Methods and compositions for modulating beta chain mediated immunity

Abstract

The present disclosure relates to bispecific molecules targeting V017 and DLLS, nucleic acids and expression vectors encoding said molecules, recombinant cells containing the vectors, and compositions comprising the molecules. Methods of making the antibodies, and methods of using the antibodies to kill cancer cells, are also provided.

Description

065768.12701 / 207W01METHODS AND COMPOSITIONS FOR MODULATING BETA CHAIN MEDIATED IMMUNITYCROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 679,967, filed August 6, 2024, the content of which is incorporated herein by reference in its entirety, and to which priority is claimed.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml file, created on August 5, 2025, is named 065768-207WO1 Sequence Listing. xml and is 85,743 bytes in size.FIELD OF THE INVENTION

[0003] The present disclosure relates to bispecific molecules targeting V017 and DLL3, nucleic acids and expression vectors encoding said molecules, recombinant cells containing the vectors, and compositions comprising the molecules. Methods of making the antibodies, and methods of using the antibodies to kill cancer cells, are also provided.BACKGROUND OF THE INVENTION

[0004] Cytotoxic T cells (e.g., CD8+ T cells) can be utilized to directly kill cancer cells. Finding a way to direct cytotoxic T cells to a cancer cell could lead to the killing of such cells and an inhibition of cancer cell propagation. It has been demonstrated that cytotoxic T cells can be activated against cancer cells expressing cancer-associated antigens, by bringing said cytotoxic T cells into close proximity to the cancer cells for an extended period of time using a bispecific antibody that binds both the cytotoxic T cell and the cancer cell. A variety of potential complications to this approach of killing cancer cells exist, such as selecting T cell and cancer cell antigens that mediate T cell activation, selecting parental antibodies that will have adequate affinity to mediate binding in the context of a bispecific antibody, and choosing a cancer cell antigen that will activate T cells to act specifically against cancer cells, rather than elicit1JBI6937065768.12701 / 207W01 nonspecific T cell activation. These complications are only compounded in the context of attempting to activate T cells to destroy cancer cells in an animal subject.SUMMARY OF THE INVENTION

[0005] The presently disclosed subject matter provides a bispecific antibody comprising: (a) a Fab binding V 17 comprising a heavy chain variable region comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 8; and (b) an spFv binding DLL3 comprising a heavy chain variable region comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 33, and a light chain variable region an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the Fab comprising a heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the spFv comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34.

[0006] The presently disclosed subject matter provides a bispecific antibody comprising: (a) a first heavy chain (HC1) comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 23, (b) a first light chain (LC1) comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 24, and (c) a second heavy chain (HC2) comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 37; wherein HC1 is associated with LC1 to form a binding site for V 17, and HC2 comprises an spFv binding DLL3.

[0007] The presently disclosed subject matter provides a bispecific antibody comprising: (a) a first heavy chain (HC1) comprising the amino acid sequence set forth in SEQ ID NO: 23, (b) a first light chain (LC1) comprising the amino acid sequence set forth in SEQ ID NO: 24, and (c) a second heavy chain (HC2) comprising the amino acid sequence set forth in SEQ ID NO: 37; wherein HC1 is associated with LC 1 to form a binding site for Vpi7, and HC2 comprises an spFv binding DLL3.2JBI6937065768.12701 / 207W01

[0008] The presently disclosed subject matter relates to a bispecific antibody comprising: (a) a first binding domain that binds Vpi7 including a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 53, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; and (b) a second binding domain that binds DLL3 comprising a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the first binding domain or the second binding domain comprises an spFv. In certain embodiments, the second binding domain comprises an spFv.

[0009] In certain embodiments, the second binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, the second binding domain comprises a light chain variable region including the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the second binding domain comprises a heavy chain variable region including the amino acid sequence set forth in SEQ ID NO: 33 and a light chain variable region including the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the second binding domain comprises the amino acid sequence set forth in SEQ ID NO: 36 or SEQ ID NO: 37. In certain embodiments, the second binding domain comprises the amino acid sequence set forth in SEQ ID NO: 37.

[0010] In certain embodiments, the first binding domain comprises a heavy chain variable region including the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 9. In certain embodiments, the first binding domain comprises a light chain variable region including the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 10. In certain embodiments, the first binding domain comprises (a) a heavy chain variable region including the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region including the amino acid sequence set forth in SEQ ID NO: 8; or (b) a heavy chain variable region including the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region including the amino acid sequence set3JBI6937065768.12701 / 207W01 forth in SEQ ID NO: 10. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the first binding domain comprises the amino acid sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 20. In certain embodiments, the first binding domain comprises the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the first binding domain comprises a heavy chain including the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the first binding domain comprises a light chain including the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the first binding domain comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0011] In certain embodiments, the first binding domain is a half antibody.

[0012] In certain embodiments, the presently disclosed subject matter relates to a bispecific antibody, wherein the first binding domain comprises an spFv. In certain embodiments, the first binding domain comprises a heavy chain variable region including the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17. In certain embodiments, the first binding domain comprises a light chain variable region including the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18.

[0013] In certain embodiments, the first binding domain comprises (a) a heavy chain variable region including the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region including the amino acid sequence set forth in SEQ ID NO: 12; (b) a heavy chain variable region including the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region including the amino acid sequence set forth in SEQ ID NO: 14; (c) a heavy chain variable region including the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region including the amino acid sequence set forth in SEQ ID NO: 16; or (d) a heavy chain variable region including the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region including the amino acid sequence set forth in SEQ ID NO: 18.

[0014] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 11 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15 and the light chain variable region4JBI6937065768.12701 / 207W01 comprises the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the first binding domain comprises the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 22.

[0015] In certain embodiments, the second comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31. In certain embodiments, the second binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the second comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the second binding domain comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38.

[0016] The presently disclosed subject matter further relates to a bispecific antibody comprising: (a) a Fab binding V017 comprising a heavy chain including the amino acid sequence set forth in SEQ ID NO: 23 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 24; and (b) an spFv binding DLL3 comprising the amino acid sequence set forth in SEQ ID NO: 36.

[0017] The presently disclosed subject matter also relates to a bispecific antibody including: (a) an Fab binding V017 comprising a heavy chain including the amino acid sequence set forth in SEQ ID NO: 23 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 24; and (b) an heavy chain binding DLL3 comprising the amino acid sequence set forth in SEQ ID NO: 37.

[0018] In addition, the presently disclosed subject matter relates to a nucleic acid encoding the bispecific antibody.

[0019] The presently disclosed subject matter relates to a vector comprising the nucleic acid disclosed herein and to a host cell comprising the nucleic acid or the vector provided herein. Moreover, the presently disclosed subject matter relates to a composition comprising the bispecific antibody, the nucleic acid, the vector, or the host cell disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.5JBI6937065768.12701 / 207W01

[0020] Additionally, the presently disclosed subject matter relates to a method for treating a cancer comprising a cell expressing DLL3 in a subject in need thereof, the method comprising administering an effective amount of the bispecific antibody disclosed herein.

[0021] In certain embodiments, the cancer is adrenal cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational trophoblastic, head and neck cancer, Hodgkin lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, neuroendocrine tumor, non-Hodgkin lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma spinal cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer endometrial cancer, vaginal cancer, or vulvar cancer. In certain embodiments, the lung cancer is small cell lung cancer, small cell carcinoma, combined small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, large-cell undifferentiated carcinoma, pulmonary nodule, metastatic lung cancer, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland-type lung carcinoma, lung carcinoid, mesothelioma, sarcomatoid carcinoma of the lung, or malignant granular cell lung tumor. In certain embodiments, the lung cancer is small cell lung cancer.

[0022] The presently disclosed subject matter relates to a method for eliminating a cancer cell expressing DLL3 in a subject in need thereof, inhibiting the growth or proliferation of cancer cells expressing DLL3 in a subject in need thereof, promoting T cell mediated killing of cancer cells expressing DLL3 in a subject in need thereof, and / or directing a Vpi7-expressing T cell to a cancer cell expressing DLL3 in a subject in need thereof. In certain embodiments, the method comprises administering an effective amount of the bispecific antibody disclosed herein.

