLK1 antibody 561-F1 induces ADCC in native and overexpressing models of DLK1 expression. This figure shows the ADCC potency of DLK1-561-F1 in (A) DLK1+CORL279 and DLK1-M202 cells, and (B) HEK293 cells engineered to overexpress the short form of DLK1 (DLK-MB). NFAT activation, indicative of induction of an ADCC response, was assessed by determining Lucia luciferase activity in the supernatant. The data show that DLK1-561-F1 antibody induced ADCC in native and overexpressing models of DLK1 expression, whereas LegoChem 18A5 antibody and hIgG1 control antibody failed to induce ADCC responses in native DLK1 expressing CORL279 cells and HEK293 cells overexpressing the short form of DLK1. [Diagram 40] 1 shows that DLK1 scFv and BiTE selectively bind to DLK1+CORL279 cells and not to DLK1-M202 cells. DLK1 scFv are 04-0547-h2scfv, 04-0561-F1scfv, and 04-0562-h10scfv, each of which further comprises a (His)6 tag at its C-terminus. DLK1 BiTE are 04-0547-h2Bs, 04-0561-F1Bs, and 04-0562-h10Bs, each of which further comprises a (His)6 tag at its C-terminus. scFv and BiTE antibodies have a terminal (His)6 epitope tag that can be detected by Alexa Fluor® 647-labeled anti-His tag secondary antibody (BioLegend catalog number: 652513). AF647 fluorescence of non-antibody treated cells and cells treated with only the AF647 anti-His tag secondary antibody served as negative controls. [Diagram 41]Figure 1 shows anti-DLK1-CD3 bispecific antibodies (BiTEs) evaluated for T cell activation using Jurkat cells with NFAT-RE reporter. Co-incubation of Jurkat T cells from 04-0547-h2Bs or 04-0561-F1Bs bispecific antibodies and Promega's T cell activation bioassay kit (NFAT-RE J1621) with DLK1+ cell lines (JR and CORL-279) resulted in significant T cell activation, whereas co-incubation with DLK1- cell line (Raji) did not result in significant T cell activation. Anti-DLK1-CD3 BiTE 04-0562-h10Bs was unable to induce T cell activation. BiTEs used herein further comprise a (His)6 tag at their C-terminus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present disclosure describes antigen binding proteins against DLK1 for treating DLK1-expressing cancers.

[0019] Antigen-binding proteins Provided herein is an antigen binding protein that binds to DLK1.The antigen binding protein of the present disclosure can take any one of the many forms of antigen binding protein known in the art.In various embodiments, the antigen binding protein of the present disclosure takes the form of antibody, or antigen-binding antibody fragment, or antibody protein preparation.

[0020] In various embodiments of the present disclosure, the antigen binding protein comprises, consists essentially of, or consists of an antibody. As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin format, including heavy and light chains, and including a variable region and a constant region. For example, the antibody may be an IgG, which is a "Y-shaped" structure consisting of two identical pairs of polypeptide chains, each pair having one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). An antibody has a variable region and a constant region. In the IgG format, the variable region is generally about 100-110 amino acids or more, includes three complementarity determining regions (CDRs), and is primarily responsible for antigen recognition and is substantially different between other antibodies that bind different antigens. The constant region allows the antibody to recruit cells and molecules of the immune system. The variable region is composed of the N-terminal region of each light chain and heavy chain, and the constant region is composed of the C-terminal portion of each heavy chain and light chain (Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes", Immunobiology: The Immune System in Health and Disease, 4 th ed.Elsevier Science Ltd. / Garland Publishing, (1999)).

[0021] The general structure and properties of antibody CDRs have been described in the art. Briefly, in an antibody scaffold, CDRs are embedded within frameworks in the heavy and light chain variable regions, where they constitute the regions primarily responsible for antigen binding and recognition. A variable region typically comprises at least three heavy or light chain CDRs (Kabat et al., 1991; see also Chothia and Lesk, 1987, supra; Chothia et al., 1989, Nature 342:877-883) within a framework region (referred to as framework regions 1-4, FR1, FR2, FR3, and FR4 by Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; see also Chothia and Lesk, 1987, supra; Chothia et al., 1989, Nature 342:877-883). CDRs can be annotated by various means, including methods according to Kabat, AbM, or IMGT. Thus, CDRs of the same antibody may contain different sequences depending on which method was used to annotate the CDR sequences. Such are illustrated in the tables presented herein. In related embodiments, framework residues are altered. The heavy chain framework regions that may be altered are within the regions designated H-FR1, H-FR2, H-FR3, and H-FR4 that surround the heavy chain CDR residues, and the light chain framework region residues that may be altered are within the regions designated L-FR1, L-FR2, L-FR3, and L-FR4 that surround the light chain CDR residues. Amino acids within the framework regions can be replaced with any suitable amino acid identified, for example, in the human framework or human consensus framework.

[0022] An antibody can include any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. Embodiments of the present disclosure include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa or lambda type light chain constant region, such as a human kappa or lambda type light chain constant region. The heavy chain constant region can be, for example, an alpha, delta, epsilon, gamma, or mu type heavy chain constant region, such as a human alpha, delta, epsilon, gamma, or mu type heavy chain constant region. Thus, in various embodiments, the antibody is of isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4. In various aspects, the antibody comprises a constant region that comprises one or more amino acid modifications relative to the naturally occurring counterpart to improve half-life / stability or to make the antibody more suitable for expression / manufacturability. In various cases, the antibody comprises a constant region in which the C-terminal Lys residue present in the naturally occurring counterpart has been removed or truncated.

[0023] The antibody may be a monoclonal antibody. In some embodiments, the antibody comprises a sequence substantially similar to a naturally occurring antibody produced by a mammal, such as a mouse, rabbit, goat, horse, chicken, hamster, human, etc. In this regard, the antibody may be considered a mammalian antibody, such as a mouse antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, human antibody, etc. In certain aspects, the antigen binding protein is an antibody, such as a human antibody.

[0024] In certain embodiments, the antigen-binding protein is a chimeric antibody or a humanized antibody. The term "chimeric antibody" refers to an antibody that contains domains from two or more different antibodies. A chimeric antibody can, for example, contain a constant domain from one species and a variable domain from a second species, or more commonly, contain a stretch of amino acid sequences from at least two species. A chimeric antibody can also contain domains from two or more different antibodies in the same species.

[0025] The term "humanized" when used in the context of an antibody refers to an antibody having at least a CDR region from a non-human source that has been engineered to have a structure and immunological function more similar to a true human antibody than the original source antibody. For example, humanizing can involve transplanting CDRs from a non-human antibody, such as a mouse antibody, into a human antibody. Humanizing can also involve selective amino acid substitutions to make the non-human sequence more similar to a human sequence. Information including sequence information of human antibody heavy and light chain constant regions is publicly available through the Uniprot database and other databases well known to those skilled in the art of antibody engineering and production. For example, IgG1 constant regions are available from the Uniprot database described below, which is incorporated herein by reference. Additionally, in another example, IgG2 constant regions are available from the Uniprot database as Uniprot number P01859, which is incorporated herein by reference.

[0026] By way of example only, the sequence of a mouse immunoglobulin kappa light chain constant region or immunoglobulin gamma-2A heavy chain constant region includes the following: [Table 1] By way of example only, the sequences of the human immunoglobulin kappa light chain constant region, the human immunoglobulin lambda constant 2 light chain region, the human immunoglobulin gamma 1 heavy chain constant region, and the human immunoglobulin gamma 2 heavy chain constant region include the following: [Table 2]

[0027] Antibodies can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves antibodies to produce two Fab fragments and a single Fc fragment. Pepsin cleaves antibodies to produce an F(ab')2 fragment and a pFc' fragment. In various aspects of the disclosure, the antigen-binding proteins of the disclosure are antigen-binding fragments of antibodies (also known as antigen-binding antibody fragments, antigen-binding fragments, or antigen-binding portions). In various cases, the antigen-binding antibody fragment is a Fab fragment or a F(ab')2 fragment.

[0028] Antibody architecture has been exploited to generate a diverse range of alternative antibody formats, spanning a molecular weight range of at least about 12-150 kDa, with a range of valencies (n) from monomers (n=1), to dimers (n=2), trimers (n=3), tetramers (n=4), and potentially even higher. Such alternative antibody formats are referred to herein as "antibody protein formulations." Antibody protein formulations include those based on the complete antibody structure, as well as those that mimic antibody fragments that retain full antigen-binding ability, such as scFv, Fab, and VHH / VH (discussed below). The smallest antigen-binding fragment that retains its complete antigen-binding site is the Fv fragment, which consists entirely of the variable (V) region. To stabilize the molecule, soluble and flexible amino acid peptide linkers are used to connect the V regions to scFv (single chain fragment variable) fragments or constant (C) domains are added to the V regions to generate Fab fragments (fragment, antigen-binding). Both scFv and Fab fragments can be easily produced in host cells, e.g., prokaryotic host cells. Other antibody protein formulations include disulfide bond stabilized scFv (ds-scFv), single chain Fab (scFab), and dimeric and multimeric antibody formats, e.g., diabodies, triabodies and tetrabodies, or minibodies (miniAbs), which include different formats consisting of scFv linked to oligomerization domains. The smallest fragments are the VHH / VH of camelid heavy chain Abs and single domain Abs (sdAbs). The most frequently used building block for creating new antibody formats is the single-chain variable (V) domain antibody fragment (scFv), which contains V domains (VH and VL domains) from heavy and light chains linked by a peptide linker of about 15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another antibody protein formulation. The structure of a peptibody consists of a biologically active peptide grafted into an Fc domain. Peptibodies are well described in the art. See, for example, Shimamoto et al., mAbs 4(5):586-591 (2012).

[0029] Other antibody protein formulations include single chain antibodies (SCAs), diabodies, triabodies, tetrabodies, bispecific or trispecific antibodies, etc. Bispecific antibodies can be divided into five major classes: BsIgG, appended IgG, bispecific antibody (BsAb) fragments, bispecific fusion proteins, and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2)Part A:97-106(2015).

[0030] In various aspects, the antigen binding proteins of the disclosure comprise, consist essentially of, or consist of any one of these antibody protein formulations. In various aspects, the antigen binding proteins of the disclosure comprise, consist essentially of, or consist of any one of the following: scFv, Fab, VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibodies, multimeric antibodies (e.g., diabodies, triabodies, tetrabodies), miniAbs, peptibodies, VHH / VH of camelid heavy chain antibodies, sdAbs, diabodies; triabodies; tetrabodies; bispecific or trispecific antibodies, BsIgG, adduct IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates.

[0031] In various cases, the antigen binding proteins of the present disclosure are antibody protein preparations in monomeric form or in polymeric, oligomeric, or multimeric form. In certain embodiments where an antibody comprises two or more distinct antigen binding region fragments, the antibody is considered bispecific, trispecific, or multispecific, or bivalent, trivalent, or multivalent, depending on the number of distinct epitopes recognized and bound by the antibody.

[0032] In various embodiments, the anti-DLK1 antibody or antibody variant thereof is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single chain antibody, a monomeric antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0033] In various aspects, the antigen binding proteins of the present disclosure are linked to a therapeutic agent. As described below, the therapeutic agent can be any known in the art, including but not limited to chemotherapeutic agents, cytokines and growth factors, cytotoxic agents, etc. See "Conjugates" below.

[0034] In various aspects, any polypeptide, e.g., an antigen binding protein, of the present disclosure may further comprise a heterologous peptide or polypeptide. In some embodiments, the heterologous peptide or polypeptide may be a marker or tag that can be detected directly (e.g., GFP) or indirectly (e.g., using a secondary antibody that binds to the tag). Examples of detectable markers include various enzymes, prosthetic groups, and tags (e.g., histidine tags, myc tags, flag tags, etc.). Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of bioluminescent materials include luciferase, luciferin, fluorescent proteins (e.g., GFP, RFP, etc.), and aequorin.

[0035] Nonfucosylated antibodies Many secreted proteins undergo post-translational glycosylation, a process in which sugar moieties (e.g., glycans, sugars) are covalently attached to specific amino acids of proteins. In eukaryotic cells, two types of glycosylation reactions occur: (1) N-linked glycosylation, in which the glycan is attached to an asparagine at the recognition sequence Asn-X-Thr / Ser (where "X" is any amino acid except proline), and (2) O-linked glycosylation, in which the glycan is attached to a serine or threonine. Regardless of the type of glycosylation (N-linked or O-linked), microheterogeneity of protein glycoforms exists due to the wide range of glycan structures associated with each site (O or N).

[0036] All N-glycans have a common core sugar sequence: Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr(Man3GlcNAc2Asn) and are classified into one of three types: (A) high mannose (HM) or oligomannose (OM) type, consisting of two N-acetylglucosamine (GalNAc) moieties and multiple (e.g., 5, 6, 7, 8 or 9) mannose (Man) residues, (B) complex type, containing three or more GlcNAc moieties and any number of other glycotypes, or (C) hybrid type, containing a Man residue on one side of the branch and a GlcNAc at the base of the complex branch.

[0037] N-linked glycans typically include one or more monosaccharides of galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (GLc), N-acetylglucosamine (GlcNAc), glucosamine (GlcN), mannose (Man), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), N0-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-doxinononic acid (Kdn), fucose (Fuc), glucuronic acid (GLcA), iduronic acid (IdoA), galacturonic acid (GalA), and mannuronic acid (ManA).

[0038] N-linked glycosylation begins in the endoplasmic reticulum (ER), where a complex series of reactions results in the attachment of a core glycan structure essentially composed of two GlcNAc and three Man residues. The glycan complexes formed in the ER are modified by the action of enzymes in the Golgi apparatus. If the sugars are relatively inaccessible to enzymes, they typically remain in their original HM form. If the sugars are accessible to enzymes, many Man residues are cleaved and the sugars are further modified to produce complex N-glycan structures. For example, mannosidase-1, located in the cis Golgi, can cleave or hydrolyze HM glycans, and fucosyltransferase FUT-8, located in the medial Golgi, fucosylates glycans (Hanrue Imai-Nishiya (2007), BMC Biotechnology, 7:84).

[0039] Thus, the sugar composition and structural arrangement of glycan structures vary depending on, among other factors, the glycosylation machinery in the ER and Golgi apparatus, the accessibility of the glycan structures to the enzymes in the machinery, the order of action of each enzyme, and the stage at which the protein is released from the glycosylation machinery.

[0040] In an exemplary embodiment of the disclosure, the antigen binding protein comprises an Fc polypeptide. The term "Fc polypeptide" as used herein includes native and mutein forms of a polypeptide derived from an Fc region of an antibody. In an exemplary aspect, the Fc polypeptide of the antigen binding protein of the disclosure comprises a glycan. In various cases, the glycan lacks fucose or is non-fucosylated. In an exemplary aspect, the antigen binding protein comprises a non-fucosylated glycan. As used herein, the term "non-fucosylated glycan" or "non-fuco glycan" or "non-fucosylated glycoform" or "Afuc" refers to a glycoform that lacks core fucose, e.g., α1,6-linked fucose at the GlcNAc residue involved in the amide bond with Asn at the N-glycosylation site. Non-fucosylated glycoforms include, but are not limited to, A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5. Further non-fucosylated glycans include, for example, A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], FO-N[H3N3]. See, e.g., Reusch and Tejada, Glycobiology 25(12):1325-1334 (2015).

[0041] The present disclosure also provides compositions, e.g., pharmaceutical compositions, comprising antigen binding proteins comprising Fc polypeptides comprising nonfucosylated glycans. In exemplary embodiments, at least or about 25% of the antigen binding proteins present in the composition are antigen binding proteins comprising Fc polypeptides comprising nonfucosylated glycans. In exemplary embodiments, at least or about 25% of the antigen binding proteins present in the composition are nonfucosylated. Optionally, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the antigen binding proteins present in the composition are nonfucosylated. Methods for making compositions comprising antigen binding proteins of specific glycoprofiles are known in the art. In exemplary embodiments, the antigen binding proteins are made recombinantly in cells that have been genetically modified to alter the activity of enzymes of the de novo or salvage pathways. In an exemplary embodiment, the cells are genetically modified to alter the activity of any one or more of fucosyl-transferases (FUTs, e.g., FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucose kinase, GDP-fucose pyrophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD), and GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase (FX). In an exemplary embodiment, the cells are genetically modified to knock out the gene encoding FX. See, e.g., International Patent Publication No. WO2017 / 079165A1; Kanda et al., J Biotechnol 130, 2007, 300-310; Yamane-Ohunuki et al., Biotechnol Bioeng 87, 2004, 614-622; Malphettes et al., Biotechnol Bioeng 106, 2010, 774-783.

[0042] Bispecific Format In exemplary aspects, the antigen binding protein is bispecific and thus capable of binding to two different and distinct antigens. In exemplary embodiments, the antigen binding protein is bispecific and binds to DLK1 and a second antigen.

[0043] In an exemplary embodiment, the second antigen is a cell surface protein expressed by a T cell. In an exemplary embodiment, the cell surface protein is a component of the T cell receptor (TCR), such as CD3. In an exemplary embodiment, the second antigen is a costimulatory molecule that supports T cell activation, such as CD40 or 4-1BB (CD137). In an exemplary embodiment, the second antigen is an Fc receptor. In various embodiments, the Fc receptor is an Fc gamma receptor, an Fc-alpha receptor, or an Fc-epsilon receptor. In an exemplary embodiment, the Fc receptor is CD64 (Fc-gamma RI), CD32 (Fc-gamma RIIA), CD16A (Fc-gamma RIIIA), CD16b (Fc-gamma RIIIb), FcεRI, CD23 (Fc-epsilon RII), CD89 (Fc-epsilon RI), Fcα / μR, or FcRn. In an exemplary embodiment, the Fc receptor is CD16A. In an exemplary case, the second antigen is an immune checkpoint molecule, e.g., a protein involved in an immune checkpoint pathway. Immune checkpoint pathways and the molecules or proteins that function therein are known in the art. See, e.g., Pardoll, Nat Rev Genet 12(4):252-264(2012). In an exemplary case, the immune checkpoint molecule is A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, NOX2, PD-1, TIM3, VISTA, or SIGLEC7. Optionally, the immune checkpoint molecule is PD-1, LAG3, TIM3, or CTLA4.

[0044] Over 50 bispecific antigen binding protein formats are known in the art, some of which are described in Kontermann and Brinkmann, Drug Discovery Today 20(7):838-847(2015); Zhang et al., Exp Hematol Oncol 6:12(2017); Spiess et al., Mol Immunol.;67(2 Pt A):95-106(2015). In exemplary aspects, the bispecific antigen binding proteins of the present disclosure are produced via chemical engineering, genetic engineering, or quadroma technology.

[0045] In exemplary embodiments, the bispecific antigen binding protein is constructed using some or all of the constant domains of an antibody. In exemplary embodiments, the bispecific antigen binding protein of the present disclosure comprises an Fc polypeptide and retains Fc-mediated effector functions. In various cases, the bispecific antigen binding protein is a bispecific monoclonal antibody formed, for example, by chemical crosslinking of two monoclonal antibodies (mab) or by knob-and-hole technology. In exemplary embodiments, the bispecific antigen binding protein is produced via the "knob-into-hole" technology, which forces H-chain heterodimerization by introducing different mutations into the two CH3 domains to generate an asymmetric antibody. The "knob" mutation is made in one HC and the "hole" mutation is made in the other HC to promote heterodimerization. In exemplary embodiments, the bispecific antigen binding protein is a bispecific antibody generated by quadroma technology, which is based on somatic cell fusion of two different hybridoma cells that produce monoclonal antibodies with the desired specificity. Zhang et al., 2017 (supra). In exemplary embodiments, the bispecific antigen binding proteins are crossMab, ortho-Fab IgG, DVD-Ig, two-in-one IgG, IgG-scFv and scFv2-Fc (Kontermann and Brinkmann, 2015, supra). In various embodiments, the bispecific antigen binding proteins are Ig-scFv fusions, e.g., IgG C-terminal scFv fusions and IgG N-terminal scFv fusions, in which new antigen-binding moieties are added to full-length IgG, resulting in a fusion protein with four valencies for two distinct antigens. In an exemplary case, the bispecific antigen binding protein is a dual variable domain IgG (DVD-IgG), in which the LC and HC variable regions of an IgG specific for one antigen are fused via a linker to the N-terminal LC and HC variable regions of an IgG specific for a second antigen to form a DVD-IgG. In an exemplary embodiment, the bispecific antigen binding protein is a diabody-Fc fusion, which involves replacing the Fab fragment of an IgG with a bispecific diabody.

[0046] In alternative cases, the bispecific antigen binding proteins of the present disclosure do not include an Fc polypeptide. In exemplary embodiments, the bispecific antigen binding proteins include the variable domains of each parent monoclonal antibody, with a linker cloned and joined to form a single chain bispecific antibody. In exemplary embodiments, the bispecific antigen binding proteins are tandem scFv, diabody formats, single chain diabodies, tandem diabodies (TandAbs), dual affinity retargeting molecules (DARTs), dock-and-lock (DNLs), and nanobodies (Fan et al., J Hematol Oncol. 2015;8:130). In various embodiments, the bispecific antigen binding proteins include bispecific F(mab 1 )2, scFv, bispecific diabody (BsDb), single chain bispecific diabody (scBsDb), single chain bispecific tandem variable domain (scBsTaFv), dock-and-lock trivalent Fab (DNL-(Fab)3), single domain antibody (sdAb), or bispecific single domain antibody (BssdAb). In an exemplary embodiment, the bispecific antigen binding protein is a tandem scFv comprising two scFv fragments linked by an additional peptide linker, such as a glycine-serine repeat motif. Optionally, the tandem scFv has the structure: VL A -Linker1-VH A -Linker2-VH B -Linker3-VL B(VL and VH are derived from single chain antibody fragments, and A and B represent parent monoclonal antibodies A and B). In an exemplary embodiment, the bispecific antigen binding protein is a TandAb, which contains two pairs of VL and VH domains connected in a single polypeptide chain (Reusch et al., MAbs. 2015;7(3):584-604). The two polypeptide preparations dimerize head-to-tail and form a homodimer of large molecular weight (about 105 kDa) when expressed. In an exemplary embodiment, the bispecific antigen binding protein is one that is produced using the crossMab technology described in PNAS 108(27):11187-92 (2011). CrossMab is produced by a method that does not have any chemical linker or connector and performs precise light chain association in a bispecific heterodimeric IgG antibody. In exemplary embodiments, the CrossMab is a bivalent (1+1), trivalent (2+1) or tetravalent (2+2) bispecific crossMab or a non-Fc tandem antigen-binding fragment (Fab) based crossMab. Fab , crossMab VH-VL , or crossMab CH1-CL It is.