[0023] In certain embodiments, the T cell is a CD8 T cell or a CD4 T cell. In certain embodiments, the cancer cell is a cell of an adrenal cancer, an anal cancer, an appendix cancer, a bile duct cancer, a bladder cancer, a bone cancer, a brain cancer, a breast cancer, a cervical cancer, a colorectal cancer, an esophageal cancer, a gallbladder cancer, a gestational trophoblastic, a head and neck cancer, a Hodgkin lymphoma, an intestinal cancer, a kidney cancer, a leukemia, a liver cancer, a lung cancer, a melanoma, a mesothelioma, a multiple myeloma, a neuroendocrine tumor, a non-Hodgkin lymphoma, an oral cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a sinus cancer, a skin cancer, a soft tissue sarcoma spinal cancer, a stomach cancer, a testicular6JBI6937065768.12701 / 207W01 cancer, a throat cancer, a thyroid cancer, a uterine cancer endometrial cancer, a vaginal cancer, or a vulvar cancer. In certain embodiments, the lung cancer is small cell lung cancer, small cell carcinoma, combined small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, large-cell undifferentiated carcinoma, pulmonary nodule, metastatic lung cancer, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland-type lung carcinoma, lung carcinoid, mesothelioma, sarcomatoid carcinoma of the lung, or malignant granular cell lung tumor. In certain embodiments, the lung cancer is small cell lung cancer.

[0024] The presently disclosed subject matter provides bispecific antibodies disclosed herein for use in treating a cancer comprising a cell expressing DLL3, eliminating a cancer cell expressing DLL3, inhibiting the growth or proliferation of cancer cells expressing DLL3, and / or promoting T cell mediated killing of cancer cells expressing DLL3. Additionally, the presently disclosed subject matter provides use of the bispecific antibody disclosed herein for the preparation of a medication for treating a cancer comprising a cell expressing DLL3, eliminating a cancer cell expressing DLL3, inhibiting the growth or proliferation of cancer cells expressing DLL3, and / or promoting T cell mediated killing of cancer cells expressing DLL3.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings.

[0026] Figure 1 shows an illustration of a bispecific antibody disclosed herein targeting V017 and DLL3.

[0027] Figures 2A and 2B show representative vectors (i.e., expression plasmid) expressing the presently disclosed bispecific antibodies.

[0028] Figure 3 shows the tumor growth profile of tumors treated with J8. Specifically, Group 1-Group 4: On day 0, V017 cells (10xl06cells / animal / O.lmL, i.p.) were injected intraperitoneally to all the animals. After 24h (day 1), the dosing was initiated. On the day of dosing, IVIG (lOmg / animal / O. lmL) and Fc (0.2mg / animal / 0.1mL) were injected i.p. in the animals with 30- minute intervals in between. Later, Ih after IVIG and Fc dosing, J8 was injected i.v. to all the7JBI6937065768.12701 / 207W01 groups. Group 5-Group 6: No Vpi 7 T cell injection, no IVIG and Fc dosing was performed, only J8.

[0029] Figures 4A-4E show IncuCyte based cytotoxicity of SHP-77 / GFP cells by J8, tarlatamab and Vpi7xNull in a co-culture assay with PBMCs from 4 donors at 1 : 1 E:T (A-D). Matching ET numbers were used for tarlatamab. The average of all four donors were plotted in Figure 4E.

[0030] Figures 5A-5E show IncuCyte based cytotoxicity of NCI-H82 / GFP cells by J8, tarlatamab and Vpi7xNull in a co-culture assay with PBMCs from 4 donors at 1 : 1 E:T (A-D). Matching ET numbers were used for tarlatamab. The average of all four donors were plotted in Figure 5E.

[0031] Figures 6A-6E show IncuCyte based cytotoxicity of NCI-H1436 / GFP cells by J8, tarlatamab and vpi7xNull in a co-culture assay with PBMCs from 4 donors at 1 : 1 E:T (A-D). Matching ET numbers were used for tarlatamab. The average of all four donors were plotted in Figure 6E.

[0032] Figures 7A-7C show IncuCyte based cytotoxicity of SHP-77 / GFP, NCI-H82 / GFP and NCI-H1436 / GFP by J8, tarlatamab and Vpi7xNull in a co-culture assay with PBMCs at 0.5: 1 E:T. Matching ET numbers were used for tarlatamab.

[0033] Figure 8 shows the kinetics of IncuCyte based cytotoxicity of SHP-77 / GFP, NCI- H82 / GFP and NCI-H1436 / GFP Cells by J8 in co-culture assay with PBMCs from 4 donors at 0.5: 1 and 1: 1 E:T at the top three concentrations of lOOnM, 33.33nM and l l.l lnM. Matching ET numbers were used for tarlatamab.DETAILED DESCRIPTION OF THE INVENTION

[0034] Discussion of documents, acts, materials, devices, articles, or the like which has been included in the present specification is for the purpose of providing context for the present disclosure. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification.

[0036] Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.”8JBI6937065768.12701 / 207W01Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

[0037] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure. Such equivalents are intended to be encompassed by the present disclosure.

[0038] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0039] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0040] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of9JBI6937065768.12701 / 207W01 the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0041] It should also be understood that the terms “about,” “approximately,” “generally,” “substantially,” and like terms, used herein when referring to a dimension or characteristic of a component of the present disclosure, indicate that the described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

[0042] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences (e.g., anti-Vpi7 / anti-DLL3 bispecific antibodies and polynucleotides that encode them, Vpi7 polypeptides and Vpi7 polynucleotides that encode them, DLL3 polypeptides and DLL3 polynucleotides that encode them), refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0043] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0044] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M.10JBI6937065768.12701 / 207W01Ausubel etal., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

[0045] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389- 3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul etal., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.Bispeci fic Molecules and Bispecific Antibodies

[0046] The present disclosure provides isolated anti-VP 17 / anti-DLL3 bispecific molecules (e.g., bispecific antibodies) targeting Vpi7 and DLL3 or antigen-binding fragments thereof, nucleic acids and expression vectors encoding the molecules, recombinant cells containing the vectors, and compositions comprising the bispecific molecules (e.g., bispecific antibodies). Methods of making the molecules (e.g., bispecific antibodies), and methods of using the same to treat diseases, including cancer, are also provided herein. The bispecific molecules (e.g., bispecific antibodies) disclosed herein possess one or more desirable functional properties including, but not limited to, high-affinity binding to VP17 and / or DLL3, high specificity to VP17 and / or DLL3, and the ability to treat or prevent cancer when administered alone or in combination with other anti-cancer therapies.

[0047] As used herein, the term "antibody" is used in a broad sense and includes immunoglobulin or antibody molecules including human, humanized, composite and chimeric antibodies and antibody fragments that are monoclonal or polyclonal. Antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG and IgM), depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further subclassified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Accordingly, the antibodies11JBI6937065768.12701 / 207W01 provided herein can be of any of the five major classes or corresponding sub-classes. In certain embodiments, the antibodies provided herein are IgGl, IgG2, IgG3, or IgG4. Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains. Accordingly, the antibodies provided herein can include a kappa or lambda light chain constant domain. In certain embodiments, the antibodies disclosed herein include heavy and / or light chain constant regions from rat or human antibodies.

[0048] In addition to the heavy and light constant domains, antibodies contain an antigenbinding region that is made up of a light chain variable region (VL) and a heavy chain variable region (VH), each of which contains three domains (i.e., complementarity determining regions 1 (CDR1), CDR2 and CDR3). A "CDR" refers to one of three hypervariable regions (HCDR1, HCDR2, or HCDR3) within the non-framework region of the immunoglobulin (Ig or antibody) VH ( -sheet framework, or one of three hypervariable regions (LCDR1, LCDR2, or LCDR3) within the non-framework region of the antibody VL (P- sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by multiple designation methods (e.g., Kabat, Chothia, AbM, IMGT, etc.).

[0049] The light chain variable region CDR1 domain is interchangeably referred to herein as LCDR1 or VL CDR1. The light chain variable region CDR2 domain is interchangeably referred to herein as LCDR2 or VL CDR2. The light chain variable region CDR3 domain is interchangeably referred to herein as LCDR3 or VL CDR3. The heavy chain variable region CDR1 domain is interchangeably referred to herein as HCDR1 or VH CDR1. The heavy chain variable region CDR2 domain is interchangeably referred to herein as HCDR2 or VH CDR2. The heavy chain variable region CDR1 domain is interchangeably referred to herein as HCDR3 or VH CDR3.

[0050] The term "hypervariable region", such as a VH or VL, when used herein refers to the regions of an antibody variable region that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3).

[0051] The term "constant region" or "constant domain" refers to a carboxy terminal portion of the light and heavy chain, which is not directly involved in binding of the antibody to antigen but exhibits various effector functions, such as interaction with the Fc receptor. The terms refer to the12JBI6937065768.12701 / 207W01 portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding site. The constant region may contain the CHI, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0052] The term "framework" or "FR" residues are those variable region residues flanking the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.

[0053] As used herein, the term an "isolated antibody" refers to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to VP 17 is substantially free of antibodies that do not bind to V 17; an isolated antibody that specifically binds to DLL3 is substantially free of antibodies that does not bind to DLL3. In addition, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0054] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. The monoclonal antibodies disclosed herein can be made by the hybridoma method, phage display technology, single lymphocyte gene cloning technology, or by recombinant DNA methods. For example, the monoclonal antibodies can be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, such as a transgenic mouse or rat, having a genome comprising a human heavy chain transgene and a light chain transgene.

[0055] As used herein, the term "antigen-binding fragment" refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv1), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a single domain antibody (sdAb) an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, an spFv or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds. In certain13JBI6937065768.12701 / 207W01 embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of the heavy chain. In certain embodiments, the antigenbinding fragment comprises Fab and F(ab').

[0056] As used herein, a “binding domain” refers to that part of a molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Binding domains include, for example, antibodies, antibody fragments thereof (such as Fab fragments, Fab '2, scFv antibodies, spFv antibodies, SMIP, domain antibodies, diabodies, minibodies, scFv-Fc, affibodies, nanobodies) and VH and / or VL domains of antibodies.

[0057] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the field, which comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single domain antibody" refers to a conventional single domain antibody in the field, which comprises a heavy chain variable region and a heavy chain constant region or which comprises only a heavy chain variable region.

[0058] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide.

[0059] As used herein, the term "humanized antibody" refers to a non-human antibody that is modified to increase the sequence homology to that of a human antibody, such that the antigenbinding properties of the antibody are retained, but its antigenicity in the human body is reduced.

[0060] As used herein, the term "chimeric antibody" refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable region of both the light and heavy chains often correspond to the variable region of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capability, while the constant regions correspond to the sequences of an antibody derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.

[0061] The term "specificity" refers to selective recognition of an antigen binding protein (such as an antibody) for a particular epitope of an antigen. Natural antibodies, for example, are14JBI6937065768.12701 / 207W01 monospecific. The term "multispecific" as used herein denotes that an antigen binding protein (such as an antibody) has two or more antigen-binding sites of which at least two bind different antigens. "Bispecific" as used herein denotes that an antigen binding protein has two different antigen-binding specificities.

[0062] As used herein, the term "multispecific antibody" refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In certain embodiments, the first and second epitopes do not overlap or do not substantially overlap. In certain embodiments, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In an embodiment, a multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In certain embodiments, a multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0063] As used herein, the terms “spFv” or “stapled Fv” refer to a stabilized scFv including a linker that contains one or two cysteine residues capable of forming a disulfide bond with an anchor point cysteine. spFv antibodies are further described in International Patent Publication No. WO 2021 / 030657, which is incorporated by reference herein in its entirety.

[0064] As used herein, the term “bispecific antibody” refers to a multispecific antibody that binds no more than two epitopes or two antigens. A bispecific antibody comprises a first binding domain and a second binding domain. In certain embodiments, the first binding domain comprises a first immunoglobulin heavy and light chain pair which has binding specificity for a first epitope (e.g., an epitope on a Vpi7 antigen). In certain embodiments, the second binding domain comprises a second immunoglobulin heavy and light chain pair that has binding specificity for a second epitope (e.g., an epitope on a DLL3 antigen). In certain embodiments, the first binding domain comprises a first immunoglobulin heavy and light chain pair which has binding specificity for VP17. In certain embodiments, the second binding domain comprises a second immunoglobulin heavy and light chain pair that has binding specificity for DLL3. In certain embodiments, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In certain embodiments, a bi specific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence15JBI6937065768.12701 / 207W01 which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope. In certain embodiments, a bispecific antibody comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In certain embodiments, a bispecific antibody comprises a scFv, or fragment thereof, having binding specificity for a first epitope, and a scFv, or fragment thereof, having binding specificity for a second epitope. In certain embodiments, a bispecific antibody comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a scFv, or fragment thereof, having binding specificity for a second epitope. In certain embodiments, a bispecific antibody comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a spFv, or fragment thereof, having binding specificity for a second epitope. In certain embodiments, a bi specific antibody comprises a scFv, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In certain embodiments, a bispecific antibody comprises a spFv, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope.

[0065] The term "half antibody" as used herein refers to one immunoglobulin heavy chain associated with one immunoglobulin light chain. One skilled in the art will readily appreciate that a half-antibody can encompass a fragment thereof and can also have an antigen binding domain consisting of a single variable domain, e g., originating from a camelidae.

[0066] As used herein, the term “Vpi7” refers to a T cell receptor, which is expressed in response to an immune response on a cytotoxic T cell. Vpi7-expressing CD8+ T cells are commonly produced in response to influenza A virus exposure in a subject. Vpi7-expressing CD8+ T cells provide great recall in response to influenza exposure in the subject. The term “Vpi7” includes any Vpi7 variant, isoform, and species homolog, which is naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNA encoding the polypeptide. Unless noted, preferably the Vpi7 is a human Vpi7. A human Vpi7 amino acid sequence is provided by GenBank Accession Number AAB49730.1.

[0067] The term “DLL3” as used herein refers to a molecule that is found on cells that acts as an inhibitory Notch pathway ligand. DLL3 can also be referred to as “Delta-like ligand 3.” The term “DLL3” includes any DLL3 variant, isoform, and species homolog, which is naturally expressed16JBI6937065768.12701 / 207W01 by cells (including tumor cells) or can be expressed on cells transfected with genes or cDNA encoding the polypeptide. In certain embodiments, the DLL3 is a human DLL3. The extracellular domain of human DLL3 consists of, according to UniProt Accession Number Q9NYJ7, amino acids 27 - 492. The term “antibody targeting DLL3,” “antibody binding DLL3,” or “anti-DLL3 antibody” as used herein relates to an antibody specifically binding to DLL3.

[0068] As used herein, an antibody that “specifically binds to VP17” refers to an antibody that binds to a V 17, preferably a human V017, with a KD of 1 x 107M or less, preferably 1 x 108M or less, more preferably 5 / |09M or less, 1 / 109M or less, 5 / 1010M or less, or | / 1010M or less. The term “KD” refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods in the art in view of the present disclosure. For example, the KD of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as an Octet RED96 system.

[0069] As used herein, an antibody that “specifically binds to DLL3” refers to an antibody that binds to a DLL3, e.g., a human DLL3, with a KD of 1x10‘7M or less, preferably 1x10'8M or less, more preferably 5x l0'9M or less, I xlO'9M or less, 5x l0'10M or less, or I x lO'10M or less.

[0070] The term “KD” refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods in the art in view of the present disclosure. For example, the KD of an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as an Octet RED96 system.

[0071] The smaller the value of the KD of an antibody, the higher affinity that the antibody binds to a target antigen.

[0072] In certain embodiments, the present disclosure relates to abispecific molecule comprising a first binding domain that binds V017 and a second binding domain that binds DLL3. In certain embodiments, the bispecific molecule is a bispecific antibody or antigen-binding fragment thereof. In certain embodiments, the bispecific antibody comprises (a) a first heavy chain (HC1), (b) a second heavy chain (HC2), (c) a first light chain (LC1), and (d) a second light chain (LC2). In17JBI6937065768.12701 / 207W01 certain embodiments, the HC1 can be associated with the LC 1 and the HC2 can be associated with LC2.

[0073] In certain embodiments, the first binding domain that binds V017 comprises a heavy chain and a light chain.