[0047] In exemplary aspects, the bispecific antigen binding protein comprises a single domain antibody or nanobody comprising a single monomeric variable antibody domain. Optionally, the variable domain is based on a heavy chain variable domain. In alternative aspects, the variable domain is based on a light chain variable domain.

[0048] In an exemplary aspect, the bispecific antigen binding protein is a bispecific T cell engager or BiTE®. A BiTE is a bivalent small molecule that contains only the variable regions of an antibody in the form of an scFv connected by a flexible peptide linker. In an exemplary aspect, the bispecific antigen binding protein comprises an scFV that contains the LC and HC variable regions of the DLK1 antibody of the present disclosure and the LC and HC variable regions of a second antibody specific for a second antigen. In some embodiments, the BiTE contains the LC and HC variable regions of a second antibody specific for CD3. In some embodiments, the CD3 is CD3E.

[0049] In an exemplary case, the bispecific antigen-binding protein is a dual affinity retargeting (DART), which differs from BiTE® in that the degree of freedom of the antigen-binding site is restricted by the covalent bond between the two chains of DART. Therefore, DART is structurally compact and can form stable contact between target and effector cells. DART comprises two engineered Fv fragments, which exchange their own VH with the VH of the other. The interchanged Fv domains advantageously free the variant fragments from the conformational constraints imposed by the short connecting peptide.

[0050] In an exemplary embodiment, the bispecific antigen binding protein is an HSABody comprising two scFvs fused to modified HSA. HSABody is described in McDonagh et al., Mol Cancer Ther. 2012;11(3):582-93.

[0051] Thus, in an exemplary embodiment, the bispecific antigen-binding protein comprises an antigen-binding fragment of any of the DLK1 antibodies of the present disclosure. In an exemplary embodiment, the antigen-binding fragment is a Fab. In an exemplary embodiment, the bispecific antigen-binding protein comprises a F(ab) of any of the DLK1 antibodies of the present disclosure. 2'. In an exemplary embodiment, the bispecific antigen binding protein comprises an scFv comprising the LC and HC variable regions of any of the DLK1 antibodies of the present disclosure. In an exemplary embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 514 or 515. In various embodiments, the antigen binding fragment is based on the heavy chain variable region, and in other embodiments, the antigen binding fragment is based on the light chain variable region. In an exemplary embodiment, the antigen binding fragment comprises at least a portion of both the HC variable region and the LC variable region. In an exemplary embodiment, the bispecific antigen binding protein comprises at least one, but not both, of the LC or HC variable region of the DLK1 antibody of the present disclosure and at least one, but not both, of the LC and HC variable regions of a second antibody specific for a second antigen. In an exemplary case, the bispecific antigen binding protein comprises an scFV comprising the LC and HC variable regions of the DLK1 antibody of the present disclosure and the LC and HC variable regions of a second antibody specific for a second antigen.

[0052] nucleic acid The present disclosure further provides a nucleic acid comprising a nucleotide sequence encoding an antigen binding protein of the present disclosure. "Nucleic acid," as used herein, includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA, or modified forms thereof, that may be single-stranded or double-stranded, that may be synthesized or obtained (e.g., isolated and / or purified) from a natural source, that may contain natural, non-natural, or modified nucleotides, and that may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters found between the nucleotides of unmodified oligonucleotides. A nucleic acid may include any nucleotide sequence that encodes any of the antigen binding proteins of the present disclosure.

[0053] The present invention further provides a nucleic acid molecule encoding an amino acid sequence corresponding to the antigen binding protein of the present invention. In some embodiments, the nucleic acid molecule is DNA (e.g., cDNA) or a hybrid thereof. Alternatively, the molecule is RNA or a hybrid thereof.

[0054] In some aspects, the nucleic acid of the present disclosure is recombinant.As used herein, the term "recombinant" refers to a molecule that is (i) constructed outside a living cell by combining a natural or synthetic nucleic acid segment with a nucleic acid molecule that can be replicated in the living cell, or (ii) resulting from the replication of the molecule described in (i) above.For the purposes of this specification, replication can be in vitro replication or in vivo replication.

[0055] Any nucleic acid of the present disclosure may be codon-optimized. The frequency of codon usage within a gene helps determine the achievable protein expression level. Certain sequences can be more easily translated by certain hosts, so selecting the appropriate codons for a given host may be necessary to maximize expression. Methods for optimizing codons are well known in the art. Many online tools exist, including, for example, the World Wide Web at codonstatsdb.unr.edu (see Subramanian et al. (2022) Mol Biol Evol 3;39(8):msac157).

[0056] Nucleic acids are constructed in some embodiments based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, e.g., Sambrook et al., supra; and Ausubel et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substituted adenines, 7-methylguanine, 5-methylammomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present disclosure can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0057] vector The nucleic acids of the present disclosure are incorporated into vectors in some aspects. In this regard, the present disclosure provides vectors that include any of the nucleic acids of the present disclosure. In various aspects, the vector is a recombinant expression vector. For the purposes of this specification, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that allows expression of an mRNA, protein, polypeptide, or peptide by a host cell when the construct includes a nucleotide sequence that encodes the mRNA, protein, polypeptide, or peptide, and the vector contacts the cell under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed in the cell. The vectors of the present disclosure are not naturally occurring in their entirety. However, portions of the vectors may be naturally occurring. The vectors of the present disclosure may be single-stranded or double-stranded, may be partially synthesized or obtained from natural sources, and may contain any type of nucleotide, including but not limited to DNA and RNA, which may contain natural, non-natural, or altered nucleotides. The vectors may include naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. In some aspects, the modified nucleotides or non-naturally occurring internucleotide linkages do not interfere with the transcription or replication of the vector.

[0058] The vector of the present disclosure can be any suitable vector and can be used to transduce, transform, or transfect any suitable host. Suitable vectors include those designed for propagation and multiplication, or for expression, or both, such as plasmids and viruses. The vector can be a plasmid-based expression vector. In various aspects, the vector is selected from the group consisting of pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGTIO, λGTl1, λZapII (Stratagene), λEMBL4, and λNMl149 can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some aspects, the vector is a viral vector, e.g., a retroviral vector. In various aspects, the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes simplex viral (HSV) vector, a vesicular stomatitis viral (VSV) vector, a vaccinia viral vector, or a lentiviral vector. See, e.g., Howarth et al., Cell Biol. Toxicol. 26(1):1-20 (2010). In various aspects, the vector is a baculoviral vector that infects arthropods, e.g., insects. In various embodiments, the baculovirus vector is Autographa californica polynuclear virus (AcMNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV).See, e.g., Khan, Adv Pharm Bull 3(2):257-263 (2013); Miller, Bioessays 11(4):91-96 (1989); Atkinson et al., Pestic Sci 28:215-224 (1990).

[0059] The vectors of the present disclosure can be prepared using standard recombinant DNA techniques, for example, as described in Sambrook et al., supra, and Ausubel et al., supra. Circular or linear expression vector constructs can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from CoIE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0060] In some aspects, vectors include regulatory sequences, such as transcriptional and translational initiation and termination codons, that are specific to the type of host (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced, as appropriate and taking into account whether the vector is DNA- or RNA-based.

[0061] Vectors can contain one or more marker genes to allow for the selection of transformed or transfected hosts. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. Suitable marker genes for expression vectors of the present disclosure include, for example, neomycin / G418 resistance gene, hygromycin resistance gene, histidinol resistance gene, tetracycline resistance gene, and ampicillin resistance gene.

[0062] The vector may comprise a natural or normative promoter operably linked to a nucleotide sequence encoding a polypeptide (including functional parts and functional variants thereof) or a nucleotide sequence that is complementary or hybridizes to the nucleotide sequence encoding the polypeptide. The selection of a promoter, such as a strong, weak, inducible, tissue-specific and development-specific promoter, is within the ordinary skill of the art. Similarly, it is also within the skill of the art to combine a nucleotide sequence with a promoter. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long terminal repeat of murine stem cell virus.

[0063] host cell A host cell comprising the nucleic acid or vector of the present disclosure is provided herein. As used herein, the term "host cell" refers to any kind of cell that can contain the vector of the present disclosure and produce the expression product (e.g., mRNA, protein) encoded by the nucleic acid. In some embodiments, the host cell is an adherent cell or a suspension cell, i.e., a cell that grows in suspension. In various embodiments, the host cell is a cultured cell or a primary cell, i.e., a cell that is directly isolated from an organism, such as a human. The host cell can be any cell type, originate from any kind of tissue, and can be at any developmental stage.

[0064] In various aspects, the antigen binding protein is a glycosylated protein and the host cell is a cell capable of glycosylation. In various aspects, the cell capable of glycosylation is a eukaryotic cell, including but not limited to a yeast cell, a filamentous fungal cell, a protozoan cell, an algae cell, an insect cell, or a mammalian cell. Such host cells are described in the art. See, for example, Frenzel, et al., Front Immunol 4:217 (2013). In various aspects, the eukaryotic cell is a mammalian cell. In various aspects, the mammalian cell is a non-human mammalian cell. In some embodiments, the cells are selected from the group consisting of Chinese hamster ovary (CHO) cells and derivatives thereof (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical carcinoma cells (e.g., HeLa), human osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonal carcinoma cells P19, mouse embryonic fibroblast cells NIH 3T3, mouse fibroblast L929, mouse neuroblastoma N2a, human breast cancer MCF-7, retinoblastoma Y79, human retinoblastoma SO-Rb50, human hepatoma Hep G2, mouse myeloma B J558L, or baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263(2013)).

[0065] For purposes of amplifying or replicating a vector, the host cell is in some aspects a prokaryotic cell, such as a bacterial cell.

[0066] Also provided by the present disclosure is a population of cells comprising at least one host cell as described herein. The population of cells is, in some aspects, a heterogeneous population comprising host cells comprising the vector as described and at least one other cell that does not comprise any vector. Alternatively, in some aspects, the population of cells is a substantially homogeneous population that comprises primarily (e.g., essentially) host cells comprising vector. The population is, in some aspects, a clonal population of cells, in which all cells of the population are clones of a single host cell comprising vector, such that all cells of the population comprise vector. In various embodiments of the present disclosure, the population of cells is a clonal population that comprises host cells comprising vector as described herein.

[0067] Manufacturing method Also provided herein are methods of making an antigen binding protein that binds DLK1. In various embodiments, the method comprises culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding an antigen binding protein as described herein in a cell culture medium and harvesting the antigen binding protein from the cell culture medium. The host cell may be any of the host cells described herein. In various aspects, the host cell is selected from the group consisting of CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In various aspects, the step of culturing the host cell comprises culturing the host cell in a growth medium that supports the growth and proliferation of the host cell. In various aspects, the growth medium increases cell density, culture viability, and productivity in a timely manner. In various aspects, the growth medium comprises amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors. In various aspects, the growth medium is a synthetic medium consisting of amino acids, vitamins, trace elements, inorganic salts, lipids, and insulin or insulin-like growth factors. In addition to nutrients, the growth medium also serves to maintain pH and osmolality. Several growth media are commercially available and described in the art, see, e.g., Arora, "Cell Culture Media: A Review" MATER METHODS 3:175 (2013).

[0068] In various embodiments, the method comprises culturing the host cells in a feed medium. In various embodiments, the method comprises fed-batch culturing in a feed medium. Methods for recombinant protein production are known in the art. See, e.g., Li et al., "Cell culture processes for monoclonal antibody production" MAbs 2(5):466-477 (2010).

[0069] The method of producing an antigen-binding protein may include one or more steps for purifying the protein from the cell culture or its supernatant, and preferably recovering the purified protein. In various embodiments, the method includes one or more chromatography steps, such as affinity chromatography (e.g., Protein A affinity chromatography), ion exchange chromatography, hydrophobic interaction chromatography. In various embodiments, the method includes purifying the protein using a Protein A affinity chromatography resin.

[0070] In various embodiments, the method further comprises a step of formulating the purified protein, etc., thereby obtaining a formulation comprising the purified protein. Such steps are described in Formulation and Process Development Strategies for Manufacturing, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0071] In various aspects, the antigen binding protein linked to the polypeptide and the antigen binding protein are part of a fusion protein. Thus, the present disclosure further provides a method of making a fusion protein comprising an antigen binding protein that binds to DLK1. In various embodiments, the method comprises culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding the fusion protein described herein in a cell culture medium and harvesting the fusion protein from the cell culture medium.

[0072] Conjugates The present disclosure also provides an antigen binding protein bound, linked or conjugated to a second moiety (e.g., a heterologous moiety, a conjugate moiety). Thus, the present disclosure provides a conjugate comprising an antigen binding protein and a heterologous moiety. As used herein, the term "heterologous moiety" is synonymous with "conjugate moiety" and refers to any molecule (naturally occurring or non-encoded chemical or biochemical) that is different from the antigen binding protein of the present disclosure. Various heterologous moieties include, but are not limited to, polymers, carbohydrates, lipids, nucleic acids, oligonucleotides, DNA or RNA, amino acids, peptides, polypeptides, proteins, therapeutic agents (e.g., cytotoxic agents, cytokines), or diagnostic agents.

[0073] In some embodiments, the heterologous moiety is a detectable marker. Examples of detectable markers include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin (PE); an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 H. As used herein, the term "labeled" with respect to an antibody is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g., fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)) to the antibody, and indirect labeling of the antibody by reactivity with a detectable substance. For example, an antibody can be labeled with a nucleic acid sequence that can be amplified and detected, or an antisense oligonucleotide that reduces the expression of a particular gene, such that expression can then be detected and measured.

[0074] In some embodiments, the heterologous moiety is a polymer. The polymer may be branched or unbranched. The polymer may be of any molecular weight. The polymer, in some embodiments, has an average molecular weight of about 2 kDa to about 100 kDa (the term "about" indicates that in preparations of water-soluble polymers, some molecules may be larger and some molecules may be smaller than the stated molecular weight). The average molecular weight of the polymer, in some aspects, is about 5 kDa to about 50 kDa, about 12 kDa to about 40 kDa, or about 20 kDa to about 35 kDa.

[0075] In some embodiments, the polymer is modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled. In some embodiments, the polymer is water-soluble, so that the protein to which the polymer is attached does not precipitate in an aqueous environment, such as a physiological environment. In some embodiments, the polymer is pharma- ceutically acceptable, for example, when the composition is used for therapeutic applications. In addition, in some aspects, the polymer is a mixture of polymers, such as a copolymer, a block copolymer.

[0076] In some embodiments, the polymer may be selected from the group consisting of polyamides, polycarbonates, polyalkylenes and their derivatives, such as polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polymers of acrylic and methacrylic esters, such as poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate), polyvinyl polymers, such as polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyhalogenated vinyl esters, and the like. and polyvinyl, poly(vinyl acetate), and polyvinylpyrrolidone, polyglycolide, polysiloxane, polyurethane and copolymers thereof, cellulose, such as alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, and sodium cellulose sulfate, polypropylene, polyethylene, such as poly(ethylene glycol), poly(ethylene oxide), and poly(ethylene terephthalate), and polystyrene.

[0077] A particularly preferred water-soluble polymer for use herein is polyethylene glycol (PEG). As used herein, polyethylene glycol is intended to encompass any form of PEG that can be used to derivatize other proteins, such as mono-(C1-C10) alkoxy or aryloxy polyethylene glycol. PEG is a linear or branched neutral polyether available in a wide range of molecular weights that is soluble in water and most organic solvents.

[0078] In some embodiments, the heterologous moiety is a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan).

[0079] In some embodiments, the heterologous moiety is a lipid. The lipid is, in some embodiments, a fatty acid, an eicosanoid, a prostaglandin, a leukotriene, a thromboxane, an N-acylethanolamine, a glycerolipid (e.g., mono-, di-, or tri-substituted glycerol), a glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), a sphingolipid (e.g., sphingosine, ceramide), a sterol lipid (e.g., steroid, cholesterol), a prenol lipid, a glycolipid, or a polyketide, an oil, a wax, a cholesterol, a sterol, a fat-soluble vitamin, a monoglyceride, a diglyceride, a triglyceride, or a phospholipid.

[0080] In some embodiments, the heterologous moiety is a therapeutic agent. The therapeutic agent can be any of those known to one of skill in the art. Examples of therapeutic agents contemplated herein include, but are not limited to, natural enzymes, proteins derived from natural sources, recombinant proteins, natural peptides, synthetic peptides, cyclic peptides, antibodies, receptor agonists, cytotoxic agents, immunoglobulins, beta-adrenergic blockers, calcium channel blockers, coronary vasodilators, cardiac glycosides, antiarrhythmics, cardiac sympathomimetics, angiotensin-converting enzyme (ACE) inhibitors, diuretics, vasopressors, cholesterol and triglyceride lowering agents, bile acid sequestrants, and the like. sequestrants, fibrates, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, niacin derivatives, antiadrenergic agents, alpha-adrenergic blocking agents, centrally acting antiadrenergic agents, vasodilators, potassium-sparing agents, thiazides and related agents, angiotensin II receptor antagonists, peripheral vasodilators, antiandrogens, estrogens, antibiotics, retinoids, insulin and analogs, alpha-glucosidase inhibitors, biguanides, meglitinides, sulfonylureas, thiazolidinediones, androgens, progestogens, bone metabolism regulators, anterior pituitary hormones, hypothalamic hormones, posterior pituitary hormones, gonadotropins, gonadotropin-releasing hormone antagonists, ovulation stimulants, selective estrogen receptor modulators -, antithyroid drugs, thyroid hormones, bulking agents, laxatives, antikinetic drugs, bacterial flora regulators, intestinal adsorbents, intestinal anti-infective drugs, appetite stimulants (antianorexics), anti-cachexia drugs, anti-hyperphagia drugs (antibulimics), appetite suppressants, anti-obesity drugs, antacids, upper gastrointestinal drugs, anticholinergic drugs, aminosalicylic acid derivatives, biological response modifiers, corticosteroids, antispasmodics, 5-HT4 partial agonists, antihistamines, cannabinoids, dopamine antagonists, serotonin antagonists, cytoprotective drugs, histamine H2-receptor antagonists, mucosal protective drugs, proton pump inhibitors, H. pylori eradication therapy drugs, erythropoiesis stimulants, hematopoietic drugs, anemia drugs, heparin, antifibrinolytic drugs, hemostatic drugs, blood coagulation factors, adenosine diphosphate inhibitors, glycoprotein receptor inhibitors, fibrinogen-platelet binding inhibitors,Thromboxane-A2 inhibitors, plasminogen activators, antithrombotic agents, glucocorticoids, mineralocorticoids, corticosteroids, selective immunosuppressants, antifungal agents, drugs involved in prophylactic therapy, AIDS-related infections, cytomegalovirus, non-nucleoside reverse transcriptase inhibitors, nucleoside analogue reverse transcriptase inhibitors, protease inhibitors, anemia, Kaposi's sarcoma, aminoglycosides, carbapenems, cephalosporins, glycopeptides, lincosamides, macrolies, oxazolidinones, penicillins, streptogramins, sulfonamides, trimethoprim and derivatives, tetracyclines, anthelmintics, amoebic drugs, biguanides, cinchona alkaloids, folate antagonists, quinoline derivatives, Pneumocystis carinii therapeutic agents, hydrazides, imidazoles, triazoles, nitroimidazoles, cyclic amines, neuraminidase inhibitors, nucleosides, phosphate binders, cholinesterase inhibitors, adjunctive therapies, barbiturates and derivatives, benzodiazepines, gamma aminobutyric acid derivatives, hydantoin derivatives, iminostilbene derivatives, succinimide derivatives, anticonvulsants, ergot alkaloids, antimigraine preparations, biological response modifiers, carbamates, tricyclic derivatives, depolarizing agents, non-depolarizing agents, neuromuscular paralytic agents, CNS stimulants, dopaminergic agents, monoamine oxidase inhibitors, COMT inhibitors, alkylsulfonates, ethylenimines, imidazotetrazines, nitrogen mustard analogues, nitrosoureas, platinum-containing compounds, alternatives antagonists, purine analogs, pyrimidine analogs, urea derivatives, anthracyclines, actinomycin d, camptothecin derivatives, epipodophyllotoxins, taxanes, vinca alkaloids and analogs, antiandrogens, antiestrogens, nonsteroidal aromatase inhibitors, protein kinase inhibitors, antineoplastic agents, azaspirodecanedione derivatives, anxiolytics, stimulants, monoamine reuptake inhibitors, selective serotonin reuptake inhibitors, antidepressants, benzisoxazole derivatives, butyrophenone derivatives, dibenzodiazepine derivatives, dibenzothiazepine derivatives, diphenylbutylpiperidine derivatives, phenothiazines, thienobenzodiazepine derivatives, thioxanthene derivatives, allergen extracts, nonsteroidal drugs,Leukotriene receptor antagonists, xanthines, endothelin receptor antagonists, prostaglandins, pulmonary surfactants, mucolytic agents, antimitotics, uricosurics, xanthine oxidase inhibitors, phosphodiesterase inhibitors, methenamine salts, nitrofuran derivatives, quinolones, smooth muscle relaxants, parasympathomimetics, halogenated hydrocarbons, esters of aminobenzoic acid, amides (e.g., lidocaine, articaine hydrochloride, bupivacaine hydrochloride), antipyretics, hypnotics and sedatives, cyclopyrrolones, pyrazolones Pyrimidines, nonsteroidal anti-inflammatory drugs, opioids, para-aminophenol derivatives, alcohol dehydrogenase inhibitors, heparin antagonists, adsorbents, emetics, opioid antagonists, cholinesterase revitalizers, nicotine replacement therapy, vitamin A analogs and antagonists, vitamin B analogs and antagonists, vitamin C analogs and antagonists, vitamin D analogs and antagonists, vitamin E analogs and antagonists, vitamin K analogs and antagonists.

[0081] The antigen binding proteins of the present disclosure can be conjugated to one or more cytokines and growth factors that are effective in inhibiting tumor metastasis, where the cytokine or growth factor has been shown to have an anti-proliferative effect on at least one cell population. Such cytokines, lymphokines, growth factors, or other hematopoietic factors include, but are not limited to, M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin.Additional growth factors for use herein include angiogenin, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein receptor IA, bone morphogenetic protein receptor IB, brain-derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor alpha, cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil, chemotactic factor 2alpha, cytokine-induced neutrophil chemotactic factor 2beta, beta endothelial cell growth factor, endothelin 1, epithelial-derived neutrophil attractant, glial cell line-derived neurotrophic factor receptor alpha 1, glial cell line-derived neurotrophic factor receptor alpha 2, growth-associated protein, growth-associated protein alpha, growth-associated protein beta ... Protein gamma, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor alpha, nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor alpha, transforming growth factor beta, transforming growth factor beta 1, transforming growth factor beta 1.2, transforming growth factor beta 2, transforming growth factor beta 3, transforming growth factor beta 5, latent transforming growth factor beta 1, transforming growth factor beta binding protein I, transforming growth factor beta binding protein II, transforming growth factor beta binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, and chimeric proteins and biologically or immunologically active fragments thereof.