[0074] In certain embodiments, the first binding domain that binds V017 comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 4, a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 5, a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0075] In certain embodiments, the first binding domain that binds V017 comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 3, a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 53, a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 5, a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 6

[0076] In certain embodiments, the first binding domain that binds VP 17 comprises one or more modification to improve antibody developability. In certain embodiments, the first binding domain that binds Vpi7 comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the first binding domain that binds V 17 comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10.18JBI6937065768.12701 / 207W01

[0077] In certain embodiments, the first binding domain that binds V017 comprises one or more modification to improve stability and lower aggregation risks. In certain embodiments, the first binding domain that binds Vpi7 comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the first binding domain that binds Vpi7 comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0078] In certain embodiments, the first binding domain that binds Vpi7 comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the first binding domain that binds VP17 comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0079] In certain embodiments, the first binding domain that binds Vpi7 comprises a singlechain antibody molecule (scFv) comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 19.

[0080] In certain embodiments, the first binding domain that binds Vpi7 comprises a singlechain antibody molecule (scFv) comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 20.

[0081] In certain embodiments, the first binding domain that binds Vpi7 comprises an spFv comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 21.

[0082] In certain embodiments, the first binding domain that binds Vpi7 comprises an spFv comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID19JBI6937065768.12701 / 207W01NO: 13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0083] In certain embodiments, the first binding domain that binds V017 comprises an spFv comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 22.

[0084] In certain embodiments, the first binding domain that binds V017 comprises an spFv comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0085] In certain embodiments, the first binding domain that binds V017 comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the first binding domain that binds Vpi7 comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the first binding domain that binds Vpi7 comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0086] SEQ ID Nos: 1-24 are provided in Table 1 below.Table 120JBI6937065768.12701 / 207W01

[0087] In certain embodiments, the second binding domain that binds DLL3 comprises a heavy chain and a light chain.

[0088] In certain embodiments, the second binding domain that binds DLL3 comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 25, a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 26, a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 27,21JBI6937065768.12701 / 207W01 a light chain complementarity determining region 1 (LCDR1 ) comprising the amino acid sequence set forth in SEQ ID NO: 28, a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 29, a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0089] In certain embodiments, the second binding domain that binds DLL3 comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32.

[0090] In certain embodiments, the second binding domain that binds DLL3 comprises one or more modification to improve stability and lower aggregation risks. In certain embodiments, the second binding domain that binds DLL3 comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34.

[0091] In certain embodiments, the second binding domain that binds DLL3 comprises a singlechain antibody molecule (scFv) comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 35.

[0092] In certain embodiments, the second binding domain that binds DLL3 comprises an spFv comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the spFv comprises the amino acid sequence set forth in SEQ ID NO: 36.

[0093] In certain embodiments, the second binding domain that binds DLL3 comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the second binding domain that binds DLL3 comprises the amino acid sequence set forth in SEQ ID NO: 37.

[0094] In certain embodiments, the second binding domain that binds DLL3 comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the second binding domain that binds DLL3 comprises the amino acid sequence set forth in SEQ ID NO: 38.

[0095] SEQ ID Nos: 25-38 are provided in Table 2 below.22JBI6937065768.12701 / 207W01Table 223JBI6937065768.12701 / 207W01

[0096] In certain embodiments, the bispecific molecule comprises a first binding domain that binds Vpi7 and a second binding domain that binds DLL3, wherein the second binding domain comprises a spFv. In certain embodiments, the first binding domain comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the second binding domain comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 37.

[0097] In certain embodiments, the bispecific molecule comprises a first binding domain that binds Vpi7 and a second binding domain that binds DLL3, wherein the second binding domain comprises a spFv. In certain embodiments, the first binding domain comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the second binding domain comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 38.

[0098] In certain embodiments, the bispecific molecule comprises a first binding domain that binds VP17 and a second binding domain that binds DLL3, wherein the second binding domain comprises a spFv. In certain embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the24JBI6937065768.12701 / 207W01 second binding domain comprises a heavy chain variable region and a light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0099] In certain embodiments, the bispecific molecule comprises a first binding domain that binds Vpi7 and a second binding domain that binds DLL3, wherein the second binding domain comprises a spFv. In certain embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the second binding domain comprises a heavy chain variable region and a light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0100] In certain embodiments, the bispecific molecule comprises a first binding domain that binds Vpi7 and a second binding domain that binds DLL3, wherein the second binding domain comprises a spFv. In certain embodiments, the first binding domain comprises the heavy chain variable region and the light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ25JBI6937065768.12701 / 207W01ID NO: 7 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the second binding domain comprises the heavy chain variable region and the light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34.

[0101] In certain embodiments, the bispecific molecule comprises a first binding domain that binds Vpi7 and a second binding domain that binds DLL3, wherein the second binding domain comprises a spFv. In certain embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises aLCDRl comprising the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the second binding domain comprises an spFv comprising the amino acid sequence set forth in SEQ ID NO: 36.

[0102] In certain embodiments, the bispecific molecule comprises a first binding domain that binds VP17 and a second binding domain that binds DLL3, wherein the second binding domain comprises a spFv. In certain embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the second binding domain comprises an spFv comprising the amino acid sequence set forth in SEQ ID NO: 36.

[0103] In certain embodiments, the bispecific molecule comprises a first binding domain that binds Vpi7 and a second binding domain that binds DLL3, wherein the second binding domain26JBI6937065768.12701 / 207W01 comprises a spFv. In certain embodiments, the first binding domain comprises the heavy chain variable region and the light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the second binding domain comprises an spFv comprising the amino acid sequence set forth in SEQ ID NO: 36.

[0104] In certain embodiments, the bispecific molecule comprises a first binding domain that binds Vpi7 and a second binding domain that binds DLL3, wherein the second binding domain comprises a spFv. In certain embodiments, the first binding domain comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the second binding domain comprises an spFv comprising the amino acid sequence set forth in SEQ ID NO: 36.

[0105] In certain embodiments, the bispecific molecule comprises a Fab binding V017 and a spFv binding DLL3. In certain embodiments, the Fab comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the spFv comprises aHCDRl comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0106] In certain embodiments, the bispecific molecule comprises a Fab binding V017 and a spFv binding DLL3. In certain embodiments, the Fab comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid27JBI6937065768.12701 / 207W01 sequence set forth in SEQ ID NO: 6. In certain embodiments, the spFv comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0107] In certain embodiments, the bispecific molecule comprises a Fab binding Vpi7 and a spFv binding DLL3. In certain embodiments, the Fab comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the spFv comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34.

[0108] In certain embodiments, the bispecific molecule comprises a Fab binding V017 and a spFv binding DLL3. In certain embodiments, the Fab comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the spFv comprises the amino acid sequence set forth in SEQ ID NO: 36.

[0109] In certain embodiments, the bispecific molecule comprises a Fab binding V017 and a spFv binding DLL3. In certain embodiments, the Fab comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the spFv comprises the amino acid sequence set forth in SEQ ID NO: 36.

[0110] In certain embodiments, the bispecific molecule comprises a Fab binding V017 and a spFv binding DLL3. In certain embodiments, the Fab comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, a LCDR2 comprising28JBI6937065768.12701 / 207W01 the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the spFv comprises the amino acid sequence set forth in SEQ ID NO: 36.[0U1] In certain embodiments, the bispecific molecule comprises a Fab binding V017 and a spFv binding DLL3. In certain embodiments, the Fab comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the spFv comprises the amino acid sequence set forth in SEQ ID NO: 36.

[0112] In certain embodiments, the bispecific molecule comprises a Fab binding V017 and a spFv binding DLL3. In certain embodiments, the Fab comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the spFv comprises the amino acid sequence set forth in SEQ ID NO: 37.

[0113] In certain embodiments, the bispecific molecule comprises a first binding domain that binds Vpi7 and a second binding domain that binds DLL3, wherein the second binding domain comprises a spFv. In certain embodiments, the first binding domain comprises the heavy chain variable region and the light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQID NO: 9 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the second binding domain comprises the heavy chain variable region and the light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34.

[0114] In certain embodiments, the bispecific molecule comprises a first binding domain that binds VP17 and a second binding domain that binds DLL3, wherein the second binding domain comprises a spFv. In certain embodiments, the first binding domain comprises the heavy chain variable region and the light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 9 and the light chain variable region comprises the amino acid sequence set forth in SEQ29JBI6937065768.12701 / 207W01ID NO: 10. In certain embodiments, the second binding domain comprises an spFv comprising the amino acid sequence set forth in SEQ ID NO: 36.

[0115] In certain embodiments, the bispecific molecule comprises a Fab binding V017 and a spFv binding DLL3. In certain embodiments, the Fab comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the spFv comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34.

[0116] In certain embodiments, the bispecific molecule comprises a Fab binding V017 and a spFv binding DLL3. In certain embodiments, the Fab comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the spFv comprises the amino acid sequence set forth in SEQ ID NO: 36.

[0117] In certain embodiments, the bispecific molecule comprises a first binding domain that binds VP17 and a second binding domain that binds DLL3, wherein the second binding domain comprises a spFv. In certain embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region in a VH-VL orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the second binding domain comprises a heavy chain variable region and a light chain variable region in a VH-VL orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises30JBI6937065768.12701 / 207W01 a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0118] In certain embodiments, the bispecific molecule comprises a Fab binding V017 and a spFv binding DLL3. In certain embodiments, the Fab comprises a heavy chain variable region and a light chain variable region. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the spFv comprises a heavy chain variable region and a light chain variable region in a VH-VL orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0119] In certain embodiments, the bispecific molecule comprises a first binding domain that binds Vpi7 and a second binding domain that binds DLL3, wherein the second binding domain comprises a spFv. In certain embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region in a VH-VL orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the heavy31JBI6937065768.12701 / 207W01 chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 9 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the second binding domain comprises a heavy chain variable region and a light chain variable region in a VH-VL orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0120] In certain embodiments, the bispecific molecule comprises a Fab binding VP17 and a spFv binding DLL3. In certain embodiments, the Fab comprises a heavy chain variable region and a light chain variable region. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 9 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the spFv comprises a heavy chain variable region and a light chain variable region in a VH-VL orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0121] In certain embodiments, the bispecific molecule comprises a first binding domain that binds VP17 and a second binding domain that binds DLL3, wherein the first binding domain comprises a spFv. In certain embodiments, the first binding domain comprises a heavy chain32JBI6937065768.12701 / 207W01 variable region and a light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 11 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the spFv comprises the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the second binding domain comprises a heavy chain variable region and a light chain variable region. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32.

[0122] In certain embodiments, the bispecific molecule comprises a spFv binding V017 and a Fab binding DLL3. In certain embodiments, the spFv comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the spFv comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the spFv comprises the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the Fab comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a33JBI6937065768.12701 / 207W01HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the Fab comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32.

[0123] In certain embodiments, the bispecific molecule comprises a first binding domain that binds Vpi7 and a second binding domain that binds DLL3, wherein the first binding domain comprises a spFv. In certain embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region in a VH-VL orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the second binding domain comprises a heavy chain variable region and a light chain variable region. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32.

[0124] In certain embodiments, the bispecific molecule comprises a spFv binding V017 and a Fab binding DLL3. In certain embodiments, the spFv comprises a HCDR1 comprising the amino34JBI6937065768.12701 / 207W01 acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the spFv comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the Fab comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the Fab comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32.

[0125] In certain embodiments, the bispecific molecule comprises a first binding domain that binds Vpi7 and a second binding domain that binds DLL3, wherein the first binding domain comprises a spFv. In certain embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the spFv comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the second binding domain comprises a heavy chain variable region and a light chain variable region. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising35JBI6937065768.12701 / 207W01 the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32.

[0126] In certain embodiments, the bispecific molecule comprises a spFv binding V017 and a Fab binding DLL3. In certain embodiments, the spFv comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the spFv comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the spFv comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the Fab comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the Fab comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32.

[0127] In certain embodiments, the bispecific molecule comprises a first binding domain that binds Vpi7 and a second binding domain that binds DLL3, wherein the first binding domain comprises a spFv. In certain embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region in a VH-VL orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth36JBI6937065768.12701 / 207W01 in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the second binding domain comprises a heavy chain variable region and a light chain variable region. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32.

[0128] In certain embodiments, the bispecific molecule comprises a spFv binding V017 and a Fab binding DLL3. In certain embodiments, the spFv comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the spFv comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the Fab comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR337JBI6937065768.12701 / 207W01 comprising the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the Fab comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32.

[0129] In certain embodiments, the bispecific molecule comprises a first binding domain that binds VP17 and a second binding domain that binds DLL3, wherein the first binding domain comprises a scFv. In certain embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the cFv comprises the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the second binding domain comprises a heavy chain variable region and a light chain variable region. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32.

[0130] In certain embodiments, the bispecific molecule comprises a first binding domain that binds VP17 and a second binding domain that binds DLL3, wherein the first binding domain comprises a scFv. In certain embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a38JBI6937065768.12701 / 207W01HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the second binding domain comprises a heavy chain variable region and a light chain variable region. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32.

[0131] In certain embodiments, the bispecific molecule comprises a first binding domain that binds Vpi7 and a second binding domain that binds DLL3. In certain embodiments, the first binding domain comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the first binding domain comprises a heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the first binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the second binding domain comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid39JBI6937065768.12701 / 207W01 sequence set forth in SEQ ID NO: 30. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the first binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0132] In certain embodiments, the bispecific molecule comprises a first binding domain that binds Vpi7 and a second binding domain that binds DLL3. In certain embodiments, the first binding domain comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the first binding domain comprises a heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the first binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the second binding domain comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31 and the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the first binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0133] In certain embodiments, the VP17 is on the surface of a CD8+or CD4+T cell. In certain embodiments, the DLL3 is on the surface of a cancer cell.

[0134] In certain embodiments, the cancer cell is a cell of an adrenal cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational trophoblastic,40JBI6937065768.12701 / 207W01 head and neck cancer, Hodgkin lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, neuroendocrine tumor, nonHodgkin lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma spinal cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer endometrial cancer, vaginal cancer, or vulvar cancer.

[0135] In certain embodiments, the cell of the lung cancer is a cell of small cell lung cancer, small cell carcinoma, combined small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, large-cell undifferentiated carcinoma, pulmonary nodule, metastatic lung cancer, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland-type lung carcinoma, lung carcinoid, mesothelioma, sarcomatoid carcinoma of the lung, or malignant granular cell lung tumor. In certain embodiments, the cell is a cell is small cell lung cancer.

[0136] In certain embodiments, the cancer is an adrenal cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational trophoblastic, head and neck cancer, Hodgkin lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, neuroendocrine tumor, non-Hodgkin lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma spinal cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer endometrial cancer, vaginal cancer, or vulvar cancer.

[0137] In certain embodiments, the lung cancer is a small cell lung cancer, small cell carcinoma, combined small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, large-cell undifferentiated carcinoma, pulmonary nodule, metastatic lung cancer, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland-type lung carcinoma, lung carcinoid, mesothelioma, sarcomatoid carcinoma of the lung, or malignant granular cell lung tumor. In certain embodiments, the lung cancer is a small cell lung cancer.

[0138] In certain embodiments, the bispecific antibodies disclosed herein can take the form of a diabody, a cross-body, or a bispecific antibody obtained via a controlled Fab arm exchange as described herein.

[0139] In certain embodiments, the bispecific antibodies include IgG-like molecules with complementary CH3 domains that promote heterodimerization; recombinant IgG-like dual41JBI6937065768.12701 / 207W01 targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; IgG fusion molecules, wherein full length IgG antibodies are fused to an extra Fab fragment or parts of Fab fragment; Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy-chain constantdomains, Fc-regions or parts thereof; Fab fusion molecules, wherein different Fab-fragments are fused together; ScFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) wherein different single chain Fv molecules or different diabodies or different heavychain antibodies (e.g. domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

[0140] In certain embodiments, IgG-like molecules with complementary CH3 domains molecules include the Triomab / Quadroma (Trion Pharma / Fresenius Biotech), the Knobs-into- Holes (Genentech), CrossMAbs (Roche) and the electrostatically-matched (Amgen), the LUZ-Y (Genentech), the Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), the Biclonic (Merus) and the DuoBody (Genmab A / S).

[0141] In certain embodiments, recombinant IgG-like dual targeting molecules include Dual Targeting (DT)-Ig (GSK / Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX / Pfizer).

[0142] In certain embodiments, IgG fusion molecules include Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific (InnClone / Eli Lilly), Ts2Ab (Medlmmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idee) and TvAb (Roche).

[0143] In certain embodiments, Fc fusion molecules can include ScFv / Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics) and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine— China).