[0082] In some embodiments, the conjugate comprises an antigen binding protein as described herein and a cytotoxic agent. A cytotoxic agent is any molecule (chemical or biochemical) that is toxic to cells. In some aspects, when a cytotoxic agent is conjugated to an antigen binding protein of the present disclosure, the results obtained are synergistic. That is, the efficacy of a combination therapy of an antigen binding protein and a cytotoxic agent is synergistic. That is, the efficacy is greater than that expected from the additive effect of each individually. Thus, the dosage of the cytotoxic agent can be reduced, thus simultaneously reducing the risk of toxicity issues and other side effects. In some embodiments, the cytotoxic agent is a chemotherapeutic agent. Chemotherapeutic agents are known in the art and include, but are not limited to, platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids, and difluoronucleosides, as described in U.S. Pat. No. 6,630,124.

[0083] In some embodiments, the chemotherapeutic agent is a platinum coordination compound. The term "platinum coordination compound" refers to any platinum coordination compound that provides platinum in ionic form and inhibits tumor cell growth.

[0084] In some embodiments, the platinum coordination complex is cis-diamminediaquoplatinum(II)-ion; chloro(diethylenetriamine)-platinum(II) chloride; dichloro(ethylenediamine)-platinum(II), diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin); spiroplatin; iproplatin; diammine(2-ethylmalonato)-platinum(II); ethylenediaminemalonatoplatinum(II); aqua(1,2-diamino (dicyclohexane)-sulfatoplatinum(II); (1,2-diaminocyclohexane)malonatoplatinum(II); (4-carboxyphthalato)(1,2-diaminocyclohexane)platinum(II); (1,2-diaminocyclohexane)-(isocitrato)platinum(II); (1,2-diaminocyclohexane)cis(pyruvato)platinum(II); (1,2-diaminocyclohexane)oxalatoplatinum(II); ormaplatin; and tetraplatin.

[0085] In some embodiments, cisplatin is the platinum coordination compound used in the compositions and methods of the present invention. Cisplatin is commercially available from Bristol Myers-Squibb Corporation under the name PLATINOL™ and is available as a powder for use with water, sterile saline or other suitable vehicles. Other platinum coordination compounds suitable for use in the present invention are known, commercially available, and / or can be prepared by conventional techniques. For many years, cisplatin or cis-dichlorodiammineplatinum II has been used successfully as a chemotherapeutic agent in the treatment of various human solid malignancies. More recently, other diamino-platinum complexes have also shown efficacy as chemotherapeutic agents in the treatment of various human solid malignancies. Such diamino-platinum complexes include, but are not limited to, spiroplatinum and carboplatinum. Cisplatin and other diamino-platinum complexes have been widely used as chemotherapeutic agents in humans, but have had to be delivered at high dosage levels that can cause toxicity problems such as kidney damage.

[0086] In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerases are enzymes that can change DNA topology in eukaryotic cells. They are important for cell function and cell proliferation. Generally, there are two classes of topoisomerases in eukaryotic cells: type I and type II. Topoisomerase I is a monomeric enzyme with a molecular weight of approximately 100,000. This enzyme binds to DNA, introduces a transient single-strand break, unwinds the double helix (or allows the double helix to unwind), and then dissociates from the DNA strand after reclosing the break. Recently, various topoisomerase inhibitors have shown clinical efficacy in treating humans with ovarian cancer, esophageal cancer, or non-small cell lung cancer.

[0087] In some embodiments, the topoisomerase inhibitor is camptothecin or a camptothecin analog. Camptothecin is a water-insoluble cytotoxic alkaloid produced by the Camptotheca accuminata tree, native to China, and the Nothapodytes foetida tree, native to India. Camptothecin exhibits tumor cell growth inhibitory activity against some tumor cells. Camptothecin analog class compounds are typically specific inhibitors of DNA topoisomerase I. The term "topoisomerase inhibitor" refers to any compound that is structurally related to camptothecin and inhibits tumor cell growth. Camptothecin analog class compounds include, but are not limited to, topotecan, irinotecan, and 9-aminocamptothecin.

[0088] In additional embodiments, the cytotoxic agent is a cytotoxic agent as described in U.S. Pat. No. 5,004,758, issued April 2, 1991, and European Patent Application No. 88311366.4, published June 21, 1989, with 20' Publication No. EP 0321122; U.S. Pat. No. 4,604,463, issued August 5, 1986, and European Patent Application Publication No. EP 0137145, published April 17, 1985; No. 4,473,692, issued Sep. 25, 1984, and European Patent Application Publication No. EP 0074256, published Mar. 16, 1983; U.S. Patent No. 4,545,880, issued Oct. 8, 1985, and European Patent Application Publication No. EP 0074256, published Mar. 16, 1983; European Patent Application Publication No. EP 0088642, published Sep. 14, 1983; Wani et al., J. Med. Chem., 29, 2358-2363 (1986); Nitta et al., Proc. 14th International Congr. Chemotherapy, Kyoto, 1985, Tokyo Press, Anticancer Section 1, p. 28-30, and in particular the compound designated CPT-11, which inhibits tumor cell growth. CPT-11 is a camptothecin analog in which a 4-(piperidino)-piperidine side chain is attached to the C-10 of 10-hydroxy-7-ethylcamptothecin via a carbamate linkage.CPT-11 is currently undergoing human clinical trials and is also called irinotecan; Wani et al, J. Med. Chem., 23, 554 (1980); Wani et al, J. Med. Chem., 23, 554 (1980); the disclosures of each of which are incorporated herein by reference in their entireties. et.al., J. Med. Chem., 30, 1774 (1987); U.S. Patent No. 4,342,776, issued August 3, 1982; U.S. Patent Application No. 581,916, filed September 13, 1990, and European Patent Application Publication No. EP 418099, published March 20, 1991; U.S. Patent No. 4,513,138, issued April 23, 1985, and European Patent Application Publication No. EP 0074770, published March 23, 1983; U.S. Patent No. 4,399,276, issued August 16, 1983, and European Patent Application Publication No. 0056692, published July 28, 1982. All of the compounds of the camptothecin analog class listed above are commercially available and / or can be prepared by conventional techniques, including those described in the references listed above. The topoisomerase inhibitor may be selected from the group consisting of topotecan, irinotecan, and 9-aminocamptothecin.

[0089] In some embodiments, the camptothecin analog is an active metabolite of irinotecan (CPT-11). In some such embodiments, the camptothecin analog is 7-ethyl-10-hydroxycamptothecin (SN-38). As a metabolite, SN-38 is formed by hydrolysis of irinotecan by carboxylesterase. In some embodiments, SN-38 has the following structure: [ka] SN-38 is described in U.S. Patent Application Nos. 7,999,083, 8,080,250, 8,759,496, 8,999,344, 10,195,288, and 9,808,537.

[0090] In some embodiments, the camptothecin analog is exatecan methanesulfonate. Exatecan methanesulfonate is a water-soluble camptothecin (CPT) that exhibits stronger topoisomerase I inhibitory activity and antitumor activity than other CPT analogs. In addition, exatecan is effective against p-glycoprotein (P-gp)-mediated multidrug resistance cells.

[0091] In some embodiments, the camptothecin analog is deruxtecan (Dxd), a potent derivative of exatecan that has 10-fold greater topoisomerase I inhibitory potency than SN-38. In some embodiments, Dxd has the following structure: [ka] Dxd is described in U.S. Patent Application No. 6,407,115, U.S. Patent Application No. 10,195,288, U.S. Patent Application No. 9,808,537, and U.S. Patent Application No. 6,407,115.

[0092] The preparation of numerous compounds of the camptothecin analog class, including pharma- ceutically acceptable salts, hydrates and solvates thereof, as well as oral and parenteral pharmaceutical compositions comprising such compounds of the camptothecin analog class and an inert, pharma- ceutically acceptable carrier or diluent, are described in detail in U.S. Pat. No. 5,004,758, issued Apr. 2, 1991, and European Patent Application No. 88311366.4, published Jun. 21, 1989, with Publication No. EP 0321122, the teachings of which are incorporated herein by reference.

[0093] In yet another embodiment of the invention, the chemotherapeutic agent is an antibiotic compound. Suitable antibiotics include, but are not limited to, doxorubicin, mitomycin, bleomycin, daunorubicin, and streptozocin.

[0094] In some embodiments, the chemotherapeutic agent is an antimitotic alkaloid. In general, antimitotic alkaloids can be extracted from Catharanthus roseus and have been shown to be effective as anticancer chemotherapy agents. A number of semi-synthetic derivatives have been studied both chemically and pharmacologically (see O. Van Tellingen et al, Anticancer Research, 12, 1699-1716 (1992)). Antimitotic alkaloids of the present invention include, but are not limited to, vinblastine, vincristine, vindesine, taxol, and vinorelbine. The latter two antimitotic alkaloids are commercially available from Eli Lilly and Company and Pierre Fabre Laboratories, respectively (see U.S. Patent No. 5,620,985). In some embodiments, the antimitotic alkaloid is vinorelbine.

[0095] In another embodiment of the invention, the chemotherapeutic agent is a difluoronucleoside. 2'-deoxy-2',2'-difluoronucleosides are known in the art to have antiviral activity. Such compounds are disclosed and taught in U.S. Patent Nos. 4,526,988 and 4,808,614. European Patent Application Publication No. 184,365 discloses that these same difluoronucleosides have oncolytic activity. In one particular aspect, the 2'-deoxy-2',2'-difluoronucleoside used in the compositions and methods of the invention is 2'-deoxy-2',2'-difluorocytidine hydrochloride, also known as gemcitabine hydrochloride. Gemcitabine is either commercially available or can be synthesized by the multi-step process disclosed and taught in U.S. Pat. Nos. 4,526,988, 4,808,614 and 5,223,608, the teachings of which are incorporated herein by reference.

[0096] In various embodiments, the chemotherapeutic agent is an antimitotic agent that inhibits cell division by inhibiting tubulin polymerization, destabilizing microtubules, or altering microtubule dynamics, such as a maytansinoid or a derivative thereof (e.g., DM1 or DM4), an auristatin or a derivative thereof. In various cases, the chemotherapeutic agent is an auristatin. For example, the auristatin is, in some embodiments, a dolastatin, monomethyl auristatin E (MMAE), monomethyl auristatin E (MMAE), or PF-06380101. Auristatins are described in the art. See, for example, Maderna, A. et al., Mol Pharmaceutics 12(6):1798-1812 (2015). In various embodiments, the conjugate comprises an antibody of the present disclosure in combination with MMAE. Optionally, the conjugate comprises a linker. In some embodiments, the linker comprises a cleavable linking moiety. In various cases, the conjugate comprises an antibody of the present disclosure linked to a linking group that is linked to a cathepsin-cleavable linker, which is linked to a spacer that is linked to MMAE. In some embodiments, the linking group is attached to the antibody via a Cys residue in the Fc region of the antibody. In an exemplary embodiment, the linking group is represented by Formula I: [ka] In an exemplary embodiment, the cathepsin-cleavable linker comprises the structure of Formula II: [ka] In an exemplary embodiment, the spacer comprises a structure of formula III: [ka] Includes the structure of.

[0097] In some embodiments, MMAE has the following structure: [ka] has.

[0098] The present disclosure also provides a conjugate that comprises the antigen-binding protein of the present disclosure linked to a polypeptide, resulting in a fusion protein.Thus, the present disclosure provides a fusion protein that comprises the antigen-binding protein of the present disclosure linked to a polypeptide.In various embodiments, the polypeptide is a diagnostic label, for example a fluorescent protein such as green fluorescent protein, or other tag, for example a Myc tag.In various aspects, the polypeptide is one of the cytokines, lymphokines, growth factors, or other hematopoietic factors listed above.

[0099] Linker In some embodiments, the conjugate is directly linked to the heterologous moiety. In alternative embodiments, the conjugate comprises a linker that connects the compound of the present disclosure to the heterologous moiety. In some aspects, the linker comprises a chain length of 1 to about 60 atoms, or 1 to 30 or more atoms, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S. The chain atoms and linkers can be selected to provide more soluble conjugates depending on their expected solubility (hydrophilicity). In some embodiments, the linker provides a functional group that is subject to cleavage by enzymes or other catalysts, or to hydrolysis conditions found in the target tissue or organ or cell. In some embodiments, the length of the linker is long enough to reduce the possibility of steric hindrance. In some embodiments, the linker is an amino acid or a peptidyl linker. Such peptidyl linkers can be of any length. Various linkers are about 1-50 amino acids in length, 5-50, 3-5, 5-10, 5-15, or 10-30 amino acids in length.

[0100] A variety of suitable linkers are known in the art. The linker can be cleavable (cleavable linker), for example, under physiological conditions, e.g., under intracellular conditions, so that the drug is released in the intracellular environment by cleavage of the linker. Alternatively, the linker can be cleavable under extracellular conditions, e.g., outside the tumor cell or in the vicinity of the tumor mass, so that the drug that preferentially penetrates the inside of the tumor cell is released by cleavage of the linker. In other embodiments, the linker is not cleavable (non-cleavable linker), and the drug is released, for example, by antibody degradation.

[0101] The linker may be attached to a chemically reactive group of the antibody moiety, such as a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., at the N- or C-terminus, the epsilon amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic or aspartic acid residues, the sulfhydryl group of one or more cysteinyl residues, or the hydroxyl group of one or more serine or threonine residues). The site at which the linker is attached may be a naturally occurring residue in the amino acid sequence of the antibody moiety, or may be introduced into the antibody moiety, for example, by recombinant DNA techniques (e.g., by introducing a cysteine ​​or a protease cleavage site into the amino acid sequence) or by protein biochemistry (e.g., reduction, pH adjustment, or proteolysis). The site at which the linker is attached may also be a non-natural amino acid. The site at which the linker is attached may also be a glycan of the antibody.

[0102] Typically, the linker is substantially inert under the conditions under which the two groups it connects are linked. The term "bifunctional crosslinker", "bifunctional linker" or "crosslinker" refers to a modifying agent having two reactive groups at each end of the linker, where one reactive group can be reacted first with a cytotoxic compound to provide a compound having a linker moiety and a second reactive group, and the second reactive group can then react with an antibody. Alternatively, one end of the bifunctional crosslinker can be reacted first with an antibody to provide an antibody having a linker moiety and a second reactive group, and the second reactive group can then react with a cytotoxic compound. The linking moiety can contain a chemical bond that allows for the release of the cytotoxic moiety at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid labile bonds, photolabile bonds, protease / peptidase labile bonds, and esterase labile bonds. See, e.g., U.S. Patent Nos. 5,208,020, 5,475,092, 6,441,163, 6,716,821, 6,913,748, 7,276,497, 7,276,499, 7,368,565, 7,388,026, and 7,414,073. In some embodiments, the linkage is a disulfide bond, a thioether, and / or a protease / peptidase labile bond. Other linkers that can be used in the present invention include non-cleavable linkers such as those described in detail in US20050169933, charged linkers such as those described in US2009 / 0274713, US2010 / 0129314, and WO2009 / 134976, or hydrophilic linkers, each of which is expressly incorporated herein by reference.

[0103] In some embodiments, the linker is a hydrophilic linker that confers hydrophilicity to the conjugate. In some embodiments, the hydrophilic linker comprises polyethylene glycol (PEG). In some embodiments, the hydrophilic linker is CLA2. In some embodiments, the CLA2 linker has the following structure: [ka] CLA2 is described in U.S. Patent Nos. 8,080,250, 8,759,496, and 10,195,288.

[0104] In some embodiments, the hydrophilic linker is CL2E. In some embodiments, CL2E has the following structure: [ka] CL2E is described in U.S. Patent Nos. 8,080,250, 8,759,496, and 10,195,288.

[0105] In some embodiments, the linker is cleavable by a cleavage factor present in the intracellular environment (e.g., in a lysosome or endosome or caveolae). The linker can be, for example, a peptide linker that is cleaved by an intracellular or extracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptide linker is composed of at least 2, at least 3, at least 4, or at least 5 amino acids in length.

[0106] In some embodiments, the peptide linker is VC-PAB, which includes valine and citrulline residues. In some such embodiments, the peptide linker is MC-VC-PAB. In some embodiments, the MC-VC-PAB linker has the following structure: [ka] MC-VC-PAB is described in U.S. Patent Nos. 7,659,241, 7,829,531, 6,884,869, 6,214,345, and 6,214,345.

[0107] In some embodiments, the peptide linker is maleimidocaproylglycine-glycine-phenylalanine-glycine (MC-GGFG). In some embodiments, the MC-GGFG linker has the following structure: [ka] MC-GGFG is described in U.S. Patent Nos. 9,808,537 and 10,195,288.

[0108] In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at a certain pH value. In some embodiments, the pH sensitive linker is hydrolyzable under acidic conditions. For example, acid labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used (see, for example, U.S. Pat. Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al, 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable below pH 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker, such as a thioether that is attached to the therapeutic agent via an acylhydrazone bond (see, eg, US Pat. No. 5,622,929).

[0109] In other embodiments, the linker is cleavable under reducing conditions (e.g., disulfide linker). Bifunctional crosslinkers that allow the antibody to be linked to a cytotoxic compound via a disulfide bond include, but are not limited to, N-succinimidyl-4-(4-nitropyridyl-2-dithio)butanoate, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB). Sulfo-SPDB is described, for example, in U.S. Pat. No. 8,236,319, which is incorporated herein by reference. Alternatively, crosslinkers that introduce thiol groups, such as 2-iminothiolane, homocysteine ​​thiolactone, or S-acetyl succinic anhydride, may be used. In other embodiments, the linker may contain a combination of one or more of the peptide linkers, pH-sensitive linkers, or disulfide linkers described above.

[0110] A "heterobifunctional crosslinker" is a bifunctional crosslinker that has two different reactive groups. Heterobifunctional crosslinkers that contain both an amine-reactive N-hydroxysuccinimide group (NHS group) and a carbonyl-reactive hydrazine group can also be used to link cytotoxic compounds to antibodies. Examples of such commercially available heterobifunctional crosslinkers include succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazid terephthalate hydrochloride (SHTH) and succinimidyl hydrazinium nicotinate hydrochloride (SHNH). Conjugates with acid-labile linkages can also be prepared using the hydrazine-containing benzodiazepine derivatives of the present invention. Examples of bifunctional crosslinkers that can be used include succinimidyl-p-formylbenzoate (SFB) and succinimidyl-p-formylphenoxyacetate (SFPA).

[0111] The linkers described herein can be used in any combination with the heterologous moieties described herein. In addition, the linkers described herein can have any chemically reactive moiety (e.g., maleimide, cysteine, etc.) that can react with any moiety of the antigen binding protein of the present disclosure (e.g., amino acids, disulfide bonds, carbohydrates (e.g., from post-translational modifications), etc.). Often, lysines or cysteines of antibodies or antigen binding proteins (e.g., cysteines from reduced disulfide bonds (e.g., from inter- or intra-chain disulfide bonds of antibodies or antigen binding proteins) or engineered unpaired cysteines) are used as sites for conjugation. All of the above-listed linkers and heterologous moieties described herein are commercially available and / or can be prepared by conventional techniques, including those described in the references listed above.

[0112] Conjugation The heterologous moiety to antigen-binding protein ratio (HAR) represents the number of heterologous moieties linked per antigen-binding molecule. In some embodiments, the HAR is in the range of 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2. In some embodiments, the HAR is in the range of 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3. In other embodiments, the HAR is about 2, about 2.5, about 3, about 4, about 5, or about 6. In some embodiments, the HAR is in the range of about 2 to about 4. The HAR can be characterized by conventional means, such as mass spectrometry, UV / Vis spectroscopy, ELISA assay, and / or HPLC.

[0113] In some embodiments, the conjugates are heterogeneous conjugates (also referred to as "conventional") in which the antigen binding proteins are conjugated to different numbers of heterologous moieties. In some embodiments, the heterogeneous conjugates follow a Gaussian or quasi-Gaussian distribution of conjugates, where the distribution is centered around the mean of the heterologous moiety loading value, with some antigen binding proteins being more conjugated than the mean and some antigen binding proteins being less conjugated than the mean.

[0114] In some embodiments, the conjugate is a homogenous conjugate in which a substantial proportion of the antigen binding protein is conjugated to a defined number of heterologous moieties. In some embodiments, the homogenous conjugate has a HAR of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the homogenous conjugate has a HAR of 2, 4, 6, or 8. In preferred embodiments, the homogenous conjugate has a HAR of 4. In other preferred embodiments, the homogenous conjugate has a HAR of 2. In some embodiments, the homogenous conjugate has a HAR of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent or more, or 100 percent of the conjugate has a defined HAR. In some embodiments, homogeneous conjugates have about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent of the conjugates have a defined HAR. In some embodiments, homogeneous conjugates have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent of the conjugates have a defined HAR. In some embodiments, homogeneous conjugate comprises a HAR distribution that is not Gaussian or quasi-Gaussian.In some embodiments, the homogeneity of homogeneous conjugate is determined by chromatogram, for example, HPLC or any suitable chromatography.In some embodiments, the chromatogram is HIC chromatogram.Homogeneous conjugate can be produced by site-specific conjugation.

[0115] In some embodiments, the heterologous moiety is site-specifically conjugated to an antigen binding protein (eg, an antibody). Various site-specific conjugation methods, such as conjugation at thiomab or TDC or unpaired cysteine ​​residues (Junutula et al. (2008) Nat. Biotechnol. 26:925-932, Dimasi et al. (2017) Mol. Pharm. 14:1501-1516, Shen et al. (2012) Nat. Biotechnol. 30:184-9), thiol cross-linker (Behrens et al. (2015) Mol. Pharm. 12:3986-98), conjugation at glutamine using transglutaminase (Dennler et al. (2013) Methods Mol. Bio. 1045:205-15, Dennler et al. (2014) Bioconjug Chem. 25:569-78), conjugation at engineered unnatural amino acid residues (Axup et al. (2012) Proc Natl Acad Sci USA 104-16101-6, Tian et al. (2014) Proc Natl Acad Sci USA 111:1766-71, VanBrunt et al. (2015) Bioconjug Chem 26:2249-60, Zimmerman et al. (2014) Bioconjug Chem 25:351-61), selenocysteine ​​conjugation (Li et al. (2017) Cell Chem Biol 24:433-442), and glycan-mediated conjugation (Okeley et al. (2013) Bioconjug Chem 24:1650-5), conjugation with galactose or GalNAc analogues (Ramakrishnan and Qasba (2002) J Biol Chem 277:20833-9; van Geel et al. (2015) Bioconjug Chem 26:2233-42), or via glycan engineering (Zhou et al. (2014) Bioconjug Chem 25:510-20; Tang et al.(2017) Nat Protoc 12:1702-1721), via short peptide tags, such as engineering glutamine tags or sortase A-mediated transpeptidation (Strop et al. (2013) Chem Biol 20:161-7; Beerli et al. (2015) PLoS One10:e0131177), and via aldehyde tags (Wu et al. (2009) Proc Natl Acad Sci USA106:3000-5) are known in the art.

[0116] Unpredictability of conjugates (e.g., ADCs) It is impossible to predict in advance which antibody-drug conjugates will be sufficiently safe and effective in clinical applications based solely on antibody profiles or drug payload profiles. For example, a particular drug payload may work perfectly well when conjugated to an antibody directed to a certain target, but may not work well at all when conjugated to an antibody directed to a different target, or even to different antibodies directed to the same target. The reasons why different antibody-drug conjugates show different antitumor activity in vivo are not well understood enough to allow accurate predictions in the design of new antibody-drug conjugates. It is speculated that the unpredictable interplay of many factors is in play. These factors may include, for example, the binding affinity of the antibody-drug conjugate to the target antigen, the ability of the conjugate to penetrate solid tumors, and the half-life in circulation to adequately expose the tumor without causing toxicity.

[0117] The complexity and unpredictability are well demonstrated by antibody affinity alone. Antibodies or antibody-drug conjugates with high affinity are associated with better uptake by cells, which results in higher levels of cytotoxic payload released inside the cell. Higher affinity is also known to enhance antibody-dependent cellular cytotoxicity (ADCC). All of these properties favor the cell killing properties of antibody-drug conjugates. However, it is also known that high affinity of antibodies or antibody-drug conjugates may hinder efficient tumor penetration through the "antigen barrier effect", suggesting that in order to achieve potent antitumor activity in vivo, the affinity of antibody-drug conjugates needs to be just right, not too high or too low. To date, there is no known method to predict what may be the most efficient or effective level of affinity for antibody-drug conjugates.

[0118] In addition, in vivo antitumor activity cannot be predicted solely by the mechanism of the linker and payload. For example, O. Ab et al, Mol. Cancer Ther. 14(&):1605-1613 (2015) demonstrated that the same antibody conjugated to the same antitubulin toxin via different linkers exhibited dramatically different antitumor activity when tested in preclinical cancer models. This example is particularly surprising because the chemical structures of the two linkers are very similar. Furthermore, the linker present in the superior conjugate contained a hydrophilic moiety. It is believed that hydrophilic metabolites are generally less membrane permeable and more slowly effluxed from lysosomes (sites of conjugate degradation), resulting in delayed antitubulin activity of the released payload. This finding claims an "ideal" kinetics of payload delivery, but to date, there is no insight into what constitutes such kinetics. Adding to this complexity is the open question of whether the ideal kinetics of payload delivery, even if elucidated for a particular cell type, can be applied to all cell types. Therefore, it is not possible to predict the most effective in vivo antitumor activity simply from the chemical composition of the linker or payload.

[0119] Compositions, pharmaceutical compositions and formulations Compositions comprising the antigen binding proteins, nucleic acids, vectors, host cells, or conjugates of the present disclosure are provided herein. The compositions, in some embodiments, comprise the antigen binding proteins in isolated and / or purified form. In some embodiments, the compositions comprise a single type (e.g., structure) of the antigen binding proteins of the present disclosure, or a combination of two or more antigen binding proteins of the present disclosure, where the combination comprises two or more antigen binding proteins of different types (e.g., structures).

[0120] In some aspects, the composition comprises an agent that enhances the chemical and physical characteristics of the antigen binding protein, e.g., through stabilizing the antigen binding protein at a certain temperature, e.g., room temperature, extending the shelf life, inhibiting degradation, e.g., oxidative protease mediated degradation, extending the half-life of the antigen binding protein, etc. In some aspects, the composition comprises any of the agents disclosed herein as a heterologous or conjugated moiety, optionally in admixture with or conjugated to the antigen binding protein of the present disclosure.

[0121] In various aspects of the present disclosure, the composition further comprises a pharma- ceutically acceptable carrier, diluent, or excipient.In some embodiments, the antigen-binding protein, nucleic acid, vector, host cell, or conjugate (hereinafter referred to as "active agent") of the present disclosure is formulated into a pharmaceutical composition comprising the active agent together with a pharma- ceutically acceptable carrier, diluent, or excipient.In this respect, the present disclosure further provides a pharmaceutical composition comprising the active agent intended for administration to a subject, for example, a mammal.

[0122] In some embodiments, the active agent is present in the pharmaceutical composition at a purity level suitable for administration to a patient. In some embodiments, the active agent has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%, and a pharma- ceutically acceptable diluent, carrier or excipient. In some embodiments, the composition contains the active agent in a concentration of about 0.001 to about 30.0 mg / ml.

[0123] In various embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier. As used herein, the term "pharma-ceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions such as oil / water or water / oil emulsions, and various types of wetting agents. This term also includes any of the agents approved by the regulatory agencies of the United States Federal Government or listed in the United States Pharmacopeia for use in animals, including humans.

[0124] Pharmaceutical compositions may contain, for example, an acidifier, an additive, an adsorbent, an aerosol propellant, an air displacing agent, an alkalizing agent, an anti-caking agent, an anticoagulant, an antimicrobial preservative, an antioxidant, a preservative, a base, a binder, a buffer, a chelating agent, a coating agent, a colorant, a drying agent, a detergent, a diluent, a disinfectant, a disintegrant, a dispersant, a dissolution enhancer, a pigment, an emollient, an emulsifier, an emulsion stabilizer, a filler, a film former, a flavor enhancer, a flavoring, a flow improver, a gel Any pharma- ceutical acceptable ingredient may be included, including: viscosifying agent, granulating agent, moisturizing agent, lubricant, mucoadhesive agent, ointment base, ointment, oily vehicle, organic vehicle, lozenge base, pigment, plasticizer, abrasive, preservative, sequestering agent, skin penetrating agent, solubilizer, solvent, stabilizer, suppository base, surface active agent, surfactant, suspending agent, sweetener, therapeutic agent, viscosity agent, tonicity agent, toxicity agent, thickener, water absorption agent, water miscible cosolvent, water softener, or wetting agent.See, for example, Handbook of Pharmaceutical Excipients, Third Edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000) (incorporated by reference in its entirety); Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) (incorporated by reference in its entirety).

[0125] In various aspects, pharmaceutical compositions include formulation materials that are non-toxic to recipients at the dosages and concentrations employed. In specific embodiments, pharmaceutical compositions include an active agent and one or more pharma- ceutically acceptable salts, polyols, surfactants, osmolality modifiers, tonicity agents, antioxidants, antibiotics, antifungals, bulking agents, lyoprotectants, antifoaming agents, chelating agents, preservatives, coloring agents, analgesics, or additional pharmaceutical agents. In various aspects, pharmaceutical compositions include one or more polyols and / or one or more surfactants, optionally in addition to one or more excipients, including, but not limited to, pharma- ceutically acceptable salts, osmolality modifiers (tonicity agents), antioxidants, antibiotics, antifungals, bulking agents, lyoprotectants, antifoaming agents, chelating agents, preservatives, coloring agents, and analgesics.

[0126] In certain embodiments, pharmaceutical compositions can contain formulation materials to alter, maintain or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, flavor, sterility, stability, rate of dissolution or release, adsorption, or permeation of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions. (e.g. sodium); preservatives (e.g. benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (e.g. glycerin, propylene glycol or polyethylene glycol); sugar alcohols (e.g. mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g. pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbatc, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (e.g. sucrose or sorbitol); tonicity enhancers (e.g. alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.See REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0127] The pharmaceutical composition may be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition may be, for example, about 4 or about 5 to about 8.0, or about 4.5 to about 7.5, or about 5.0 to about 7.5. In various embodiments, the pH of the pharmaceutical composition is 5.5 to 7.5.

[0128] The present disclosure provides methods of making pharmaceutical compositions. In various aspects, the methods include combining an antigen binding protein, a conjugate, a fusion protein, a nucleic acid, a vector, a host cell, or a combination thereof with a pharma- ceutically acceptable carrier, diluent, or excipient.

[0129] Route of administration In the present disclosure, the active agent or pharmaceutical composition comprising it can be administered to a subject via any suitable administration route.For example, the active agent can be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal or rectal administration.The following discussion of administration routes is provided merely to illustrate various embodiments and should not be construed as limiting in any way.

[0130] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizing agents, viscosity agents, stabilizers, and preservatives. The term "parenteral" means not through the digestive tract, but by some other route such as subcutaneous, intramuscular, intraspinal, or intravenous. The active agents of the present disclosure may be administered with a physiologically acceptable diluent in a pharmaceutical carrier such as a sterile liquid or mixture of liquids including water, saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, dimethyl sulfoxide, glycerol, ketals such as 2,2-dimethyl-153-dioxolane-4-methanol, ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides, with or without pharma- ceutical acceptable surfactants, such as soaps or detergents, suspending agents, such as pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0131] The oil that can be used in parenteral formulations includes petroleum, animal, vegetable, or synthetic oils.Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum oil, and mineral oil.The fatty acid that is suitable for use in parenteral formulations includes oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0132] Suitable soaps for use in parenteral formulations include fatty acid alkali metal, ammonium, and triethanolamine salts, and suitable detergents include: (a) cationic detergents, such as dimethyldialkylammonium halides, and alkylpyridinium halides; (b) anionic detergents, such as alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates and sulfosuccinates; (c) nonionic detergents, such as fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers; (d) amphoteric detergents, such as alkyl-β-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts; and (e) mixtures thereof.

[0133] Parenteral formulations, in some embodiments, contain from about 0.5% to about 25% by weight of the active agent of the present disclosure in solution. Preservatives and buffers may be used. To minimize or eliminate irritation at the injection site, such compositions may contain one or more non-ionic surfactants having a hydrophilic-lipophilic balance (HLB) of from about 12 to about 17. The amount of surfactant in such formulations may typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. Parenteral formulations, in some embodiments, are provided in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored for injection in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid excipient, e.g., water, immediately prior to use. Extemporaneous injection solutions and suspensions, in some embodiments, are prepared from sterile powders, granules, and tablets of the type described above.

[0134] Injectable formulations are subject to the present disclosure. The requirements of effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, JBLippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

[0135] Dosage The active agents of the present disclosure are believed to be useful in other methods further described herein, including methods of inhibiting tumor growth and methods of treating or preventing cancer. For purposes of this disclosure, the amount or dose of the active agent administered should be sufficient, for example, to provide a therapeutic or prophylactic response in a subject or animal over a reasonable time frame. For example, a dose of the active agent of the present disclosure should be sufficient to treat a cancer as described herein for a period of about 1-4 minutes, 1-4 hours, or 1-4 weeks or longer from the time of administration, for example, 5-20 weeks or longer. In certain embodiments, the period may be even longer. The dose will be determined by the efficacy of the particular active agent and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.

[0136] Many assays for determining the dose to be administered are known in the art.For the purposes of this specification, the starting dose to be administered to a mammal can be determined by using an assay that includes comparing the degree to which cancer is treated when a given dose of the active agent of the present disclosure is administered to a mammal among a group of mammals, each group being administered a different dose of the active agent.The degree to which cancer is treated when a certain dose is administered can be represented, for example, by the degree of tumor regression achieved by the active agent in a mouse xenograft model.Methods for assaying tumor regression are known in the art.

[0137] The dosage of the active agent of the present disclosure will also be determined by the presence, nature and extent of any adverse side effects that may accompany the administration of a particular active agent of the present disclosure. Typically, the attending physician will determine the dosage of the active agent of the present disclosure for treating each individual patient, taking into account a variety of factors, such as age, body weight, general health, diet, sex, the active agent of the present disclosure to be administered, the route of administration, and the severity of the condition to be treated. By way of example, and not intended to limit the present disclosure, the dosage of the active agent of the present disclosure may be about 0.0001 to about 1 g / kg body weight (of the subject to be treated) / day, about 0.0001 to about 0.001 g / kg body weight / day, or about 0.01 mg to about 1 g / kg body weight / day.

[0138] Controlled-release formulations In some embodiments, the active agent described herein can be modified into a depot form, so that the manner in which the active agent of the present disclosure is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150). The active agent of the present disclosure in a depot form can be, for example, an implantable composition comprising the active agent and a porous or non-porous material, such as a polymer, where the active agent is encapsulated by or diffuses throughout the material and / or diffuses through the degradation of the non-porous material. In this case, the depot is implanted into the subject's body at a desired location, and the active agent is released from the implant at a predetermined rate.

[0139] In certain embodiments, the pharmaceutical composition comprising the active agent is modified to have any kind of in vivo release profile. In some embodiments, the pharmaceutical composition is an immediate release, controlled release, sustained release, extended release, delayed release, or biphasic release formulation. Methods for formulating peptides for controlled release are known in the art. See, for example, Qian et al., J Pharm 374:46-52 (2009) and International Patent Publication Nos. WO2008 / 130158, WO2004 / 033036, WO2000 / 032218, and WO1999 / 040942.

[0140] The compositions may further comprise, for example, micelles or liposomes, or some other encapsulated form, or may be administered in an extended release form to achieve a retention and / or delivery effect over a prolonged period of time.

[0141] use The antigen binding proteins of the present disclosure are useful for inhibiting tumor growth. Without being bound to a particular theory, the inhibitory effect of the antigen binding proteins provided herein makes such entities useful in methods of treating cancer.

[0142] Thus, methods of inhibiting tumor growth in a subject and methods of reducing tumor size in a subject are provided herein. In various embodiments, the methods include administering to the subject an amount of the pharmaceutical composition of the present disclosure that is effective to inhibit tumor growth or reduce tumor size in the subject. In various aspects, the growth of ovarian tumors, melanoma tumors, bladder tumors, or endometrial tumors is inhibited. In various aspects, the size of ovarian tumors, melanoma tumors, bladder tumors, or endometrial tumors is reduced.

[0143] As used herein, the terms "inhibit" or "reduce" and words derived therefrom may not be 100% or complete inhibition or reduction. Rather, there are various degrees of inhibition or reduction that one of skill in the art would recognize as having potential benefit or therapeutic effect. In this regard, the antigen binding proteins of the present disclosure may inhibit tumor growth or reduce tumor size to any amount or level. In various embodiments, the inhibition provided by the methods of the present disclosure is at least or about 10% inhibition (e.g., at least or about 20% inhibition, at least or about 30% inhibition, at least or about 40% inhibition, at least or about 50% inhibition, at least or about 60% inhibition, at least or about 70% inhibition, at least or about 80% inhibition, at least or about 90% inhibition, at least or about 95% inhibition, at least or about 98% inhibition). In various embodiments, the reduction provided by the methods of the present disclosure is at least or about a 10% reduction (e.g., at least or about a 20% reduction, at least or about a 30% reduction, at least or about a 40% reduction, at least or about a 50% reduction, at least or about a 60% reduction, at least or about a 70% reduction, at least or about a 80% reduction, at least or about a 90% reduction, at least or about a 95% reduction, at least or about a 98% reduction).

[0144] In addition, provided herein is a method for treating a subject with cancer, for example, DLK1-expressing cancer.In various embodiments, the method comprises administering to the subject the pharmaceutical composition of the present disclosure in an amount effective for treating cancer in the subject.

[0145] For purposes herein, the cancer of the methods disclosed herein can be any cancer, for example, any malignant growth or tumor caused by abnormal and uncontrolled cell division that can spread to other parts of the body via the lymphatic system or bloodstream. Cancers, in some aspects, include acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin's disease, and the like. In a particular embodiment, the cancer is selected from the group consisting of head and neck cancer, ovarian cancer, ovarian cancer, cervical cancer, bladder and esophageal cancer, pancreatic cancer, gastric cancer, breast cancer, endometrial cancer and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer, such as non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma, and ovarian cancer, pancreatic cancer, bladder cancer, esophageal cancer, pancreatic cancer, gastrointestinal cancer, gastric cancer, breast cancer, endometrial cancer and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer, such as non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma. In various embodiments, the cancer is pancreatic cancer, gastrointestinal cancer, bladder cancer, colon cancer, lung cancer, liver cancer, endometrial cancer.In various embodiments, the cancer is any cancer characterized by moderate to high expression of DLK1.In various embodiments, the cancer is acute myeloid leukemia, large B-cell lymphoma, gastric cancer, prostate cancer, melanoma, colon cancer, rectal cancer, bladder cancer, cervical cancer, liver cancer, breast cancer, renal clear cell carcinoma, head and neck cancer, sarcoma, chromophobe renal carcinoma, low-grade glioma, adrenocortical carcinoma, glioblastoma, papillary renal cell carcinoma, lung squamous cell carcinoma, thyroid cancer, lung adenocarcinoma, pancreatic cancer, endometrioid carcinoma, uterine carcinosarcoma, or ovarian cancer. In various aspects, the cancer is selected from pancreatic cancer, gastrointestinal cancer, bladder cancer, colon cancer, lung cancer, liver cancer, ovarian cancer, endometrioid cancer, uterine cancer, lung cancer, gastric cancer, breast cancer, head and neck squamous cell carcinoma (HNSCC), and cervical cancer.

[0146] As used herein, the term "treat" and related words do not necessarily mean 100% or complete treatment. Rather, there are various degrees of treatment that one skilled in the art will recognize as having potential benefits or therapeutic effects. In this regard, the disclosed method of treating cancer may provide any amount or level of treatment. Furthermore, the treatment provided by the disclosed method may include treatment of one or more conditions or symptoms or signs of the cancer being treated. The treatment provided by the disclosed method may also include slowing the progression of the cancer. For example, the method may treat the cancer by enhancing T cell activity or immune response against the cancer, inhibiting tumor or cancer growth, inhibiting metastasis of tumor cells, and increasing cell death of tumor or cancer cells, etc. In various embodiments, the method treats to delay the onset or recurrence of the cancer for at least 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, 2 months, 3 months, 4 months, 6 months, 1 year, 2 years, 3 years, or 4 years or more. In various aspects, the methods treat to prolong survival of a subject.

[0147] The antigen binding protein of the present disclosure can also be used to detect DLK1 in a sample or diagnose DLK1-positive cancer. Thus, the present disclosure provides a method for detecting DLK1 in a sample. In various embodiments, the method comprises contacting a sample with an antigen binding protein, conjugate, or fusion protein described herein, and assaying an immune complex comprising the antigen binding protein, conjugate, or fusion protein bound to DLK1. The present disclosure also provides a method for diagnosing DLK1-positive cancer in a subject. In various embodiments, the method comprises contacting a biological sample comprising cells or tissue obtained from a subject with an antigen binding protein, conjugate, or fusion protein described herein, and assaying an immune complex comprising the antigen binding protein, conjugate, or fusion protein bound to DLK1.

[0148] subject In some embodiments of the present disclosure, the subject is a mammal, including but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals of the order Carnivora, including Feline (cats) and Canine (dogs), mammals of the order Artiodactyla, including Bovine (cows) and Swine (pigs), or mammals of the order Perssodactyla, including Equine (horses). In some aspects, the mammal is a mammal of the order Primates, Ceboids, or Simoids (monkeys), or a mammal of the order Anthropoids (humans and apes). In some aspects, the mammal is a human.

[0149] kit In some embodiments, the antigen binding proteins of the present disclosure are provided in a kit. In various aspects, the kit includes the antigen binding protein(s) as a unit dose. For purposes herein, a "unit dose" refers to a discrete amount dispersed in a suitable carrier. In various aspects, the unit dose is an amount sufficient to produce a desired effect in a subject, e.g., inhibiting tumor growth, reducing tumor size, treating cancer. Thus, kits are provided herein that include the antigen binding proteins of the present disclosure, optionally provided in a unit dose. In various aspects, the kit includes several unit doses, e.g., weekly or monthly unit doses, each of which is optionally packaged individually or otherwise separated from other unit doses. In some embodiments, the components of the kit / unit dose are packaged with instructions for administration to a patient. In some embodiments, the kit includes one or more devices for administration to a patient, e.g., needles and syringes, etc. In some aspects, the antigen binding protein of the present disclosure, a pharma- ceutically acceptable salt thereof, a conjugate comprising the antigen binding protein, or a multimer or dimer comprising the antigen binding protein is pre-packaged in a ready-to-use form, e.g., a syringe, an infusion bag, etc. In some aspects, the kit further comprises other therapeutic or diagnostic agents or a pharma- ceutically acceptable carrier (e.g., a solvent, a buffer, a diluent, etc.), including any of those described herein. In certain aspects, the kit comprises the antigen binding protein of the present disclosure together with an agent, e.g., a therapeutic agent, used in chemotherapy or radiation therapy.

[0150] Various embodiments In various embodiments of the disclosure, the antigen binding protein binds to human DLK1 protein (SEQ ID NO: 19 or 20).

[0151] In some embodiments, the antigen binding proteins of the disclosure comprise an Fc polypeptide. In some embodiments, the antigen binding proteins of the disclosure comprise an Fc polypeptide comprising a non-fucosylated glycan.

[0152] In various embodiments, the antigen binding protein of the present disclosure is an antibody, e.g., a monoclonal antibody. In various cases, the antigen binding protein is an IgG. In various embodiments, the antigen binding protein inhibits colony growth by at least about 50% in a soft agar 3D growth assay or inhibits tumor growth in xenograft mice injected with human cancer cells. In various embodiments, the antigen binding protein inhibits tumor growth in xenograft mice injected with ovarian cancer cells, melanoma cancer cells, bladder cancer cells, or endometrial cancer cells. In various cases, the antigen binding protein inhibits tumor growth by at least 50% in xenograft mice injected with ovarian cancer cells, bladder cancer cells, or endometrial cancer cells.

[0153] The present disclosure provides a bispecific antigen binding protein that binds to DLK1 and a second antigen, wherein the antigen binding protein that binds to DLK1 is any one of the antigen binding proteins described herein. In some embodiments, the bispecific antigen binding protein comprises (a) a heavy chain variable region amino acid sequence set forth in Table 1 and / or Figures 19-30, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity, (b) a light chain variable region amino acid sequence set forth in Table 1 and / or Figures 19-30, or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity, or (c) both (a) and (b). In some embodiments, the variant sequences have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the bispecific antigen binding protein comprises an Fc polypeptide. In some embodiments, the bispecific antigen binding protein comprises an Fc polypeptide comprising a non-fucosylated glycan.

[0154] In various aspects, the bispecific antigen-binding protein binds to DLK1 and a second antigen. In some embodiments, the bispecific antigen-binding protein comprises an antigen-binding fragment of an antibody specific for the second antigen. In various embodiments, the second antigen is a cell surface protein expressed by T cells, optionally a component of the T cell receptor (TCR), such as CD3. In some embodiments, the second antigen is CD3. In some embodiments, the second antigen is CD3E.