[0144] In certain embodiments, Fab fusion bispecific antibodies include F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech). ScFv-, diabody- based, and domain antibodies, include but are not limited to, Bispecific T Cell Engager (BiTE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT,42JBI6937065768.12701 / 207W01ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain only domain antibodies.

[0145] Full length bispecific antibodies disclosed herein can be generated for example using Fab arm exchange (or half molecule exchange) between two mono specific bivalent antibodies by introducing substitutions at the heavy chain CH3 interface in each half molecule to favor heterodimer formation of two antibody half molecules having distinct binding specificity either in vitro in cell-free environment or using co-expression. The Fab arm exchange reaction is the result of a disulfide-bond isomerization reaction and dissociation-association of CH3 domains. The heavy-chain disulfide bonds in the hinge regions of the parent mono specific antibodies are reduced. The resulting free cysteines of one of the parent monospecific antibodies form an inter heavy-chain disulfide bond with cysteine residues of a second parent mono specific antibody molecule and simultaneously CH3 domains of the parent antibodies release and reform by dissociation-association. The CH3 domains of the Fab arms can be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody having two Fab arms or half molecules, each binding a distinct epitope, i.e., an epitope on Vpi7 and an epitope on a tumor antigen.

[0146] ‘ ‘Homodimerization” as used herein refers to an interaction of two heavy chains having identical CH3 amino acid sequences. “Homodimer” as used herein refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

[0147] ‘ ‘Heterodimerization” as used herein refers to an interaction of two heavy chains having non-identical CH3 amino acid sequences. “Heterodimer” as used herein refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

[0148] The “knob-in-hole” strategy (see, e.g., International Patent Publication No. WO 2006 / 028936) can be used to generate full length bispecific antibodies. Briefly, selected amino acids forming the interface of the CH3 domains in human IgG can be mutated at positions affecting CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into a heavy chain of an antibody specifically binding a first antigen and an amino acid with a large side chain (knob) is introduced into a heavy chain of an antibody specifically binding a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain with a “hole” with the heavy chain with a “knob”. Exemplary CH3 substitution pairs forming a knob and a hole are (expressed43JBI6937065768.12701 / 207W01 as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S andT366W / T366 S_L368 A_Y407 V.

[0149] Other strategies such as promoting heavy chain heterodimerization using electrostatic interactions by substituting positively charged residues at one CH3 surface and negatively charged residues at a second CH3 surface can be used, as described in US Pat. Publ. No. US2010 / 0015133; US Pat. Publ. No. US2009 / 0182127; US Pat. Publ. No. US2010 / 028637; or US Pat. Publ. No. US2011 / 0123532. In other strategies, heterodimerization can be promoted by the following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): L351 Y_F405AY407V / T394W, T366I_K392M_T394W / F405A_Y407V,T366L_K392M_T394W / F405A_Y407V, L351 Y_Y407A / T366A_K409F,L351Y_Y407A / T366V K409F Y407A / T366A_K409F, or T350V_L351Y_F405A Y407V / T350V_T366L_K392L_T394W as described in U.S. Pat. Publ. No. US2012 / 0149876 or U.S. Pat. Publ. No. US2013 / 0195849.

[0150] In addition to methods described above, bispecific antibodies disclosed herein can be generated in vitro in a cell-free environment by introducing asymmetrical mutations in the CH3 regions of two mono specific homodimeric antibodies and forming the bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies in reducing conditions to allow disulfide bond isomerization according to methods described in International Patent Publication No. WO 2011 / 131746. In the methods, the first monospecific bivalent antibody (and the second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promotes heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; thereby generating the bispecific antibody by Fab arm exchange. The incubation conditions can optionally be restored to non-reducing conditions. Exemplary reducing agents that may be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), di thioerythritol (DTE), glutathione, tris (2-carb oxy ethyl) phosphine (TCEP), L-cysteine and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol and tris (2-carboxy ethyl) phosphine. For example, incubation for at least 90 min at44JBI6937065768.12701 / 207W01 a temperature of at least 20° C in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH from 5-8, for example at pH of 7.0 or at pH of 7.4 may be used.

[0151] In certain embodiments, the present disclosure relates to an anti-Vpi7 / anti-DLL3 bispecific antibody that induces antibody-dependent cell-mediated cytotoxicity (ADCC). The anti-Vpi7 / anti-DLL3 bispecific antibody can, for example, induce ADCC in vitro.

[0152] In certain embodiments, immune effector properties of the bispecific antibodies provided herein can be enhanced or silenced through Fc modifications by techniques known to those skilled in the art. For example, Fc effector functions such as Clq binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. can be provided and / or controlled by modifying residues in the Fc responsible for these activities.

[0153] “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a cell-mediated reaction in which non-specific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.

[0154] The ability of antibodies to induce ADCC can be enhanced by engineering their oligosaccharide component. Human IgGl or IgG3 are N-glycosylated at Asn297 with the majority of the glycans in the well-known biantennary GO, G0F, Gl, GIF, G2 or G2F forms. Antibodies produced by non-engineered CHO cells typically have a glycan fucose content of about at least 85%. The removal of the core fucose from the biantennary complex-type oligosaccharides attached to the Fc regions enhances the ADCC of antibodies via improved FcyRIIIa binding without altering antigen-binding or CDC activity. Such Abs can be achieved using different methods reported to lead to the successful expression of relatively high defucosylated antibodies bearing the biantennary complex-type of Fc oligosaccharides such as control of culture osmolality (Konno et al., Cytotechnology 64:249-65, 2012), application of a variant CHO line Lecl3 as the host cell line (Shields et al., J Biol Chem 277:26733-26740, 2002), application of a variant CHO line EB66 as the host cell line (Olivier et al., MAbs; 2(4), 2010; Epub ahead of print; PMID:20562582), application of a rat hybridoma cell line YB2 / 0 as the host cell line (Shinkawa et al., J Biol Chem 278:3466-3473, 2003), introduction of small interfering RNA specifically against the a-1,6- fucosyltrasferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88:901-908, 2004), or coexpression45JBI6937065768.12701 / 207W01 of P-l,4-N-acetylglucosaminyltransferase III and golgi a-mannosidase II or a potent alpha- mannosidase I inhibitor, kifunensine (Ferrara et al., J Biol Chem 281 :5032-5036, 2006, Ferrara et al., Biotechnol Bioeng 93:851-861, 2006; Xhou et al., Biotechnol Bioeng 99:652-65, 2008).

[0155] In certain embodiments, ADCC elicited by the bispecific antibodies provided herein can also be enhanced by certain substitutions in the antibody Fc. Exemplary substitutions include, for example, substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430 (residue numbering according to the EU index) as described in U.S. Pat. No. 6,737,056.

[0156] In certain embodiments, the presently disclosed bispecific antibody is chimeric. In certain embodiments, the presently disclosed bispecific antibody is human. In certain embodiments, the presently disclosed bispecific antibody is humanized.

[0157] Furthermore, in certain non-limiting embodiments, the present disclosure relates to nucleic acids encoding the antibodies and molecules disclosed herein. In certain embodiments, the disclosure relates to isolated nucleic acids encoding the bispecific antibodies or antigenbinding fragments thereof disclosed herein. It will be appreciated by those skilled in the art that the coding sequence of a protein can be changed (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding monoclonal antibodies and / or bispecific antibodies disclosed herein can be altered without changing the amino acid sequences of the proteins.

[0158] In certain embodiments, the nucleic acids encoding the antibodies and molecules disclosed herein comprise one or more nucleotide sequence set forth in SEQ ID Nos: 39-52, which are provided in Table 3 below.Table 346JBI6937065768.12701 / 207W0147JBI6937065768.12701 / 207W0148JBI6937065768.12701 / 207W0149JBI6937065768.12701 / 207W0150JBI6937065768.12701 / 207W0151JBI6937065768.12701 / 207W01

[0159] In certain embodiments, the present disclosure relates to vectors comprising the isolated nucleic acids disclosed herein. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector. In certain embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible or repressible promoter. A number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of an antibody or antigen-binding fragment thereof in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments disclosed herein. Such techniques are well known to those skilled in the art in view of the present disclosure. Exemplary vectors of the present disclosure are depicted in Figures 2A and 2B.52JBI6937065768.12701 / 207W01

[0160] In certain embodiments, the present disclosure relates to host cells comprising the isolated nucleic acids encoding the monoclonal antibodies and / or bispecific antibodies or antigenbinding fragments thereof disclosed herein. Any host cell known to those skilled in the art in view of the present disclosure can be used for recombinant expression of antibodies or antigen-binding fragments thereof disclosed herein. In some embodiments, the host cells are E. coli TGI or BL21 cells (for expression of, e.g., an scFv or Fab antibody), CHO-DG44 or CHO-K1 cells or HEK293 cells (for expression of, e.g., a full-length IgG antibody). According to particular embodiments, the recombinant expression vector is transformed into host cells by conventional methods such as chemical transfection, heat shock, or electroporation, where it is stably integrated into the host cell genome such that the recombinant nucleic acid is effectively expressed.