[0155] In various embodiments, the second antigen is a costimulatory molecule that assists in T cell activation, such as CD40 or 4-1BB (CD137). In various embodiments, the second antigen is an Fc receptor, optionally an Fc gamma receptor, an Fc-alpha receptor, or an Fc-epsilon receptor. In some embodiments, the Fc receptor is CD64 (Fc-gamma RI), CD32 (Fc-gamma RIIA), CD16A (Fc-gamma RIIIA), CD16b (Fc-gamma RIIIb), FcεRI, CD23 (Fc-epsilon RII), CD89 (Fc-epsilon RI), Fcα / μR, or FcRn. In some embodiments, the Fc receptor is CD16A.

[0156] In various embodiments, the second antigen is an immune checkpoint molecule, e.g., a protein involved in an immune checkpoint pathway, optionally A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, NOX2, PD-1, TIM3, VISTA, or SIGLEC7. In some embodiments, the immune checkpoint molecule is PD-1, LAG3, TIM3, or CTLA4. In various embodiments, the bispecific antigen binding protein comprises an scFv, Fab, or F(ab)2' of any of the DLK1 antibodies of the present disclosure.

[0157] In various embodiments, the bispecific antigen binding protein comprises an antigen binding protein comprising a sequence set forth in Table 1 and / or Figures 19-30, or a variant sequence thereof that differs by only 1-5 amino acids or has at least or about 70% sequence identity. In some embodiments, the variant sequence has at least or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In various embodiments, the bispecific antigen binding protein comprises a nanobody, diabody, BiTE®, DART, TandAb, CrossMab, or HSAbody structure. The present disclosure provides a conjugate comprising an antigen binding protein or bispecific antigen binding protein described herein and a heterologous moiety. In some embodiments, the antigen binding protein comprises an amino acid sequence set forth in Table 1 and / or Figures 19-30. In some embodiments, the conjugate comprises a cytotoxic or chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is an antimitotic agent that inhibits cell division by inhibiting tubulin polymerization. In some embodiments, the antimitotic agent is an auristatin. In some embodiments, the auristatin is MMAE.

[0158] In various embodiments, the conjugates of the present disclosure are conjugated to the antigen binding protein via a cleavable linker. In some embodiments, the cleavable linker is VC-PAB.

[0159] In some embodiments, the conjugate comprises an antigen-binding protein that is an antibody, where the antibody is a monoclonal antibody, optionally, the monoclonal antibody is an IgG antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0160] In various embodiments, the conjugates of the present disclosure have an average number of units of drug conjugated per antigen binding protein in the range of 1-8, preferably an average number of units of drug conjugated per antigen binding protein in the range of 3-8. In some embodiments, the conjugates are heterogeneous conjugates. In other embodiments, the conjugates are homogeneous conjugates. In some embodiments, the conjugates comprise a heterologous moiety or drug, where the drug is conjugated to a specific site on the antigen binding protein. In some embodiments, the specific site is an unpaired cysteine ​​residue.

[0161] The present disclosure also provides a fusion protein comprising the antigen-binding protein or bispecific antigen-binding protein described herein.The present disclosure further provides a nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein, bispecific antigen-binding protein, conjugate, or fusion protein of the present disclosure.The present disclosure provides a vector comprising a nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein, conjugate, or fusion protein of the present disclosure.The present disclosure further provides a host cell comprising the nucleic acid or vector of the present disclosure.

[0162] The present disclosure provides a method of making an antigen-binding protein or bispecific antigen-binding protein that binds to DLK1 protein, comprising (i) culturing a host cell of the present disclosure, comprising a nucleic acid comprising a nucleotide sequence encoding the antigen-binding protein or bispecific antigen-binding protein described herein, in a cell culture medium, and (ii) harvesting the antigen-binding protein or bispecific antigen-binding protein from the cell culture medium. Also provided is a method of making a fusion protein comprising an antigen-binding protein or bispecific antigen-binding protein that binds to DLK1 protein, comprising (i) culturing a host cell of the present disclosure, comprising a nucleic acid comprising a nucleotide sequence encoding the fusion protein of the present disclosure, in a cell culture medium, and (ii) harvesting the fusion protein from the cell culture medium.

[0163] The present disclosure further provides a method of making a pharmaceutical composition comprising combining an antigen binding protein, bispecific antigen binding protein, conjugate, fusion protein, nucleic acid, vector, host cell, or combination thereof of the present disclosure with a pharma- ceutically acceptable carrier, diluent or excipient. Also provided is a pharmaceutical composition comprising an antigen binding protein, bispecific antigen binding protein, conjugate, fusion protein, nucleic acid, vector, host cell, or combination thereof of the present disclosure with a pharma- ceutically acceptable carrier, diluent or excipient.

[0164] Provided herein is a method for treating a subject with DLK1-expressing cancer, comprising administering to the subject an amount of a pharmaceutical composition described herein that is effective for treating cancer.Also provided is a method for inhibiting tumor growth in a subject, comprising administering to the subject an amount of a pharmaceutical composition described herein that is effective for inhibiting tumor growth.The present disclosure provides a method for reducing tumor size in a subject, comprising administering to the subject an amount of a pharmaceutical composition described herein that is effective for reducing tumor size.Further provided is a method for preventing the recurrence of cancer in a subject, comprising administering to the subject an amount of a pharmaceutical composition described herein that is effective for preventing the recurrence of cancer.

[0165] The present disclosure provides a method for detecting DLK1 (CLD18.2) in a sample, comprising contacting the sample with an antigen-binding protein, bispecific antigen-binding protein, conjugate, or fusion protein of the present disclosure, and assaying an immune complex comprising the antigen-binding protein, conjugate, or fusion protein bound to CLD18.2. Also provided herein is a method for diagnosing DLK1-positive cancer in a subject, comprising contacting a biological sample comprising cells or tissue obtained from the subject with an antigen-binding protein, bispecific antigen-binding protein, conjugate, or fusion protein of the present disclosure, and assaying an immune complex comprising the antigen-binding protein, conjugate, or fusion protein bound to DLK1.

[0166] The present disclosure also provides a method for treating cancer in a subject diagnosed as having low overexpression of DLK1. In various embodiments, the method comprises administering to the subject an amount of the pharmaceutical composition of the present disclosure that is effective for preventing recurrence of the cancer. In some aspects, the administration induces apoptosis in tumor cells. Optionally, the administration induces apoptosis in cells that express DLK1. In various aspects, the subject has a tumor, and the tumor is semi-quantitatively classified into one of four groups: high expresser, medium expresser, low expresser, and non-expresser.

[0167] Exemplary embodiments 1.a. CDRs 1-3 derived from a heavy chain variable region comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFSISDYYMAWVRQAPGKGLEWVANINYDGTNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRSYYYYGMEYWGQGTTVTVSS (SEQ ID NO: 45) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 70% sequence identity; and / or b. CDR1-3 from a light chain variable region comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASHDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYRIPLTFGQGTKLEIK (SEQ ID NO: 46) or a variant sequence thereof differing in only 1-5 amino acids or having at least or about 70% sequence identity. 2. An antigen-binding protein comprising:

[0168] 2.a. CDRs 1-3 derived from a heavy chain variable region comprising the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSLSIYSVHWVRQPPGKGLEWIGLIWGGGSTDYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCARKEGNYLWFAYWGQGTLVTVSS (SEQ ID NO: 47) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 70% sequence identity; and / or b. CDRs 1-3 from a light chain variable region comprising the amino acid sequence: DIVMTQSPDSLAVSLGERVTMNCKSSQSLLQSSNQKNYLAWYQQKPGQPPKLLVYFASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQHYSIPLTFGQGTKLEIK (SEQ ID NO: 48) or a variant sequence thereof differing in only 1-5 amino acids or having at least or about 70% sequence identity. 2. An antigen-binding protein comprising:

[0169] 3.a. CDRs 1-3 derived from a heavy chain variable region comprising the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVSWVRQPPGKGLEWIGVIWGDGSTSYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCAKPDGPLGQGTLVTVSS (SEQ ID NO: 49) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 70% sequence identity; and / or b. CDR1-3 from a light chain variable region comprising the amino acid sequence: DIVMTQSPLSLPVTPGEPASISCRSSQSLVHINGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTTHVPWTFGQGTKLEIK (SEQ ID NO: 50) or a variant sequence thereof differing in only 1-5 amino acids or having at least or about 70% sequence identity. 2. An antigen-binding protein comprising:

[0170] 4.a. A heavy chain CDR1 comprising the amino acid sequence: GFSISDYY (SEQ ID NO:1) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; b. A heavy chain CDR2 comprising the amino acid sequence: INYDGTNT (SEQ ID NO:2) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; c. A heavy chain CDR3 comprising the amino acid sequence: VRSYYYYGMEY (SEQ ID NO:3) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; d. A light chain CDR1 comprising the amino acid sequence: HDVSTA (SEQ ID NO: 4) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; e. A light chain CDR2 comprising the amino acid sequence: SAS (SEQ ID NO:5) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; f. a light chain CDR3 comprising the amino acid sequence: QQHYRIPLT (SEQ ID NO:6) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; or g. A combination of two or more of (a) to (f) 2. An antigen-binding protein comprising:

[0171] 5.a. A heavy chain CDR1 comprising the amino acid sequence: GFSLSIYS (SEQ ID NO: 7) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; b. Heavy chain CDR2 amino acid sequence: IWGGGST (SEQ ID NO: 8) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; c. A heavy chain CDR3 comprising the amino acid sequence: ARKEGNYLWFAY (SEQ ID NO: 9) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; d. A light chain CDR1 comprising the amino acid sequence: QSLLQSSNQKNY (SEQ ID NO: 10) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; e. A light chain CDR2 comprising the amino acid sequence: FAS (SEQ ID NO: 11) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; f. The light chain CDR3 amino acid sequence: QQHYSIPLT (SEQ ID NO: 12) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; or g. A combination of two or more of (a) to (f) 2. An antigen-binding protein comprising:

[0172] 6.a. Heavy chain CDR1 amino acid sequence: GFSLTSYG (SEQ ID NO: 13) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; b. A heavy chain CDR2 comprising the amino acid sequence: IWGDGST (SEQ ID NO: 14) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; c. A heavy chain CDR3 comprising the amino acid sequence: AKPDGP (SEQ ID NO: 15) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; d. A light chain CDR1 comprising the amino acid sequence: QSLVHINGNTY (SEQ ID NO: 16) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; e. Light chain CDR2 amino acid sequence: KVS (SEQ ID NO: 17) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; f. a light chain CDR3 comprising the amino acid sequence: SQTTHVPWT (SEQ ID NO: 18) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; or g. A combination of two or more of (a) to (f) 2. An antigen-binding protein comprising:

[0173] 7. The antigen binding protein of any one of 1 to 7, wherein said variant sequence has at least about 80%, at least about 85%, at least about 90% sequence identity, or at least or about 95% sequence identity.

[0174] 8.a. A heavy chain FR1 comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 21) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70%, 80%, 85%, 90% or 95% sequence identity; b. A heavy chain FR2 comprising the amino acid sequence: MAWVRQAPGKGLEWVAN (SEQ ID NO: 22) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70%, 80%, 85%, 90% or 95% sequence identity; c. A heavy chain FR3 comprising the amino acid sequence: YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 23) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; d. A heavy chain FR4 comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 24) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; e. a light chain FR1 comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 25) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; f. a light chain FR2 comprising the amino acid sequence: VAWYQQKPGKAPKLLIY (SEQ ID NO: 26) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; g. A light chain FR3 comprising the amino acid sequence: YRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 27) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; h. a light chain FR4 comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 28) or a variant sequence thereof differing by only one or two amino acids or having at least or about 785%, 90% or 95% sequence identity; or i. A combination of two or more of (a) to (h) 5. The antigen-binding protein of claim 4, further comprising:

[0175] 9.a. A heavy chain FR1 comprising the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO: 29) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; b. A heavy chain FR2 comprising the amino acid sequence: VHWVRQPPGKGLEWIGL (SEQ ID NO: 30) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; c. A heavy chain FR3 comprising the amino acid sequence: DYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYC (SEQ ID NO: 31) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; d. A heavy chain FR4 comprising the amino acid sequence: WGQGTLVTVSS (SEQ ID NO: 32) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; e. a light chain FR1 comprising the amino acid sequence: DIVMTQSPDSLAVSLGERVTMNCKSS (SEQ ID NO: 33) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; f. a light chain FR2 comprising the amino acid sequence: LAWYQQKPGQPPKLLVY (SEQ ID NO: 34) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; g. A light chain FR3 comprising the amino acid sequence: TRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYC (SEQ ID NO: 35) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; h. a light chain FR4 comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 36) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; or i. A combination of two or more of (a) to (h) 6. The antigen-binding protein of 5, further comprising:

[0176] 10.a. A heavy chain FR1 comprising the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO: 37) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; b. A heavy chain FR2 comprising the amino acid sequence: VSWVRQPPGKGLEWIGV (SEQ ID NO: 38) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; c. A heavy chain FR3 comprising the amino acid sequence: SYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYC (SEQ ID NO: 39) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; d. A heavy chain FR4 comprising the amino acid sequence: LGQGTLVTVSS (SEQ ID NO: 40) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; e. a light chain FR1 comprising the amino acid sequence: DIVMTQSPLSLPVTPGEPASISCRSS (SEQ ID NO: 41) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; f. A light chain FR2 comprising the amino acid sequence: LHWYLQKPGQSPQLLIY (SEQ ID NO: 42) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; g. A light chain FR3 comprising the amino acid sequence: NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 43) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; h. Light chain FR4 amino acid sequence: FGQGTKLEIK (SEQ ID NO:44) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 85%, 90% or 95% sequence identity; or i. A combination of two or more of (a) to (h) 7. The antigen binding protein of 6, further comprising:

[0177] 11.a. a heavy chain variable region comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFSISDYYMAWVRQAPGKGLEWVANINYDGTNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRSYYYYGMEYWGQGTTVTVSS (SEQ ID NO: 45), or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or b. A light chain variable region comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASHDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYRIPLTFGQGTKLEIK (SEQ ID NO: 46), or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity. 9. The antigen-binding protein according to any one of 1, 4, 7, and 8, comprising:

[0178] 12.a. a heavy chain variable region comprising the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSLSIYSVHWVRQPPGKGLEWIGLIWGGGSTDYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCARKEGNYLWFAYWGQGTLVTVSS (SEQ ID NO: 47), or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or b. A light chain variable region comprising the amino acid sequence: DIVMTQSPDSLAVSLGERVTMNCKSSQSLLQSSNQKNYLAWYQQKPGQPPKLLVYFASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQHYSIPLTFGQGTKLEIK (SEQ ID NO: 48), or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity. 10. The antigen-binding protein according to any one of 2, 5, 7, and 9, comprising:

[0179] 13.a. a heavy chain variable region comprising the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVSWVRQPPGKGLEWIGVIWGDGSTSYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCAKPDGPLGQGTLVTVSS (SEQ ID NO: 49), or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or b. A light chain variable region comprising the amino acid sequence: DIVMTQSPLSLPVTPGEPASISCRSSQSLVHINGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTTHVPWTFGQGTKLEIK (SEQ ID NO:50), or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity. 13. The antigen-binding protein according to any one of 3, 6, 7, and 10, comprising:

[0180] 14.a. Amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSLSIYSVHWVRQPPGKGLEWIGLIWGGGSTDYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCARKEGNYLWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC An antibody heavy chain comprising: PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:51); and b. An antibody light chain comprising the amino acid sequence: DIVMTQSPDSLAVSLGERVTMNCKSSQSLLQSSNQKNYLAWYQQKPGQPPKLLVYFASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQHYSIPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:52). An antigen-binding protein that specifically binds to human Delta-like non-canonical Notch ligand 1 (DLK1), comprising:

[0181] 15.a. Binds to human DLK1 protein isoform 1 having the amino acid sequence set forth in SEQ ID NO: 19 (UniProtKB ID: P80370-1); b. binds to human DLK1 protein isoform 2 having the amino acid sequence set forth in SEQ ID NO: 69 (UniProtKB ID: P80370-2); c) a polypeptide having a dissociation constant (K) of less than about 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, 0.25 nM, 100 pM, 50 pM, 25 pM, 10 pM, or 5 pM with the extracellular domain of human DLK1; D ) or d. does not bind to mouse DLK1 protein having the amino acid sequence set forth in SEQ ID NO: 20 (UniProtKB ID: Q09163), or binds to said mouse DLK1 protein with an affinity 100-fold lower than that of said human DLK1 protein; or e. combinations thereof 2. The antigen binding protein of any one of the preceding embodiments,

[0182] 16. An antigen binding protein according to any one of the preceding embodiments, which binds to human DLK1 isoform 1 (UniProt ID: P80370-1) and isoform 2 (UniProt ID: P80370-2).

[0183] 17. Human DLK1 isoform 1 (UniProt ID: P80370-1) and isoform 2 (UniProt ID: P80370-2), a. less than about 6 nM; b. Approximately 5nM, c. about 2 nM, or d. About 1nM 17. The antigen-binding protein according to 16, which binds with a KD of

[0184] 18.a. an epitope within the EGF-like 2 and EGF-like 3 domains of the human DLK1 isoform 1 protein (UniProt ID: P80370-1), and / or b. A polypeptide comprising amino acid residues 55 to 113 of the human DLK1 isoform 1 protein (UniProt ID: P80370-1). 2. The antigen binding protein of any one of the preceding embodiments, which binds to:

[0185] 19. An antigen binding protein according to any one of the preceding embodiments, which binds to the human DLK1 protein and induces (a) an antibody-dependent cell-mediated cytotoxicity (ADCC) response, and / or (b) a complement-dependent cytotoxicity (CDC) response in DLK1-positive cells.

[0186] 20. The antigen-binding protein of any one of the preceding embodiments, which is an antibody or an antigen-binding antibody fragment.

[0187] 21. The antigen-binding protein according to claim 20, wherein the antibody is a monoclonal antibody.

[0188] 22. The antigen-binding protein according to 20, wherein the antibody is a chimeric antibody, a human antibody, or a humanized antibody.

[0189] 23. The antigen-binding protein of any one of 20 to 22, wherein the antibody is an IgG.

[0190] 24. The antigen-binding protein according to 23, wherein the IgG is selected from IgG1, IgG2, IgG3 and IgG4.

[0191] 25. The antigen-binding protein of 23 or 24, wherein the IgG is IgG1.

[0192] 26. The antigen-binding protein according to claim 20, wherein the antigen-binding antibody fragment is selected from the group consisting of scFv, F(ab')2, Fab, Fab' and Fv.

[0193] 27. The antigen-binding protein according to 20, wherein the antigen-binding antibody fragment is a single-chain variable fragment (scFv).

[0194] 28. The scFv, a. The variable light (V L) chain and variable weight (V H )chain, b. The amino acid sequence set forth in SEQ ID NO: 45 and SEQ ID NO: 46; c. The amino acid sequence set forth in SEQ ID NO: 47 and SEQ ID NO: 48; d. the amino acid sequence set forth in SEQ ID NO: 49 and SEQ ID NO: 50; or e. The amino acid sequence set forth in SEQ ID NO: 57, 58, or 59, or an antigen-binding portion thereof. 28. The antigen-binding protein according to 27, comprising:

[0195] 29. The antigen-binding protein according to 27, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 58, or an antigen-binding portion thereof.

[0196] 30. The antigen binding protein of any one of the preceding embodiments, which is a bispecific antigen binding protein or a bispecific T cell engager (BiTE).

[0197] 31. The bispecific antigen-binding protein or the BiTE, a. SEQ ID NO:45 and SEQ ID NO:46, b. SEQ ID NO: 47 and SEQ ID NO: 48, or c. SEQ ID NO: 49 and SEQ ID NO: 50 31. The antigen-binding protein according to claim 30, comprising the amino acid sequence set forth in

[0198] 32. The antigen-binding protein of 30 or 31, wherein said bispecific antigen-binding protein or said BiTE comprises an scFv of 28 or 29.

[0199] 33. The antigen binding protein of any one of 30 to 32, wherein said bispecific antigen binding protein or said BiTE binds to DLK1 and a T cell surface marker.

[0200] 34. The antigen binding protein according to 33, wherein the T cell surface marker is CD3 protein.

[0201] 35. The antigen binding protein of any one of 30 to 34, wherein said bispecific antigen binding protein or BiTE comprises an amino acid sequence as set forth in SEQ ID NO: 60, 61, or 62, or an antigen-binding portion thereof.

[0202] 36. The antigen binding protein of any one of 30 to 35, wherein said bispecific antigen binding protein or BiTE comprises the amino acid sequence set forth in SEQ ID NO: 60, or an antigen-binding portion thereof.

[0203] 37. The antigen binding protein of any one of 30 to 35, wherein said bispecific antigen binding protein or BiTE comprises the amino acid sequence set forth in SEQ ID NO: 61 or an antigen-binding portion thereof.

[0204] 38. The antigen binding protein of any one of 30 to 35, wherein said bispecific antigen binding protein or said BiTE induces T cell activation in the presence of DLK positive cells.

[0205] 39. An antigen binding protein according to any one of the preceding embodiments, which inhibits tumor growth in xenograft mice injected with human cancer cells.

[0206] 40. The antigen binding protein of any one of the preceding embodiments, comprising an Fc polypeptide comprising a non-fucosylated glycan.

[0207] 41. An antigen binding protein according to any one of the preceding embodiments, or a conjugate comprising an antigen binding protein as described herein.

[0208] 42. The conjugate according to 41, comprising a detectable marker, a cytotoxic agent, or a chemotherapeutic agent.

[0209] 43. The conjugate according to 42, wherein the chemotherapeutic agent is an antimitotic agent that inhibits cell division by inhibiting tubulin polymerization.

[0210] 44. The conjugate according to 43, wherein the antimitotic agent is an auristatin.

[0211] 45. The conjugate according to 44, wherein the auristatin is monomethylauristatin E or MMAE.

[0212] 46. ​​The conjugate according to any one of 42 to 45, wherein the drug or marker is conjugated to the antigen binding protein via a cleavable or non-cleavable linker.

[0213] 47. The conjugate according to 46, wherein the cleavable linker is VC-PAB.

[0214] 48. The conjugate according to any one of 41 to 47, wherein the antigen-binding protein is an antibody.

[0215] 49. The conjugate according to 48, wherein the antibody is a monoclonal antibody.

[0216] 50. The conjugate according to 49, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

[0217] 51. The conjugate according to 50, wherein the antibody is an IgG antibody, optionally wherein the IgG is IgG1, IgG2, IgG3, or IgG4.

[0218] 52. The conjugate according to any one of 41 to 52, wherein the average number of units of said drug conjugated per antigen binding protein is in the range of 1 to 8, preferably, said average number of units of said drug conjugated per antigen binding protein is in the range of (a) 3 to 8, or (b) 4.