[0161] In certain embodiments, the present disclosure relates to a method of producing a bispecific antibody disclosed herein. The methods comprise culturing a cell comprising a nucleic acid encoding the bispecific antibody under conditions to produce a bispecific antibody disclosed herein, and recovering the antibody or antigen-binding fragment thereof from the cell or cell culture (e.g., from the supernatant). Expressed antibodies or antigen-binding fragments thereof can be harvested from the cells and purified according to conventional techniques known in the art and as described herein.Pharmaceutical Compositions

[0162] In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising an isolated bispecific antibody provided herein and a pharmaceutically acceptable carrier. Also provided is a method of producing the pharmaceutical composition, comprising combining the antibody with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition. In certain embodiments, provided herein is a pharmaceutical composition comprising a comprising: (a) a first binding domain that binds to V 17, and (b) a second binding domain that binds to DLL3, and a pharmaceutically acceptable carrier. The term “pharmaceutical composition,” as used herein, means a product comprising an antibody provided herein together with a pharmaceutically acceptable carrier. Antibodies provided herein and compositions comprising them are also useful in the manufacture of a medicament for therapeutic applications mentioned herein.

[0163] Also provided herein are methods of producing compositions comprising the bispecific antibodies or antigen-binding fragments disclosed herein, such as buffered compositions or53JBI6937065768.12701 / 207W01 purified compositions and the like. For example, the methods may comprise combining the bispecific antibody with a buffer acceptable that is acceptable for storage and use of the bispecific antibody.

[0164] As used herein, the term “carrier” refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the effectiveness of a composition according to the invention or the biological activity of a composition provided herein. In certain embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in an antibody pharmaceutical composition can be used herein.

[0165] The formulation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, e.g., Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005), and any later editions). Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity regulating agents, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used in formulating the pharmaceutical compositions provided herein.

[0166] In certain embodiments, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e., a formulation comprising water. The liquid formulation can comprise a solution, a suspension, an emulsion, a microemulsion, a gel, and the like. An aqueous formulation typically comprises at least 50% w / w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w / w of water.

[0167] In certain embodiments, the pharmaceutical composition can be formulated as an injectable which can be injected, for example, via an injection device (e.g., a syringe or an infusion pump). The injection can be delivered subcutaneously, intramuscularly, intraperitoneally, intravitreally, or intravenously, for example.

[0168] In certain embodiments, the pharmaceutical composition is a solid formulation, e.g., a freeze-dried or spray-dried composition, which can be used as is, or whereto the physician or the patient adds solvents, and / or diluents prior to use. Solid dosage forms can include tablets, such as compressed tablets, and / or coated tablets, and capsules (e.g., hard or soft gelatin capsules). The54JBI6937065768.12701 / 207W01 pharmaceutical composition can also be in the form of sachets, dragees, powders, granules, lozenges, or powders for reconstitution, for example.

[0169] The dosage forms can be immediate release, in which case they can comprise a water- soluble or dispersible carrier, or they can be delayed release, sustained release, or modified release, in which case they can comprise water insoluble polymers that regulate the rate of dissolution of the dosage form in the gastrointestinal tract or under the skin.

[0170] In certain embodiments, the pharmaceutical composition can be delivered intranasally, intrabuccally, or sublingually.

[0171] The pH in an aqueous formulation can be between pH 3 and pH 10. In one embodiment provided herein, the pH of the formulation is from about 7.0 to about 9.5. In another embodiment provided herein, the pH of the formulation is from about 3.0 to about 7.0.

[0172] In certain embodiments, the pharmaceutical composition comprises a buffer. Nonlimiting examples of buffers include arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tricine, and tris(hydroxymethyl)-aminomethane, and mixtures thereof. The buffer can be present individually or in the aggregate, in a concentration from about 0.01 mg / ml to about 50 mg / ml, for example from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific buffers constitute alternative embodiments provided herein.

[0173] In certain embodiments, the pharmaceutical composition comprises a preservative. Nonlimiting examples of preservatives include: benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4- hydroxybenzoate, chlorobutanol, chlorocresol, chlorohexidine, chlorphenesin, o- cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidurea, methyl 4- hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4- hydroxybenzoate, sodium dehydroacetate, thiomerosal, and mixtures thereof. The preservative can be present individually or in the aggregate, in a concentration from about 0.01 mg / ml to about 50 mg / ml, for example from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific preservatives constitute alternative embodiments provided herein.

[0174] In certain embodiments, the pharmaceutical composition comprises an isotonic agent. Non-limiting examples of isotonic agents include a salt (such as sodium chloride), an amino acid (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine),55JBI6937065768.12701 / 207W01 an alditol (such as glycerol, 1,2- propanediol propyleneglycol), 1,3 -propanediol, and 1 ,3- butanediol), polyethyleneglycol (e.g., PEG400), and mixtures thereof. Another example of an isotonic agent includes a sugar. Non-limiting examples of sugars can include mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta- HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethyl-cellulose. Another example of an isotonic agent is a sugar alcohol, wherein the term “sugar alcohol” is defined as a C(4-8) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. The isotonic agent can be present individually or in the aggregate, in a concentration from about 0.01 mg / ml to about 50 mg / ml, for example from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific isotonic agents constitute alternative provided herein.

[0175] In certain embodiments, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, salts of ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. The chelating agent can be present individually or in the aggregate, in a concentration from about 0.01 mg / ml to about 50 mg / ml, for example from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific chelating agents constitute alternative embodiments of the invention.

[0176] In certain embodiments, the pharmaceutical composition comprises a stabilizer. Nonlimiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and / or one or more protease inhibitors.

[0177] In certain embodiments, the pharmaceutical composition comprises a stabilizer, wherein said stabilizer is carboxy - / hydroxycellulose and derivates thereof (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, 2- methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinyl pyrrolidone, salts (such as sodium chloride), sulphur- containing substances such as monothioglycerol), or thioglycolic acid. The stabilizer can be present individually or in the aggregate, in a concentration from about 0.01 mg / ml to about 50 mg / ml, for example from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific stabilizers constitute alternative embodiments provided herein.56JBI6937065768.12701 / 207W01

[0178] In certain embodiments, the pharmaceutical composition comprises one or more surfactants, preferably a surfactant, at least one surfactant, or two different surfactants. The term “surfactant” refers to any molecules or ions that are comprised of a water-soluble (hydrophilic) part, and a fat-soluble (lipophilic) part. The surfactant can, for example, be selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitterionic surfactants. The surfactant can be present individually or in the aggregate, in a concentration from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific surfactants constitute alternative embodiments provided herein.

[0179] In certain embodiments, the pharmaceutical composition comprises one or more protease inhibitors, such as, e.g., EDTA, and / or benzamidine hydrochloric acid (HC1). The protease inhibitor can be present individually or in the aggregate, in a concentration from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific protease inhibitors constitute alternative embodiments provided herein.

[0180] In certain embodiments, the present disclosure also relates to a method of producing a pharmaceutical composition comprising a bispecific antibody disclosed herein, comprising combining a bispecific antibody with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.Methods of use

[0181] The present disclosure provides a method of directing a T cell expressing V017 to a target cell expressing DLL3. In certain embodiments, the method comprises contacting the T cell with a bispecific antibody provided herein. In certain embodiments, the contacting directs the T cell to the target cell.

[0182] Also provided is a method of targeting DLL3 expressed on the surface of a target cell, the method comprising exposing the target cell to the bispecific molecules disclosed herein or a pharmaceutical composition thereof.