[0219] 53. The conjugate according to any one of 41 to 52, which is a heterogeneous conjugate.

[0220] 54. The conjugate according to any one of 41 to 52, which is a homogeneous conjugate.

[0221] 55. The conjugate according to any one of 42 to 54, wherein the agent is conjugated to a specific site on the antigen-binding protein.

[0222] 56. The conjugate according to 55, wherein the specific site is an unpaired cysteine ​​residue.

[0223] 57. A conjugate according to any one of 41 to 56, comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 45 and SEQ ID NO: 46 conjugated to VC-PAB-MMAE.

[0224] 58. A conjugate according to any one of 41 to 56, comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 47 and SEQ ID NO: 48 conjugated to VC-PAB-MMAE.

[0225] 59. A conjugate according to any one of 41 to 56, comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 49 and SEQ ID NO: 50 conjugated to VC-PAB-MMAE.

[0226] 60. A conjugate according to any one of 41 to 56, comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 51 and SEQ ID NO: 52 conjugated to VC-PAB-MMAE.

[0227] 61. A fusion protein comprising the antigen binding protein of any one of the preceding embodiments.

[0228] 62. A nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein according to any one of 1 to 40, a conjugate according to any one of 41 to 60, or a fusion protein according to 61.

[0229] 63. The nucleic acid according to 62, which is a cDNA.

[0230] 64. A vector (eg, an expression vector) comprising the nucleic acid of 62 or 63.

[0231] 65. The vector according to 64, further comprising an internal ribosome entry site (IRES).

[0232] 66. A host cell comprising a nucleic acid according to 62 or 63, or a vector according to 64 or 65.

[0233] 67. The host cell according to 66, which is a bacterial cell.

[0234] 68. The host cell according to 66, which is a eukaryotic cell.

[0235] 69. The host cell according to 68, wherein the eukaryotic cell is a mammalian cell.

[0236] 70. The host cell according to 69, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

[0237] 71. A method for producing an antigen binding protein that binds to Delta-like non-canonical Notch ligand 1 (DLK1) protein, the method comprising: (i) culturing a host cell according to any one of 66 to 70 in a cell culture medium; and (ii) recovering the antigen binding protein from the cell culture medium.

[0238] 72. A method for producing a fusion protein comprising an antigen binding protein that binds to a Delta-like non-canonical Notch ligand 1 (DLK1) protein, the method comprising: (i) culturing a host cell according to any one of 66 to 70 in a cell culture medium; and (ii) recovering the fusion protein from the cell culture medium.

[0239] 73. A method of making a pharmaceutical composition, comprising combining (a) an antigen binding protein according to any one of 1 to 40, a conjugate according to any one of 41 to 60, a fusion protein according to 61, a nucleic acid according to 62 or 63, a vector according to 64 or 65, a host cell according to any one of 66 to 70, or any combination thereof, and (b) a pharma- ceutically acceptable carrier, diluent and / or excipient.

[0240] 74. A pharmaceutical composition comprising an antigen binding protein according to any one of 1 to 40, a conjugate according to any one of 41 to 60, a fusion protein according to 61, a nucleic acid according to 62 or 63, a vector according to 64 or 65, a host cell according to any one of 66 to 70, or any combination thereof, and (b) a pharma- ceutically acceptable carrier, diluent and / or excipient.

[0241] 75. A method of treating a subject having a DLK1-expressing cancer, comprising administering to the subject a pharmaceutical composition according to 74 to treat the cancer.

[0242] 76. A method for inhibiting tumor growth in a subject, comprising administering to the subject a pharmaceutical composition according to 74, thereby inhibiting tumor growth.

[0243] 77. A method for reducing tumor size in a subject, comprising administering to the subject a pharmaceutical composition according to 74, thereby reducing tumor size.

[0244] 78. A method for preventing recurrence of cancer in a subject, comprising administering to the subject a pharmaceutical composition according to 74 to prevent recurrence of the cancer.

[0245] 79. A method for treating cancer in a subject diagnosed with low overexpression of DLK1, comprising administering to the subject a pharmaceutical composition according to 74 to prevent recurrence of the cancer.

[0246] 80. The method of any one of 75 to 79, wherein said administration induces apoptosis in tumor cells.

[0247] 81. The method of any one of 75 to 79, wherein said administering induces apoptosis in cells expressing Delta-like noncanonical Notch ligand 1 (DLK1).

[0248] 82. A method for detecting Delta-like non-canonical Notch ligand 1 (DLK1) in a sample, comprising contacting the sample with an antigen binding protein described in any one of 1 to 40, a conjugate described in any one of 41 to 60, or a fusion protein described in 61, and assaying an immune complex comprising the antigen binding protein, the conjugate or the fusion protein bound to DLK1.

[0249] 83. A method for diagnosing Delta-like noncanonical Notch ligand 1 (DLK1) positive cancer in a subject, comprising contacting a biological sample comprising cells or tissue obtained from the subject with an antigen binding protein described in any one of 1 to 40, a conjugate described in any one of 41 to 60, or a fusion protein described in 61, and assaying an immune complex comprising the antigen binding protein, the conjugate or the fusion protein bound to DLK1.

[0250] 84. The method of claim 83, further comprising treating the subject diagnosed with DLK1-positive cancer by administering to the subject an antigen binding protein described in any one of 1 to 40, a conjugate described in any one of 41 to 60, or a fusion protein described in 61.

[0251] 85. A method for activating T cells targeting DLK1-expressing cancer cells in a subject, the method comprising administering to the subject a bispecific T cell engager (BiTE) described in any one of 30 to 38.

[0252] 86. A method of inducing an antibody-dependent cell-mediated cytotoxicity (ADCC) response against DLK1-expressing cancer cells in a subject, comprising administering to the subject an antigen binding protein that binds DLK1, wherein the antigen binding protein comprises an Fc effector function and a VH region and a VL region of an antigen binding protein of any one of the preceding embodiments or an antigen binding protein described herein.

[0253] 87. The method of any one of the preceding embodiments, wherein the subject is a mammal, optionally a dog, a cat, a mouse, or a human.

[0254] 88. A kit comprising: (a) an antigen binding protein according to any one of 1 to 40, a conjugate according to any one of 41 to 60, a fusion protein according to 61, a nucleic acid according to 62 or 63, a vector according to 64 or 65, a host cell according to any one of 66 to 70, or any combination thereof; and (b) instructions for use.

[0255] The following examples are offered merely to illustrate the present disclosure and are not intended to limit its scope in any way. EXAMPLES

[0256] Example 1 In this example, the generation of DLK1-specific antibodies is described.

[0257] A mammalian expression vector encoding the human DLK1-myc-DDK fusion protein was used to immunize mice with 3T3 cells overexpressing epitope-tagged full-length human DLK1 protein. The amino acid sequence of the human DLK1 protein is shown in UniProtKB accession number P80370-1 and is designated MTATEALLRVLLLLLAFGHSTYGAECFPACNPQNGFCEDDNVCRCQPGWQGPLCDQCVTSPGCLHGLCGEPGQCICTDGWDGELCDRDVRACSSAPCANNRTCVSLDDGLYECSCAPGYSGKDCQKKDGPCVINGSPCQHGGTCVDDEGRASHASCLCPPGFSGNFCEIVANSCTPNPC It has the amino acid sequence of ENDGVCTDIGGDFRCRCPAGFIDKTCSRPVTNCASSPCQNGGTCLQHTQVSYECLCKPEFTGLTCVKKRALSPQQVTRLPSGYGLAYRLTPGVHELPVQQPEHRILKVSMKELNKKTPLLTEGQAICFTILGVLTSLVVLGTVGIVFLNKCETWVSNLRYNHMLRKKKNLLLQYNSGEDLAVNIIFPEKIDMTTFSKEAGDEEI (SEQ ID NO: 19).

[0258] Spleen cells were harvested from immunized mice and fused with myeloma lines by BTX Electrofusion (BTX, Holliston, MA) to generate hybridomas. Primary hybridoma cultures were generated and cultured in 384-well plates. The ability of antibodies to bind to peptide and / or DLK1-expressing human cancer cells was assessed by ELISA assay and / or flow cytometry. Potential positive antibodies were redistributed in 96-well plates and further screened by flow cytometry against endogenous and artificial cell line models.

[0259] Hybridomas positive for the production of antibodies binding to human DLK1 protein were identified, and nucleic acid sequences encoding immunoglobulin light and heavy chains against human DLK1 protein were isolated. These nucleic acid sequences were used to generate DLK1 antibodies formatted as full-length IgG antibodies (e.g., human IgG1) using ExpiCHO™ expression. The heavy and light chain variable regions of the antibodies were cloned into a laboratory-engineered antibody expression vector based on the pcDNA™ 3.4-TOPO® vector (Cat. No.: A14697, ThermoFisher Scientific, USA). Transfection of the antibody expression vector into CHO cells according to the protocol provided in the kit (ExpiCHO™ Expression System, Cat. No.: A29133, ThermoFisher Scientific, USA) resulted in the production of a bicistronic mRNA in which an IRES drives the expression of the second immunoglobulin chain. The produced antibodies were purified using Protein A / G resin. Cell surface binding of antibodies to DLK1 was determined by FACS using DLK1 antibodies directly conjugated with Alexa Fluor® 647 NHS ester (succinimidyl ester), catalog number A20106 (ThermoFisher Scientific) according to the manufacturer's protocol.

[0260] DLK1-expressing cells were used in FACS assay to determine the ability of DLK1 antibodies to bind to DLK1 on the surface of cells. HEK293T cells engineered to express human, monkey, mouse or rat DLK1 fused with fluorescent protein (mGFP) were used as artificial models of DLK1 expression. COR-L279 (human small cell lung cancer cell line), JR (human rhabdomyosarcoma cell line), and H524 (human small cell lung cancer cell line) were used as endogenous models of DLK1 expression, and SNU-C1 (human colorectal cancer cell line), LS513 (human colorectal cancer cell line), and M202 (human melanoma cell line) were used as endogenous models lacking DLK1 expression.

[0261] For each cell type and mAb tested, cells were detached from the surface of the culture flask by versene (not trypsin) to protect cell surface proteins. The detached cells were then incubated with Alexa Fluor® 647-labeled DLK1 mAb for 30 minutes on ice in the dark at the indicated concentrations. DLK1 mAb was directly labeled with Alexa Fluor® 647 NHS ester (succinimidyl ester). Alternatively, unlabeled DLK1 antibody may be detected using a fluorescently labeled secondary antibody. After washing, cells were read on a BD Accuri™ flow cytometer C6 to detect antibody-antigen protein binding in channel FL4H.

[0262] Example 2 This example demonstrates humanization of an antibody of the disclosure.

[0263] The antibody was selected for humanization analysis. The heavy chain variable (VH) and light chain variable (VL) sequences of the mouse monoclonal anti-DLK1 antibody were transformed to human VH and VL sequences. H Genes and Human V L Comparison was made with a library of known human germline sequences from the kappa gene (IMGT® the international ImMunoGeneTics information system® www.imgt.org, Founder and Director: Marie-Paule Lefranc, Montpellier, France). The database used was the IMGT human V H Genes (F+ORF, 273 germline sequences) and IMGT human V L The kappa gene (F+ORF, germline sequence of 74) was the acceptor human germline, selected from those whose sequences were closest to the parent antibody.

[0264] Changes in human germline framework (i.e., non-CDR residues in VH and VL; abbreviated as FR) positions to the corresponding parental murine sequences may be necessary to optimize binding of the humanized antibody. Sequences of several versions of the humanized antibodies are shown in Table 1 and Figures 19-33.

[0265] Humanized antibodies DLK1-547-h2, DLK1-548-h4, DLK1-557-h3, DLK1-557-f2, DLK1-559-h4, DLK1-559-f2, DLK1-561-f1, DLK1-562-h10, and DLK1-565-f2 ("DLK1-" may be replaced with "04-0", "mab-" or "DLK1-mab-", which refer to the same humanized antibodies, e.g., DLK1-547- h2 is the same as 04-0547-h2, mab-547-h2 and DLK1-mab-547-h2, respectively; further note that the expression plasmid DNA may be indicated by a lower case "p" preceding the antibody name, e.g., p04-0547-h2 refers to the expression plasmid encoding anti-DLK1 antibody 04-0547-h2) were constructed and expressed essentially as described in Example 1. FACS assays were performed to determine the relative antigen binding strength of the humanized antibodies (either 1 μg or 0.2 μg) for binding to DLK1 overexpressed in HEK297T cell line. The corresponding parental antibody (antibody before humanization) was used as a control and may be referred to as "chim".

[0266] Based on the in vitro antigen binding data, three humanized antibodies (04-547-h2 (also referred to as DLK1-547-h2, 04-0547-h2), 04-561-F1 (also referred to as DLK1-561-F1, 04-0561-F1), and 04-562-h10 (also referred to as DLK1-562-h10, 04-0562-h10)) were selected for further testing and development. These antibodies were derived from the murine monoclonal antibodies DLK1-547, DLK1-561, and DLK1-562.

[0267] In vivo binding studies of humanized versions of monoclonal anti-DLK1 antibodies, i.e., DLK1-547-h2, DLK1-548-h4, DLK1-557-h3, DLK1-557-f2, DLK1-559-h4, DLK1-559-f2, DLK1-561-f1, DLK1-562-h10, and DLK1-565-f2, as well as the original murine monoclonal anti-DLK1 antibodies, i.e., DLK1-547, DLK1-548, DLK1-557, and DLK1-559, were performed as well as in vivo binding studies of humanized versions of monoclonal anti-DLK1 antibodies, i.e., DLK1-547, DLK1-548, DLK1-557, and DLK1-559, as well as in vivo binding studies as antibody-drug conjugates (ADCs; antibody-MMAE conjugates). In vivo binding studies were performed in xenograft mice injected with DLK1-positive human liver cancer cell line HepG2, DLK1-positive small cell lung cancer cell lines COR-L279 (also referred to as CORL279) and NCI-H524 (also referred to as H524), DLK1-positive human rhabdomyosarcoma cell line JR-1 (also referred to as JR), DLK1-negative colorectal cancer cell lines SNU-C1 (also referred to as SNUC1) and LS513, and DLK1-negative human melanoma cell line M202. Briefly, xenograft models of different human cancer cell lines were established in 6-week-old CD-1 athymic nude mice (Charles River Laboratories). Tumors were 150-450 mm 3 Mice were randomized into treatment groups after reaching an average size of 1 mg / mL. Humanized antibodies were diluted in sterile saline for intravenous tail vein (IV) injection to a working concentration of 1 mg / mL. Tumor xenografts were measured three times weekly with calipers and measured in mm 3 Tumor volume in units was determined by multiplying height x width x length. Mice were treated for 2-7 weeks. At the end of the study, animals were euthanized and tumor tissue was excised, sectioned, and stored as snap frozen or formalin-fixed paraffin-embedded (FFPE) tissue for biomarker analysis.

[0268] The results of the xenograft assay are shown in Figures 1 to 5.

[0269] Figure 1 shows the tumor growth inhibition properties of four different mouse monoclonal antibodies against DLK1 protein (DLK1-547, DLK1-548, DLK1-557 and DLK1-559) in mice bearing xenografts of DLK1-positive HepG2 human hepatocellular carcinoma cells. Figure 1A shows HEPG2 (DLK1+) cell line xenografts treated with a non-targeting IgG control antibody or mouse monoclonal antibodies against DLK1. All mice were treated by IV tail vein injection of 10 mg antibody / kg once a week for four repeated doses. Six mice per group. Lines represent mean tumor volume ± SEM. Figure 1B shows a bar graph representing the mean (± SEM) change in tumor volume in each treatment group over 23 days of treatment. All work was performed under an IACUC-approved protocol.

[0270] FIG. 2 shows the efficacy of a panel of humanized DLK1 mAbs to inhibit the growth of DLK1-positive CORL279 human small cell lung cancer (SCLC) cell line xenografts without adversely affecting body weight. FIG. 2A shows CORL279 (DLK1+) cell line xenografts treated with a non-targeting IgG control antibody, or nine different humanized antibodies against DLK1. All mice were treated by IV tail vein injection of 10 mg antibody / kg once a week for three repeated doses. Six mice per group. Lines represent mean tumor volume ± SEM. FIG. 2B is a bar graph depicting the mean (± SEM) change in tumor volume in each treatment group over 21 days of treatment. FIG. 2C is a graph depicting percent body weight change.

[0271] Figure 3 shows the selective efficacy of DLK1-ADC (humanized mAb) in human cancer cell line xenografts. Figures 3A-C show the results of CORL279, JR and H524 (all DLK1+) cell line xenografts treated with a non-targeting IgG control antibody, DLK1-561-f1 (humanized mAb). Mice were treated with 10 mg / kg control antibody or 5 mg / kg ADC, all via IV tail vein injection, as repeated weekly doses for three doses. Tumor volume (mm 3Note that a dramatic reduction in tumor volume was observed, indicating the great efficacy of DLK1-ADC against DLK1-positive tumors. In contrast, similar treatment of DLK1-negative tumors did not result in a dramatic reduction in tumor volume. Figures 3D-F show SNUC1, LS513 and M202 (all DLK1-) cell line xenografts treated with control or DLK1-ADC as described above. 6-8 mice per group. Lines represent mean tumor volume ± SEM.

[0272] Figure 4 shows the efficacy of humanized DLK1-ADC in inhibiting the growth of DLK1-positive CORL279 human SCLC cell line xenografts and reducing tumor volume. Figure 5A shows the results of treatment of CORL279 (DLK1+) cell line xenografts with a non-targeting IgG control antibody, or three different DLK1-ADCs generated from humanized DLK1-mAb. Mice were treated by IV tail vein injection with control antibody or ADC at 5 mg / kg once a week for three repeated doses. Seven mice per group. Lines represent mean tumor volume ± SEM. Figure 4B is a bar graph representing the mean (± SEM) change in tumor volume in each treatment group over the 13-day study (last day of control group). Figure 4C is a graph showing the percent change in body weight from the start of treatment. Body weight was initially reduced by the ADC, but then recovered by day 23 for the humanized DLK-ADC. Due to significant tumor growth in animals treated with a non-targeted IgG-ADC control antibody, the average size of the xenografts was approximately 1200 mm 3 The animals were sacrificed on day 13 after immunization.

[0273] Figure 5 shows that humanized DLK1-ADCs have no antitumor activity in DLK1-negative M202 human melanoma cell line xenografts. Figure 5A shows the absence of antitumor activity in M202 (DLK1) with a non-targeting IgG control antibody or three different DLK1-ADCs generated from humanized DLK1-mAbs. -5B shows the results of treatment of cell line xenografts. Mice were treated with control antibody or ADC at 5 mg / kg once a week for three repeated doses by IV tail vein injection. Eight mice per group. Lines represent mean tumor volume ± SEM. FIG. 5B is a bar graph representing the mean (± SEM) change in tumor volume in each treatment group over the 31-day study (last day of control group).

[0274] The DLK1 antibody drug conjugate (ADC) presented herein is conjugated to the cytotoxic agent MMAE via the cleavable linker VC-PAB (thus comprising VC-PAB-MMAE). The average number of MMAE per antibody is 4 as measured by HIC and / or MS. The ADC is a heterogeneous conjugate in which approximately 90% of the MMAE is conjugated to the interchain disulfide between the heavy and light chains of IgG (reacting with the thiol of the cysteine ​​from the reduced interchain disulfide). The ADC was prepared at Wuxi Bio using their DAR4 technology.

[0275] Figures 6, 8 and 9 provide an overview of the biochemical, biophysical and cell biology properties of the three lead humanized anti-DLK1 antibodies 04-0561-F1, 04-0562-h10 and 04-0547-h2. These three antibodies bind more strongly to DLK1-positive COR-L279 human small cell lung cancer cells than to DLK1-negative M202 human melanoma cells. Antibody binding affinity to human DLK1 overexpressed on the surface of HEK293T cells or DLK1-positive COR-L279 cells shows dissociation constants (KD) of about 0.5 nM to 4 nM for the 04-0561-F1 and 04-0562-h10 antibodies and a slightly higher KD of about 5 nM to 9 nM for the 04-0547-h2 antibody. For recombinant fusion proteins (DLK1 ECD-mFc) containing human DLK1 extracellular domain (ECD) and monomeric immunoglobulin Fc domain, the measured KD ranges from 2 pM to 20 pM for the first two antibodies and is approximately 300 pM for the 04-0547-h2 antibody. Similarly, EC50 determined using human DLK1 ECD-mFc fusion protein in an ELISA format showed that the 04-0561-f1 and 04-0562-h10 antibodies have similar binding affinities of approximately 0.5 nM to the recombinant protein, while the 04-0547-h2 antibody has a lower binding affinity, approximately 20-fold lower. Thus, of the top three humanized anti-DLK1 antibodies, the 04-0561-F1 and 04-0562-h10 antibodies have lower KD and EC50 values ​​than the 04-0547-h2 antibody.

[0276] Size exclusion chromatography reveals that all three purified antibodies are predominantly monomeric, with little antibody aggregation or fragmentation (Figure 6). Figure 8 shows that long-term storage (5 weeks) and temperature stress (RT or 37°C) did not cause significant antibody aggregation and degradation (SEC) at any concentration of 10, 20, 50, or 75 mg / ml. Specifically, after 5 weeks of storage at 37°C, less than 3.5% degradation (all four concentrations) and approximately 4% aggregation (75 mg / ml only) was observed for 04-0561-F1, and less than 8% degradation was observed for 04-562-h10. When analyzed by non-reducing SDS-PAGE, long-term storage (5 weeks) and temperature stress (RT or 37°C) did not significantly affect antibody integrity at any concentration of 10, 20, 50, or 75 mg / ml. However, at all concentrations (10, 20, 50, and 75 mg / ml), some degree of antibody degradation was detected after 5 weeks of storage at 37° C.; 04-0561-F1 had a lower level of degradation compared to 04-0562-h10.

[0277] The effect of different temperatures and storage for 7 days on the stability of humanized anti-DLK1 antibody 04-0547-h2 at 10 mg / mL is shown in Figure 9, where 100% of the purified antibody remained as monomer with no detectable aggregates or fragments, as confirmed by SDS-PAGE analysis after storage at -80°C, 4°C, RT or 37°C for 1 week.

[0278] When cross-reactivity against human and non-human DLK1 proteins was examined, it was demonstrated that all three humanized anti-DLK1 antibodies bound to human and monkey DLK1 overexpressed in HEK293T cells, but the ability of these antibodies to bind to rodent DLK1 proteins was not uniform (Figure 6). In particular, the 04-0547-h2 antibody bound to mouse and rat DLK1 proteins, whereas the 04-0562-h10 antibody bound to mouse DLK1 proteins less than rat DLK1 proteins, and the 04-0561-F1 antibody did not appear to bind to any of the rodent DLK1 proteins. With regard to cross-species reactivity, the 04-0547-h2 antibody bound to all four DLK1 proteins, whereas the 04-0562-h10 preferred binding to human, monkey, and rat DLK1 proteins over mouse DLK1 proteins. The best humanized anti-DLK1 antibody with the least cross-reactivity was 04-0561-F1, which bound to human and monkey DLK1 proteins, but not to mouse or rat DLK1 proteins. Further cross-reactivity data is shown in FIG.

[0279] The similarity in amino acid sequence of human, monkey, mouse and rat DLK1 proteins is shown in the sequence alignment of Figure 10. The human DLK1 protein sequence is shown above and can be obtained using UniProtKB accession number P80370-1. The mouse DLK1 protein can be obtained using UniProtKB accession number Q09163 and can be obtained using UniProtKB accession number MIATGALLRVLLLLLAFGHSTYGAECDPPCDPQYGFCEADNVCRCHVGWEGPLCDKCVTAPGCVNGVCKEPWQCICKDGWDGKFCEIDVRACTSTPCANNGTCVDLEKGQYECSCTPGFSGKDCQHKAGPCVINGSPCQHGGACVDDEGQASHASCLCPPGFSGNFCEIVAATNSCTPN The amino acid sequence of PCENDGVCTDIGGDFRCRCPAGFVDKTCSRPVSNCASGPCQNGGTCLQHTQVSFECLCKPPFMGPTCAKKRGASPVQVTHLPSGYGLTYRLTPGVHELPVQQPEQHILKVSMKELNKSTPLLTEGQAICFTILGVLTSLVVLGTVAIVFLNKCETWVSNLRYNHTFRKKKNLLLQYNSGEELAVNIIFPEKIDMTTFNKEAGDEEI (SEQ ID NO: 20).

[0280] The antibody internalization kinetics determined against naturally DLK1 positive COR-L279 cells showed that the 50% internalization time was about 10 min and internalization was complete in about 1 h (Figure 6). Figure 12 shows the DLK1 internalization kinetics of chimeric anti-DLK1 antibodies 04-0557m and 04-0559m, in which the light and heavy chain variable regions of human IgG1 were replaced with the mouse anti-DLK1 monoclonal antibody light and heavy chain variable regions, respectively. +Internalization data into COR-L279 cells is shown. Internalization of benchmark antibodies KO_DLK1-hIgG1 and Li-DLK1-hIgG1 is also shown. Uptake of humanized anti-DLK1 antibodies 04-0547-h2, 04-0548-h4, 04-0557-h3 and 04-557-F2 into HEPG2 cells is shown in FIG. 13 and into COR-L279 cells in FIG. 14. Additionally, internalization data into COR-L279 cells for humanized anti-DLK1 antibodies 04-0559-h4, 04-559-F2 (also referred to as 04-0559-F2), 04-0561-F1, 04-0562-h10, and 04-565-F2 (also referred to as 04-0565-F2), as well as the benchmark KO_DLK1-hIgG1 antibody and the non-targeting hIgG1 control and no antibody material are shown in Figures 15 and 16. The appearance of COR-L279 cells after 48 hours of incubation with AF-647 labeled anti-DLK1 antibodies is shown in FIG. 17 for 04-0547-h2, 04-0548-h4, 04-0557-h3, 04-557-F2, 04-0559-h4, 04-559-F2, 04-0567-F1, 04-0562-h10, and 04-565-F2, as well as the benchmark KO_DLK1-hIgG1 antibody and negative controls using a non-targeting hIgG1 or no antibody. Similarly, internalization of AF-647 labeled MMAE conjugated lead antibodies (04-0547-h2-MMAE-AF647, 04-0561-F1-MMAE-AF647 and 04-0562-h10-MMAE-AF647) is shown in Figure 7 along with internalization of lead antibody 04-0561-F1 (04-0561-F1-AF647) labeled with AF-647 without MMAE as a conjugate. Internalization of the ADC was complete within about 1.5 hours in COR-L279 cells (see also Figure 18).

[0281] The amino acid sequences of the framework (FR) and complementarity determining regions (CDR) of the heavy and light chain variable regions of the humanized DLK1 antibody (04-0547-h2, 04-0561-F1, 04-0562-h10, 04-0548-h4, 04-0557-F2, 04-0557-h3, 04-0559-F2, 04-0559-h4, 04-0565-F2) and the heavy and light chain variable regions (V H and V LThe amino acid sequence of the entire sequence is shown in Figures 19-29, with the Kabat and AbM representations shown in Figures 19-27 and the IMGT representation shown in Figures 28-29. Note the presence of the "RTV" tripeptide in the light chain variable region framework 4 (LFR4) in the Kabat and AbM representations using the algorithm in abYsis (Swindells MB, Porter CT, Couch M, Hurst J, Abhinandan KR, Nielsen JH, Macindoe G, Hetherington J, Martin AC. abYsis: Integrated Antibody Sequence and Structure-Management, Analysis, and Prediction. J Mol Biol. 2017 Feb 3; 429(3): 356-364, World Wide Web abysis.org). For the light chain variable regions identified in Figures 19-27, the light chain variable regions bind to a kappa light chain constant region comprising the sequence: AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, which corresponds to the sequence of the kappa light chain constant region shown in UniProt ID: P01834, except for the presence of the three amino acids "RTV" at the amino terminus of UniProt ID: P01834. For the light chain variable region sequences shown in Figures 28 and 29, the entire kappa light chain can be obtained by adding to the C-terminus of the variable light chain region the amino acid sequence shown in UniProt ID: P01834, i.e.: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0282] The amino acid sequences of the constant regions of human IgG1 and IgG2 used in humanizing the anti-DLK1 antibody are shown in FIG.

[0283] The DNA sequences encoding the humanized DLK1 antibody light and heavy chain variable regions are shown in Figure 31, and the DNA sequences encoding the human light and heavy chain constant regions are shown in Figure 32. Combining the DNA sequences encoding the light or heavy chain variable regions of Figure 31 with the subsequent DNA sequences of the light or heavy chain constant regions of Figure 32 results in a DNA sequence encoding the full-length light or heavy chain, respectively.

[0284] The heavy and light chain variable region coding sequences of the humanized anti-DLK1 antibody (04-0561-F1) and the murine monoclonal anti-DLK1 antibody (04-0561-m) are shown in FIG.

[0285] Analysis of cross-reactivity data for one of the three lead anti-DLK1 antibodies showed that 04-0561-F1 (also called DLK-561-F1) bound to human and monkey DLK1 proteins, but not to mouse or rat DLK1 proteins (Figure 6). Although all members of the DLK1 family are related to each other, human and mouse DLK1 proteins shared about 85% amino acid sequence identity (see Figure 10). DLK proteins are membrane-bound proteins that contain a signal peptide that is cleaved from the mature protein, six EGF-like repeats clustered in the N-terminal half of the extracellular domain, a helical transmembrane domain, and a cytoplasmic domain (see Figure 36A). To map the location of DLK1 antibody binding epitopes in the long form of human DLK1 protein (long form or isoform 1) that are not present in the long form of mouse DLK1 protein, the applicants aligned the amino acid sequences of the two proteins and replaced consecutive stretches of amino acid sequences in the human DLK1 protein with the corresponding stretches from mouse DLK1 protein (see FIG. 35 for the amino acid differences between the long form of human DLK1 protein and mouse DLK1 protein and the location of light blue regions 1-8 (top two pairs of arrows)). By dividing the extracellular domain of human DLK1 protein (amino acid residues 24-303) into four regions (regions 1-4) and replacing each region with the corresponding region from mouse DLK1 protein, the location of the epitopes in human DLK1 protein was restricted to regions 1 and 2, or amino acids 24-169. DLK1-561-F1 conjugated with the AF-647 fluorophore (DLK1-561-F1-AF647) was unable to bind to HEK293T cells expressing mGFP protein fusions with the long form of the chimeric human DLK1 protein in which region 1 (834 HEK293T DLK1HM1-mGFP) or region 2 (835 HEK293T DLK1HM2-mGFP) were replaced with the corresponding region from the long form of the mouse DLK1 protein, as analyzed by flow cytometry (see Figure 34).Binding of DLK1-561-F1-AF647 to these two HEK293T cells, namely 834 and 835, was greatly reduced compared to binding to HEK293T cells expressing the long form of the parent human DLK1 protein as a fusion with the mGFP protein (HEK293T DLK1 mGFP G06 (long form)), or to HEK293T cells expressing fusion proteins in which regions 3 or 4 were used to exchange the human DLK1 protein sequence for the mouse DLK1 protein sequence (836 HEK293T DLK1HM3-mGFP and 837 HEK293T DLK1HM4-mGFP).

[0286] To further define the location of the epitope in the long form of the human DLK1 protein, regions 1 and 2 were further divided into smaller regions 5-8 and similar exchange experiments were performed. Exchange of regions 5 or 8 in Figure 35 did not affect binding of the DLK1-561-F1-AF647 antibody to HEK293T cell-expressed mGFP protein fusions with the long form of chimeric human DLK1 with mouse region 5 (850 HEK293T DLK1HM5-mGFP) or region 8 (849 HEK293T DLK1HM8-mGFP) sequences, indicating that the epitope for DLK1-561-F1 antibody binding is not present in region 5 or 8. In contrast, replacement of regions 6 or 7 of the long form of the chimeric human DLK1 protein with the mouse sequence resulted in loss of DLK1-561-F1-AF647 antibody to HEK293T cells (see 851 HEK293T DLK1 HM6-GFP and 848 HEK293T DLK1 HM7-mGFP in Figure 34). Thus, the binding site of the DLK1-561-F1 antibody is present in regions 6 and 7 of the long form of the human DLK1 protein.

[0287] Analysis of GFP fluorescence indicates that the loss of binding of DLK1-561-F1-AF647 antibody to 834, 835, 848 and 851 cells was not the result of lack of expression of the chimeric fusion protein. Negative control experiments using a non-targeting hIgG1 antibody conjugated to the AF647 fluorophore (hIgG1-AF647) and no antibody treatment (no ab) showed that AF647 fluorescence was dependent on the presence of DLK1 binding activity.

[0288] Based on the epitope mapping experiments in Figures 34 and 35, the epitope of the humanized DLK1 antibody DLK1-561-F1 (also referred to as 04-0561-F1) is located within approximately amino acid residues 56 to 113 of the long-chain form of the human DLK1 protein (isoform 1) that forms the EGF-like 2 and EGF-like 3 structural domains (see Figure 36A).

[0289] In addition to the long (or full-length) form of DLK1 protein with 383 amino acids (isoform 1; also called DLK1-FL; see UniProt ID: P80370-1), expression of the human DLK1 gene can also result in an alternatively spliced ​​mRNA that produces a short form of DLK1 protein with 310 amino acids (isoform 2; also called DLK1-MB; see UniProt ID: P80370-2), which lacks the 73 amino acids at amino acid positions 229-301 of isoform 1. The shorter DLK1-MB lacks the juxtamembrane region that contains the cleavage site for the disintegrin and metalloprotease ADAM17, also known as TNFα-converting enzyme (TACE). In FIG. 36B, the large bolt identifies the ADAM17-mediated juxtamembrane cleavage site in DLK1-FL, but is missing in DLK1-MB. The smaller bolt identifies other protease cleavage sites. The short form, DLK1-MB, is more expressed in cancer than the long (full-length) form, DLK1-FL. DLK1 isoform 2 (UniProt ID: P80370-2) contains the following sequence: MTATEALLRVLLLLLAFGHSTYGAECFPACNPQNGFCEDDNVCRCQPGWQGPLCDQCVTSPGCLHGLCGEPGQCICTDGWDGELCDRDVRACSSAPCANNRTCVSLDDGLYECSCAPGYSGKDCQKKDGPCVINGSPCQHGGTCVDDEGRASHASCLCPPGFSGNFCEIVANSCTPNPCENDGVCTDIGGDFRCRCPAGFIDKTCSRPVTNCASSPCQNGGTCLQHTQGQAICFTILGVLTSLVVLGTVGIVFLNKCETWVSNLRYNHMLRKKKNLLLQYNSGEDLAVNIIFPEKIDMTTFSKEAGDEEI (SEQ ID NO: 69). [Table 3]

[0290] Since amino acids 56-113 of the DLK1-FL protein are present in the human DLK1-MB protein, the ability of the lead anti-DLK1 antibody DLK1-561-F1 to bind to the shorter DLK1-MB protein was analyzed in a flow cytometry assay (Figure 37). The binding of the benchmark anti-DLK1 antibody LegoChem 18A5 as well as a negative control non-targeting human IgG1 antibody (hIgG1) was also analyzed. These antibodies were either directly labeled with Alexa Fluor® 647 (ThermoFisher Scientific Catalog No.: A20006) or indirectly labeled with an AF-647-conjugated anti-His tag secondary antibody (BioLegend Catalog No.: 652513) that detects the (His)6 epitope at the C-terminus of the unlabeled primary antibody. Parental HEK293T cells (HEK293T parent) or HEK293T cells expressing mGFP protein fusions with DLK1-FL (HEK293T DLK1 mGFP G06 (long form)) or DLK1-MB (832-HEK293T DLK1 Iso2-mGFP large population (short form)) were incubated with either DLK1-561-F1 antibody (561-F1-AF647 or 561-F1), LegoChem 18A5 (LegoChem 18A5-AF647 or LegoChem 18A5), or hIgG1 (hIgG1-AF647 or hIgG1). In the case of unlabeled primary antibodies, a further incubation with AF647-labeled anti-His tag secondary antibody was performed. After washing, cells were analyzed for AF647 fluorescence by flow cytometry on an iQue platform (Sartorius) (FL4-H channel). As expected, DLK1-561-F1 binds to both isoforms of the human DLK1 protein, i.e., DLK-FL and DLK-MB. However, unlike the DLK1-561-F1 anti-DLK1 antibody, the LegoChem A185 benchmark anti-DLK1 antibody only binds to the long form of the DLK1 protein (isoform 1 or DLK1-FL).Binding of the LegoChem A185 benchmark anti-DLK1 antibody to the short chain form of human DLK1 protein is comparable to that observed with a negative control non-targeting human IgG1 antibody (hIgG1). Thus, when compared to the benchmark anti-DLK1 antibody LegoChem 18A5, Applicants' lead antibody DLK-561-F1 bound to both isoforms of human DLK1, whereas LegoChem 18A5 failed to bind or bound weakly to human DLK1-MB, an isoform of human DLK1 frequently expressed in human cancers.

[0291] To determine the EC50 of the interaction between the two isoforms of human DLK1 protein and DLK1-561-F1 or ChemLego 18A antibodies, ELISA assays were performed using plates coated with either DLK1 ECD-mFc long or DLK1 ECD-mFc short (1 μg / 50 μl PBS / well; overnight at 4° C.; blocking: 5% BSA / PBS, 200 μl / well, 2 h at 37° C.). Plates were incubated for 1.5 h at RT with DLK1 Ab in 2-fold serial dilutions starting at 400 ng / ml in 5% BSA / PBS. A 1:3000 dilution of mouse anti-human IgG Fc secondary antibody conjugated to HRP (Novus catalog number: NBP1-78623H) was added to each well at 50 μl / well and further incubated for 1 h at RT. The ELISA was developed first with Thermo Scientific Pierce 1-Step™ Ultra TMB-ELISA substrate solution, then with the addition of sulfuric acid stop solution. The ELISA plate was read at 450 nm in a plate reader. Figure 38 shows the absorbance at 450 nm (OD450nm) as a function of anti-DLK1 antibody concentration. The DLK1-561-F1 antibody showed a dose-response curve in which higher antibody concentrations resulted in greater absorbance at 450 nm in wells coated with either the long or short forms of human DLK1 protein (i.e., DLK1-FL or DLK1-MB, respectively), with EC50 values ​​of about 0.81-0.87 nM. In contrast, the LegoChem 18A5 antibody showed a shallower dose-response curve, with an EC50 value of about 17.9 nM for binding to the long form of human DLK1 protein (i.e., DLK1-FL or isoform 1). No binding of LegoChem 18A5 antibody to human DLK1-MB, the shorter form or isoform 2 of human DLK1, was observed in ELISA.Thus, the DLK1-561-F1 antibody binds to both isoforms of the human DLK1 protein, and its binding affinity to the long form of the human DLK1 protein (DLK1-FL or isoform 1) is significantly higher (about 20-fold higher) than the binding affinity of the LegoChem 18A5 antibody to human DLK1-FL. Furthermore, DLK1-561-F1 binds to the shorter form of the human DLK1 protein (DLK1-MB or isoform 2) with an affinity equivalent to that of the long form of the human DLK1 protein (DLK1-FL or isoform 1), whereas LegoChem 18A5 was unable to bind to the shorter human DLK1-MB or isoform 2 of the human DLK1 protein.

[0292] The antibody-dependent cellular cytotoxicity (ADCC) function of the lead anti-DLK1 antibody, DLK1-561-F1 (also referred to as 04-0561-F1 and 561-F1), was evaluated in native cells positive for DLK1 expression (COR-L279) or negative for DLK1 expression (M202), and in HEK293 cells engineered to overexpress the short form of the human DLK1 protein (DLK1-MB or isoform 2). These native or engineered cells were incubated with the 561-F1 anti-DLK1 antibody over a range of antibody concentrations or a human IgG1 non-targeting antibody control for 18-20 hours, followed by co-incubation with Jurkat-Lucia™ NFAT-CD16 effector cells (InvivoGen, San Diego, USA) for 6 hours. NFAT activation, indicative of induction of an ADCC response, was assessed by determining Lucia luciferase activity in the supernatant. As shown in FIG. 39, the DLK1-561-F1 antibody exhibited significant ADCC activity compared to native DLK1. + EC of approximately 2.16 nM in COR-L279 cells 50 EC value of approximately 101 ng / ml in HEK293 cells expressing the short form of the human DLK1 protein (Figure 39A). 50The strong ADCC activity observed with the DLK-561-F1 antibody is dependent on the expression of DLK1 protein, since DLK1-negative M202 cells produced the smallest ADCC response, with the largest response being comparable to that observed with the non-targeting hIgG1 control antibody (Figure 39A). Unlike the DLK1-561-F1 antibody, the LegoChem 18A5 benchmark antibody failed to produce an ADCC response in the target HEK293 cells engineered to express the short form of the human DLK1 protein (DLK1-MB or isoform 2) (Figure 39B). Thus, the DLK1-561-F1 antibody induces ADCC in native and overexpressing models of DLK1 expression.

[0293] The light and heavy chain variable regions of the three lead humanized antibodies 04-0547-h2, 04-0561-F1 and 04-0562-h10 were used to generate single chain variable fragments (scFv). In addition, each of the anti-DLK1 scFvs was used to generate a bispecific antibody containing both anti-DLK1 scFv and anti-CD3 scFv to generate an anti-DLK1-CD3 bispecific T cell engager (BiTE). The amino acid sequences of the three scFvs, i.e. 04-0547-h2scfv, 04-0561-F1scFv and 04-0562-h10scFv, along with the DNA sequences encoding these scFvs, are shown in Table 1 (SEQ ID NOs: 57-59 and 63-65). Table 1 also shows the amino acid and DNA sequences of the three respective BiTEs, 04-0547-h2Bs, 04-0561-F1Bs and 04-0562-h10Bs (SEQ ID NOs: 60-62 and 66-68). Each scFv and BiTE further comprises a (His)6 epitope tag at the C-terminus.

[0294] DLK1-dependent binding of scFv and BiTE was assessed by flow cytometry on an iQue platform (Sartorius). Briefly, each scFv or BiTE antibody was incubated with either DLK1+COR-L279 cells or DLK1-M202 cells, followed by incubation with a secondary antibody, i.e., AF-647-labeled anti-His tag antibody. AF-647 fluorescence was captured in the FL4-H channel and the average was reported in the top table shown in Figure 40. Background fluorescence due to the secondary antibody in the absence of primary antibody and fluorescence due to bound cells are reported in the bottom table. Strong AF-647 fluorescence was observed in DLK1 cells incubated with DLK1 scFv and BiTE antibodies. + COR-L279 cells, which was - This was significantly higher than that observed in M202 cells or COR-L279 cells incubated only with AF-647-labeled secondary antibody. Thus, DLK1 scFv and BiTE selectively bound to DLK1-positive COR-L279 cells and did not bind to DLK1-negative M202 cells.

[0295] The T cell activation activity of anti-DLK1 BiTEs was assayed using Jurkat cells and an NFAT-RE reporter construct. T cell activation was measured using Promega's T cell activation bioassay kit (NFAT-RE J1621). Multiple cell lines (Raji, JR, CORL-279) were seeded into white 96-well plate wells at a density of 25,000-40,000 cells / well and incubated overnight at 37°C, after which ready-to-use Jurkat T cells, provided in the assay kit, were added to the seeded cells (1:1 cell ratio) and treated with DLK1 bispecific antibodies. Treated plates were incubated at 37°C for 6 hours, after which Bio-Glo reagent (provided in the kit) was added and read immediately on a Varioskan LUX plate reader. Units were RLU. As shown in Figure 41, cell lines overexpressing DLK1 (JR and COR-L279) induced T cell activation when treated with either 04-0547-h2Bs or 04-0561-F1Bs BiTEs, whereas the DLK1 negative Raji cell line had reduced levels of activation. 04-0562-h10Bs BiTE did not induce T cell activation. Each BiTE further contains a (His)6 epitope tag at the C-terminus.

[0296] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0297] The use of the terms "a," "an," and "the" and similar referents in the context of describing this disclosure (particularly in the context of the claims which follow) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open ended terms (i.e., meaning "including but not limited to"), unless otherwise indicated.

[0298] The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand notation for referring individually to each separate value and each endpoint falling within the range, and each separate value and endpoint is incorporated herein as if it were individually recited herein. As used herein, the term "about," when used before numerical designations including ranges, e.g., temperature, time, amount, concentration, etc., indicates approximations that may vary by (+) or (-) 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0299] All methods described herein can be carried out in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any examples or use of various terms (e.g., "such as") provided herein are merely intended to better illustrate the present disclosure, and do not claim limitations on the scope of the present disclosure unless specifically claimed. No term in this specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure.

[0300] Preferred embodiments of the present disclosure are described herein, including the best mode for carrying out the disclosure known to the inventors. Variations of these preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that those skilled in the art will employ such variations as appropriate, and intend for the present disclosure to be carried out in ways other than those specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11]

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

Table 4-19

Table 4-20

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 6

Claims

1. a. CDRs 1-3 from a heavy chain variable region comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFSISDYYMAWVRQAPGKGLEWVANINYDGTNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRSYYYYGMEYWGQGTTVTVSS (SEQ ID NO: 45) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 70% sequence identity; and / or b. CDRs 1-3 from a light chain variable region comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASHDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYRIPLTFGQGTKLEIK (SEQ ID NO: 46) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 70% sequence identity.

1. An antigen-binding protein comprising:

2. a. CDRs 1-3 from a heavy chain variable region comprising the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSLSIYSVHWVRQPPGKGLEWIGLIWGGGSTDYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCARKEGNYLWFAYWGQGTLVTVSS (SEQ ID NO: 47) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 70% sequence identity; and / or b. CDRs 1-3 from a light chain variable region comprising the amino acid sequence: DIVMTQSPDSLAVSLGERVTMNCKSSQSLLQSSNQKNYLAWYQQKPGQPPKLLVYFASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQHYSIPLTFGQGTKLEIK (SEQ ID NO: 48) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 70% sequence identity.

1. An antigen-binding protein comprising:

3. a. CDRs 1-3 from a heavy chain variable region comprising the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVSWVRQPPGKGLEWIGVIWGDGSTSYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCAKPDGPLGQGTLVTVSS (SEQ ID NO: 49) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 70% sequence identity; and / or b. CDRs 1-3 from a light chain variable region comprising the amino acid sequence: DIVMTQSPLSLPVTPGEPASISCRSSQSLVHINGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTTHVPWTFGQGTKLEIK (SEQ ID NO:50) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 70% sequence identity.

1. An antigen-binding protein comprising:

4. a. a heavy chain CDR1 comprising the amino acid sequence: GFSISDYY (SEQ ID NO: 1) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; b. A heavy chain CDR2 comprising the amino acid sequence: INYDGTNT (SEQ ID NO:2) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; c. A heavy chain CDR3 comprising the amino acid sequence: VRSYYYYGMEY (SEQ ID NO: 3) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; d. A light chain CDR1 comprising the amino acid sequence: HDVSTA (SEQ ID NO: 4) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; e. A light chain CDR2 comprising the amino acid sequence: SAS (SEQ ID NO:5) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; f. a light chain CDR3 comprising the amino acid sequence: QQHYRIPLT (SEQ ID NO: 6) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; or g. A combination of two or more of (a) to (f).

1. An antigen-binding protein comprising:

5. a. a heavy chain CDR1 comprising the amino acid sequence: GFSLSIYS (SEQ ID NO: 7) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; b. Heavy chain CDR2 amino acid sequence: IWGGGST (SEQ ID NO: 8) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; c. A heavy chain CDR3 comprising the amino acid sequence: ARKEGNYLWFAY (SEQ ID NO: 9) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; d. A light chain CDR1 comprising the amino acid sequence: QSLLQSSNQKNY (SEQ ID NO: 10) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; e. A light chain CDR2 comprising the amino acid sequence: FAS (SEQ ID NO: 11) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; f. Light chain CDR3 amino acid sequence: QQHYSIPLT (SEQ ID NO: 12) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 70% sequence identity; or g. A combination of two or more of (a) to (f).

1. An antigen-binding protein comprising:

6. a. Heavy chain CDR1 amino acid sequence: GFSLTSYG (SEQ ID NO: 13) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; b. A heavy chain CDR2 comprising the amino acid sequence: IWGDGST (SEQ ID NO: 14) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; c. A heavy chain CDR3 comprising the amino acid sequence: AKPDGP (SEQ ID NO: 15) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; d. A light chain CDR1 comprising the amino acid sequence: QSLVHINGNTY (SEQ ID NO: 16) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; e. Light chain CDR2 amino acid sequence: KVS (SEQ ID NO: 17) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; f. a light chain CDR3 comprising the amino acid sequence: SQTTHVPWT (SEQ ID NO: 18) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70% sequence identity; or g. A combination of two or more of (a) to (f).

1. An antigen-binding protein comprising:

7. 8. The antigen binding protein of any one of claims 1 to 7, wherein the variant sequence has at least about 80%, at least about 85%, at least about 90% sequence identity, or at least or about 95% sequence identity.

8. a. a heavy chain FR1 comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 21) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70%, 80%, 85%, 90% or 95% sequence identity; b. A heavy chain FR2 comprising the amino acid sequence: MAWVRQAPGKGLEWVAN (SEQ ID NO: 22) or a variant sequence thereof differing by only one or two amino acids or having at least or about 70%, 80%, 85%, 90% or 95% sequence identity; c. A heavy chain FR3 comprising the amino acid sequence: YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 23) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; d. A heavy chain FR4 comprising the amino acid sequence: WGQGTTVTVSS (SEQ ID NO: 24) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; e. A light chain FR1 comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 25) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; f. a light chain FR2 comprising the amino acid sequence: VAWYQQKPGKAPKLLIY (SEQ ID NO: 26) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; g. A light chain FR3 comprising the amino acid sequence: YRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 27) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; h. a light chain FR4 comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO:28) or a variant sequence thereof differing by only one or two amino acids or having at least or about 785%, 90% or 95% sequence identity; or i. A combination of two or more of (a) to (h).

5. The antigen binding protein of claim 4, further comprising:

9. a. a heavy chain FR1 comprising the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO:29) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; b. A heavy chain FR2 comprising the amino acid sequence: VHWVRQPPGKGLEWIGL (SEQ ID NO: 30) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; c. A heavy chain FR3 comprising the amino acid sequence: DYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYC (SEQ ID NO: 31) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; d. A heavy chain FR4 comprising the amino acid sequence: WGQGTLVTVSS (SEQ ID NO: 32) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; e. A light chain FR1 comprising the amino acid sequence: DIVMTQSPDSLAVSLGERVTMNCKSS (SEQ ID NO: 33) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; f. a light chain FR2 comprising the amino acid sequence: LAWYQQKPGQPPKLLVY (SEQ ID NO: 34) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; g. A light chain FR3 comprising the amino acid sequence: TRESGVPDRFSGSGSGTDFTLTISVQAEDVAVYYC (SEQ ID NO: 35) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; h. a light chain FR4 comprising the amino acid sequence: FGQGTKLEIK (SEQ ID NO: 36) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; i. A combination of two or more of (a) to (h).

6. The antigen binding protein of claim 5, further comprising:

10. a. a heavy chain FR1 comprising the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO: 37) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; b. A heavy chain FR2 comprising the amino acid sequence: VSWVRQPPGKGLEWIGV (SEQ ID NO: 38) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; c. A heavy chain FR3 comprising the amino acid sequence: SYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYC (SEQ ID NO: 39) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; d. A heavy chain FR4 comprising the amino acid sequence: LGQGTLVTVSS (SEQ ID NO: 40) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; e. A light chain FR1 comprising the amino acid sequence: DIVMTQSPLSLPVTPGEPASISCRSS (SEQ ID NO: 41) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; f. a light chain FR2 comprising the amino acid sequence: LHWYLQKPGQSPQLLIY (SEQ ID NO: 42) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; g. A light chain FR3 comprising the amino acid sequence: NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 43) or a variant sequence thereof differing by only one or two amino acids or having at least or about 85%, 90% or 95% sequence identity; h. Light chain FR4 amino acid sequence: FGQGTKLEIK (SEQ ID NO:44) or a variant sequence thereof that differs by only one or two amino acids or has at least or about 85%, 90% or 95% sequence identity; or i. A combination of two or more of (a) to (h).

7. The antigen binding protein of claim 6, further comprising:

11. a. a heavy chain variable region comprising the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFSISDYYMAWVRQAPGKGLEWVANINYDGTNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRSYYYYGMEYWGQGTTVTVSS (SEQ ID NO: 45) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or b. A light chain variable region comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASHDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYRIPLTFGQGTKLEIK (SEQ ID NO:46) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

9. The antigen-binding protein of any one of claims 1, 4, 7, and 8, comprising:

12. a. a heavy chain variable region comprising the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSLSIYSVHWVRQPPGKGLEWIGLIWGGGSTDYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCARKEGNYLWFAYWGQGTLVTVSS (SEQ ID NO: 47) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or b. A light chain variable region comprising the amino acid sequence: DIVMTQSPDSLAVSLGERVTMNCKSSQSLLQSSNQKNYLAWYQQKPGQPPKLLVYFASTRESGVPDRFSGSGSGTDFTLTISVQAEDVAVYYCQQHYSIPLTFGQGTKLEIK (SEQ ID NO:48) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

10. The antigen-binding protein of any one of claims 2, 5, 7, and 9, comprising:

13. a. a heavy chain variable region comprising the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVSWVRQPPGKGLEWIGVIWGDGSTSYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCAKPDGPLGQGTLVTVSS (SEQ ID NO:49) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity; and / or b. A light chain variable region comprising the amino acid sequence: DIVMTQSPLSLPVTPGEPASISCRSSQSLVHINGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTTHVPWTFGQGTKLEIK (SEQ ID NO:50) or a variant sequence thereof differing by only 1-5 amino acids or having at least or about 85%, 90%, 95%, 98% or 99% sequence identity.

11. The antigen-binding protein of any one of claims 3, 6, 7, and 10, comprising:

14. Amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGFSLSIYSVHWVRQPPGKGLEWIGLIWGGGSTDYNPSLKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCARKEGNYLWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP an antibody heavy chain comprising: CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:51); and b. An antibody light chain comprising the amino acid sequence: DIVMTQSPDSLAVSLGERVTMNCKSSQSLLQSSNQKNYLAWYQQKPGQPPKLLVYFASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQHYSIPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:52).

1. An antigen binding protein that specifically binds to human Delta-like non-canonical Notch ligand 1 (DLK1), comprising:

15. a. binds to human DLK1 protein isoform 1 having the amino acid sequence set forth in SEQ ID NO: 19 (UniProtKB ID: P80370-1); b. binds to human DLK1 protein isoform 2 having the amino acid sequence set forth in SEQ ID NO: 69 (UniProtKB ID: P80370-2); c. A dissociation constant (K) of less than about 10 nM, 5 nM, 2.5 nM, 1 nM, 0.5 nM, 0.25 nM, 100 pM, 50 pM, 25 pM, 10 pM, or 5 pM with the extracellular domain of human DLK1. D ) or d. does not bind to mouse DLK1 protein having the amino acid sequence set forth in SEQ ID NO: 20 (UniProtKB ID: Q09163), or binds to said mouse DLK1 protein with an affinity 100-fold or less compared to said human DLK1 protein; or e. Combinations thereof 2. An antigen-binding protein according to any one of the preceding claims,

16. 10. An antigen-binding protein according to any one of the preceding claims, which binds to human DLK1 isoform 1 (UniProt ID: P80370-1) and isoform 2 (UniProt ID: P80370-2).

17. Human DLK1 isoform 1 (UniProt ID: P80370-1) and isoform 2 (UniProt ID: P80370-2), a. less than about 6 nM; b. Approximately 5 nM, c. about 2 nM, or d. Approximately 1nM 17. The antigen-binding protein of claim 16, which binds with a KD of:

18. a. an epitope within the EGF-like 2 and EGF-like 3 domains of the human DLK1 isoform 1 protein (UniProt ID: P80370-1), and / or b. A polypeptide comprising amino acid residues 55 to 113 of the human DLK1 isoform 1 protein (UniProt ID: P80370-1).

2. An antigen-binding protein according to any one of the preceding claims which binds to

19. 10. An antigen-binding protein according to any one of the preceding claims, which binds to the human DLK1 protein and induces (a) an antibody-dependent cell-mediated cytotoxicity (ADCC) response, and / or (b) a complement-dependent cytotoxicity (CDC) response in DLK1 positive cells.

20. 2. An antigen-binding protein according to any one of the preceding claims which is an antibody or an antigen-binding antibody fragment.

21. 21. The antigen binding protein of claim 20, wherein the antibody is a monoclonal antibody.

22. 21. The antigen binding protein of claim 20, wherein the antibody is a chimeric antibody, a human antibody, or a humanized antibody.

23. 23. The antigen binding protein of any one of claims 20 to 22, wherein the antibody is an IgG.

24. 24. The antigen binding protein of claim 23, wherein the IgG is selected from IgG1, IgG2, IgG3 and IgG4.

25. 25. The antigen binding protein of claim 23 or 24, wherein the IgG is IgG1.

26. The antigen-binding antibody fragment is an scFv, F(ab') 2 21. The antigen binding protein of claim 20, which is selected from the group consisting of Fab, Fab' and Fv.

27. 21. The antigen-binding protein of claim 20, wherein the antigen-binding antibody fragment is a single-chain variable fragment (scFv).

28. The scFv is a. any variable light (V) of the antigen binding protein according to any one of the preceding claims L ) chain and variable weight (V H )chain, b. the amino acid sequences set forth in SEQ ID NO: 45 and SEQ ID NO: 46; c. the amino acid sequences set forth in SEQ ID NO: 47 and SEQ ID NO: 48; d. the amino acid sequence set forth in SEQ ID NO: 49 and SEQ ID NO: 50; or e. An amino acid sequence set forth in SEQ ID NO: 57, 58, or 59, or an antigen-binding portion thereof.

28. The antigen binding protein of claim 27, comprising:

29. 28. The antigen binding protein of claim 27, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO:58, or an antibody-binding portion thereof.

30. 10. The antigen-binding protein of any one of the preceding claims, which is a bispecific antigen-binding protein or a bispecific T cell engager (BiTE).

31. the bispecific antigen binding protein or the BiTE, a. SEQ ID NO:45 and SEQ ID NO:46; b. SEQ ID NO: 47 and SEQ ID NO: 48, or c. SEQ ID NO: 49 and SEQ ID NO: 50 31. The antigen-binding protein of claim 30, comprising the amino acid sequence set forth in

32. 32. The antigen-binding protein of claim 30 or 31 , wherein the bispecific antigen-binding protein or BiTE comprises an scFv of claim 28 or 29 .

33. 33. The antigen binding protein of any one of claims 30 to 32, wherein the bispecific antigen binding protein or BiTE binds to DLK1 and a T cell surface marker.

34. 34. The antigen binding protein of claim 33, wherein the T cell surface marker is a CD3 protein.

35. 35. The antigen-binding protein of any one of claims 30 to 34, wherein the bispecific antigen-binding protein or BiTE comprises an amino acid sequence as set forth in SEQ ID NO: 60, 61, or 62, or an antigen-binding portion thereof.

36. 36. The antigen-binding protein of any one of claims 30 to 35, wherein the bispecific antigen-binding protein or BiTE comprises the amino acid sequence set forth in SEQ ID NO: 60 or an antigen-binding portion thereof.

37. 36. The antigen-binding protein of any one of claims 30 to 35, wherein the bispecific antigen-binding protein or BiTE comprises the amino acid sequence set forth in SEQ ID NO: 61 or an antigen-binding portion thereof.

38. 36. The antigen binding protein of any one of claims 30 to 35, wherein the bispecific antigen binding protein or BiTE induces T cell activation in the presence of DLK positive cells.

39. 10. An antigen-binding protein according to any one of the preceding claims which inhibits tumour growth in xenograft mice injected with human cancer cells.

40. 2. The antigen binding protein of any one of the preceding claims, comprising an Fc polypeptide comprising non-fucosylated glycans.

41. 13. A conjugate comprising an antigen-binding protein according to any one of the preceding claims or an antigen-binding protein as described herein.

42. 42. The conjugate of claim 41, comprising a detectable marker, a cytotoxic agent, or a chemotherapeutic agent.

43. 43. The conjugate of claim 42, wherein the chemotherapeutic agent is an antimitotic agent that inhibits cell division by inhibiting tubulin polymerization.

44. 44. The conjugate of claim 43, wherein the antimitotic agent is an auristatin.

45. 45. The conjugate of claim 44, wherein the auristatin is monomethylauristatin E or MMAE.

46. 46. ​​The conjugate of any one of claims 42 to 45, wherein the drug or marker is conjugated to the antigen binding protein via a cleavable or non-cleavable linker.

47. The conjugate of claim 46, wherein the cleavable linker is VC-PAB.

48. 48. The conjugate of any one of claims 41 to 47, wherein the antigen binding protein is an antibody.

49. The conjugate of claim 48, wherein the antibody is a monoclonal antibody.

50. The conjugate of claim 49 , wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

51. 51. The conjugate of claim 50, wherein the antibody is an IgG antibody, optionally wherein the IgG is IgG1, IgG2, IgG3, or IgG4.

52. 53. The conjugate of any one of claims 41 to 52, wherein the average number of units of the drug conjugated per antigen binding protein is in the range of 1 to 8, preferably the average number of units of the drug conjugated per antigen binding protein is (a) in the range of 3 to 8, or (b) 4.

53. 53. The conjugate of any one of claims 41 to 52, which is a heterogeneous conjugate.

54. 53. The conjugate of any one of claims 41 to 52, which is a homogeneous conjugate.

55. 55. The conjugate of any one of claims 42 to 54, wherein the agent is conjugated to a specific site on the antigen binding protein.

56. 56. The conjugate of claim 55, wherein the specific site is an unpaired cysteine ​​residue.

57. 57. The conjugate of any one of claims 41 to 56, comprising a polypeptide comprising the amino acid sequences set forth in SEQ ID NO: 45 and SEQ ID NO: 46 conjugated to VC-PAB-MMAE.

58. 57. The conjugate of any one of claims 41 to 56, comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 47 and SEQ ID NO: 48 conjugated to VC-PAB-MMAE.

59. 57. The conjugate of any one of claims 41 to 56, comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 49 and SEQ ID NO: 50 conjugated to VC-PAB-MMAE.

60. 57. The conjugate of any one of claims 41 to 56, comprising a polypeptide comprising the amino acid sequences set forth in SEQ ID NO:51 and SEQ ID NO:52 conjugated to VC-PAB-MMAE.

61. A fusion protein comprising an antigen-binding protein according to any one of the preceding claims.

62. 62. A nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein according to any one of claims 1 to 40, a conjugate according to any one of claims 41 to 60, or a fusion protein according to claim 61.

63. 63. The nucleic acid of claim 62 which is a cDNA.

64. 64. A vector (e.g., an expression vector) comprising the nucleic acid of claim 62 or 63.

65. 65. The vector of claim 64, further comprising an internal ribosome entry site (IRES).

66. 66. A host cell comprising a nucleic acid according to claim 62 or 63 or a vector according to claim 64 or 65.

67. 67. The host cell of claim 66 which is a bacterial cell.

68. 67. The host cell of claim 66, which is a eukaryotic cell.

69. 69. The host cell of claim 68, wherein the eukaryotic cell is a mammalian cell.

70. 70. The host cell of claim 69, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

71. 71. A method of making an antigen binding protein that binds to Delta-like non-canonical Notch ligand 1 (DLK1) protein, the method comprising: (i) culturing a host cell of any one of claims 66 to 70 in a cell culture medium; and (ii) harvesting the antigen binding protein from the cell culture medium.

72. 71. A method for making a fusion protein comprising an antigen binding protein that binds to a Delta-like non-canonical Notch ligand 1 (DLK1) protein, the method comprising: (i) culturing a host cell described in any one of claims 66 to 70 in a cell culture medium; and (ii) harvesting the fusion protein from the cell culture medium.

73. 11. A method of making a pharmaceutical composition, the method comprising combining (a) an antigen binding protein according to any one of claims 1 to 40, a conjugate according to any one of claims 41 to 60, a fusion protein according to claim 61, a nucleic acid according to claim 62 or 63, a vector according to claim 64 or 65, a host cell according to any one of claims 66 to 70, or any combination thereof, and (b) a pharma- ceutically acceptable carrier, diluent and / or excipient.

74. 70. A pharmaceutical composition comprising an antigen binding protein according to any one of claims 1 to 40, a conjugate according to any one of claims 41 to 60, a fusion protein according to claim 61, a nucleic acid according to claim 62 or 63, a vector according to claim 64 or 65, a host cell according to any one of claims 66 to 70, or any combination thereof; and (b) a pharma- ceutically acceptable carrier, diluent and / or excipient.

75. 75. A method of treating a subject having a DLK1-expressing cancer, comprising administering to the subject a pharmaceutical composition of claim 74 to treat the cancer.

76. 75. A method of inhibiting tumor growth in a subject, comprising administering to the subject the pharmaceutical composition of claim 74, thereby inhibiting tumor growth.

77. 75. A method of reducing tumor size in a subject, comprising administering to the subject the pharmaceutical composition of claim 74, thereby reducing tumor size.

78. 75. A method of preventing recurrence of cancer in a subject, comprising administering to the subject the pharmaceutical composition of claim 74 to prevent recurrence of the cancer.

79. 75. A method for treating cancer in a subject diagnosed as having low overexpression of DLK1, comprising administering to the subject a pharmaceutical composition described in claim 74 to prevent recurrence of the cancer.

80. 80. The method of any one of claims 75 to 79, wherein said administration induces apoptosis in tumor cells.

81. 80. The method of any one of claims 75 to 79, wherein said administration induces apoptosis in cells expressing Delta-like non-canonical Notch ligand 1 (DLK1).

82. 11. A method for detecting Delta-like non-canonical Notch ligand 1 (DLK1) in a sample, the method comprising contacting the sample with an antigen binding protein of any one of claims 1 to 40, a conjugate of any one of claims 41 to 60, or a fusion protein of claim 61, and assaying an immune complex comprising the antigen binding protein, the conjugate or the fusion protein bound to DLK1.

83. 62. A method of diagnosing Delta-like non-canonical Notch ligand 1 (DLK1) positive cancer in a subject, the method comprising contacting a biological sample comprising cells or tissue obtained from the subject with an antigen binding protein described in any one of claims 1 to 40, a conjugate described in any one of claims 41 to 60, or a fusion protein described in claim 61, and assaying for immune complexes comprising the antigen binding protein, the conjugate or the fusion protein bound to DLK1.

84. 84. The method of claim 83, further comprising treating the subject diagnosed with DLK1 positive cancer by administering to the subject an antigen binding protein of any one of claims 1 to 40, a conjugate of any one of claims 41 to 60, or a fusion protein of claim 61.

85. 40. A method of activating T cells that target DLK1-expressing cancer cells in a subject, the method comprising administering to the subject a bispecific T cell engager (BiTE) according to any one of claims 30 to 38.

86. 13. A method of inducing an antibody-dependent cell-mediated cytotoxicity (ADCC) response against DLK1 expressing cancer cells in a subject, comprising administering to the subject an antigen binding protein that binds DLK1, said antigen binding protein comprising an Fc effector function and the VH and VL regions of an antigen binding protein according to any one of the preceding claims or an antigen binding protein described herein.

87. 10. The method of any one of the preceding claims, wherein the subject is a mammal, optionally a dog, a cat, a mouse, or a human.

88. 13. A kit comprising: (a) an antigen binding protein according to any one of claims 1 to 40, a conjugate according to any one of claims 41 to 60, a fusion protein according to claim 61, a nucleic acid according to claim 62 or 63, a vector according to claim 64 or 65, a host cell according to any one of claims 66 to 70, or any combination thereof; and (b) instructions for use.

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