[0183] The functional activity of bispecific antibodies and antigen-binding fragments thereof disclosed herein can be characterized by methods known in the art and as described herein. Methods for characterizing antibodies and antigen-binding fragments thereof that bind Vpi7 and DLL3 include, but are not limited to, affinity and specificity assays including Biacore, ELISA, and OctetRed analysis; binding assays to detect the binding of antibodies to target cells by FACS; binding assays to detect the binding of antibodies to Vpi7 on T cells.57JBI6937065768.12701 / 207W01

[0184] Also provided is a method of directing Vpi7-expressing T cells to a cell expressing DLL3. The methods can comprise contacting the Vpi7-expressing T cell with a bispecific antibody disclosed herein,

[0185] Also provided is a method for inhibiting growth or proliferation of target cells expressing DLL3. The methods can comprise contacting the Vpi7-expressing T cells with the presently disclosed bispecific antibody or antigen binding fragment thereof, wherein contacting the target cells expressing DLL3 with the presently disclosed bispecific antibody or composition thereof inhibits the growth or proliferation of the target cells. Also provided is a method of inhibiting growth or proliferation of target cells expressing a second target antigen on the cell surface, the method comprising contacting the target cells with a bispecific antibody provided herein, wherein contacting the target cells with the pharmaceutical composition inhibits growth or proliferation of the target cells.

[0186] Moreover, provided herein is a method for eliminating target cells expressing DLL3 in a subject, comprising administering an effective amount of a bispecific antibody, as provided herein, to the subject. In certain non-limiting embodiments, the present disclosure relates to a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a bispecific antibody or antigen binding fragment thereof that specifically binds Vpi7 and DLL3 presented on the surface of a target cell, or a pharmaceutical composition disclosed herein. In certain embodiments, provided herein is a method for eliminating target cells expressing DLL3 or treating a disease caused all or in part by target cells expressing DLL3 in a subject, comprising administering an effective amount of a bispecific antibody provided herein to the subject. In certain embodiments, the subject is a subject in need thereof. In certain embodiments, the subject is a human.

[0187] Also provided are methods of directing a Vpi7-expressing CD8+ or CD4+ T cell to a cancer cell expressing DLL3. The methods comprise contacting a Vpi7-expressing CD8+ or CD4+ T cell with an anti-Vpi7 / anti-DLL3 bispecific antibodies or composition thereof disclosed herein. Contacting the Vpi7-expressing CD8+ or CD4+ T cell with the anti-Vpi7 / anti-DLL3 bi specific antibodies or compositions thereof can direct the Vpi7-expressing CD8+ or CD4+ T cell to a cancer cell expressing DLL3. Also provided are methods for inhibiting growth or proliferation of cancer cells. The methods comprise contacting the cancer cells with the bispecific antibodies disclosed herein. Contacting the cancer cells with the described antibodies can, for58JBI6937065768.12701 / 207W01 example, inhibit the growth or proliferation of the cancer cells, or promote T cell mediated killing of the cancer cells.

[0188] In certain embodiments, the cancer cell is a cell of an adrenal cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational trophoblastic, head and neck cancer, Hodgkin lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, neuroendocrine tumor, nonHodgkin lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma spinal cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer endometrial cancer, vaginal cancer, or vulvar cancer. In certain embodiments, the adrenal cancer is an adrenocortical carcinoma (ACC), adrenal cortex cancer, pheochromocytoma, or neuroblastoma. In certain embodiments, the anal cancer is a squamous cell carcinoma, cloacogenic carcinoma, adenocarcinoma, basal cell carcinoma, or melanoma. In certain embodiments, the appendix cancer is a neuroendocrine tumor (NET), mucinous adenocarcinoma, goblet cell carcinoid, intestinal-type adenocarcinoma, or signet-ring cell adenocarcinoma. In certain embodiments, the bile duct cancer is an extrahepatic bile duct cancer, adenocarcinomas, hilar bile duct cancer, perihilar bile duct cancer, distal bile duct cancer, or intrahepatic bile duct cancer. In some embodiments, the bladder cancer is transitional cell carcinoma (TCC), papillary carcinoma, flat carcinoma, squamous cell carcinoma, adenocarcinoma, small-cell carcinoma, or sarcoma. In certain embodiments, the bone cancer is a primary bone cancer, sarcoma, osteosarcoma, chondrosarcoma, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, or metastatic bone cancer. In certain embodiments, the brain cancer is an astrocytoma, brain stem glioma, glioblastoma, meningioma, ependymoma, oligodendroglioma, mixed glioma, pituitary carcinoma, pituitary adenoma, craniopharyngioma, germ cell tumor, pineal region tumor, medulloblastoma, or primary CNS lymphoma. In certain embodiments, the breast cancer is a breast adenocarcinoma, invasive breast cancer, noninvasive breast cancer, breast sarcoma, metaplastic carcinoma, adenocystic carcinoma, phyllodes tumor, angiosarcoma, HER2- positive breast cancer, triple-negative breast cancer, or inflammatory breast cancer. In certain embodiments, the cervical cancer is a squamous cell carcinoma, or adenocarcinoma. In some embodiments, the colorectal cancer is a colorectal adenocarcinoma, primary colorectal lymphoma, gastrointestinal stromal tumor, leiomyosarcoma, carcinoid tumor, mucinous adenocarcinoma,59JBI6937065768.12701 / 207W01 signet ring cell adenocarcinoma, gastrointestinal carcinoid tumor, or melanoma. In certain embodiments, the esophageal cancer is an adenocarcinoma or squamous cell carcinoma. In certain embodiments, the gall bladder cancer is an adenocarcinoma, papillary adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma, or sarcoma. In certain embodiments, the gestational trophoblastic disease (GTD) is a hydatidiform mole, gestational trophoblastic neoplasia (GTN), choriocarcinoma, placental-site trophoblastic tumor (PSTT), or epithelioid trophoblastic tumor (ETT). In certain embodiments, the head and neck cancer is a laryngeal cancer, nasopharyngeal cancer, hypopharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, salivary gland cancer, oral cancer, oropharyngeal cancer, or tonsil cancer. In certain embodiments, the Hodgkin lymphoma is a classical Hodgkin lymphoma, nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte-depleted, or nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). In certain embodiments, the intestinal cancer is a small intestine cancer, small bowel cancer, adenocarcinoma, sarcoma, gastrointestinal stromal tumors, carcinoid tumors, or lymphoma. In certain embodiments, the kidney cancer is a renal cell carcinoma (RCC), clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, unclassified RCC, transitional cell carcinoma, urothelial cancer, renal pelvis carcinoma, or renal sarcoma. In certain embodiments, the leukemia is an acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), or a myelodysplastic syndrome (MDS). In certain embodiments, the liver cancer is a hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, or liver metastasis. In certain embodiments, the lung cancer is a small cell lung cancer, small cell carcinoma, combined small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, large-cell undifferentiated carcinoma, pulmonary nodule, metastatic lung cancer, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland-type lung carcinoma, lung carcinoid, mesothelioma, sarcomatoid carcinoma of the lung, or malignant granular cell lung tumor. In certain embodiments, the melanoma is a superficial spreading melanoma, nodular melanoma, acral-lentiginous melanoma, lentigo maligna melanoma, amelanotic melanoma, desmoplastic melanoma, ocular melanoma, or metastatic melanoma. In certain embodiments, the mesothelioma is a pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or testicular mesothelioma. In certain embodiments, the multiple myeloma is an active myeloma or smoldering myeloma. In certain embodiments, the60JBI6937065768.12701 / 207W01 neuroendocrine tumor, is a gastrointestinal neuroendocrine tumor, pancreatic neuroendocrine tumor, or lung neuroendocrine tumor. In certain embodiments, the non-Hodgkin’s lymphoma is an anaplastic large-cell lymphoma, lymphoblastic lymphoma, peripheral T cell lymphoma, follicular lymphoma, cutaneous T cell lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, MALT lymphoma, small-cell lymphocytic lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), precursor T- lymphoblastic leukemia / lymphoma, acute lymphocytic leukemia (ALL), adult T cell lymphoma / leukemia (ATLL), hairy cell leukemia, B-cell lymphomas, diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, primary central nervous system (CNS) lymphoma, mantle cell lymphoma (MCL), marginal zone lymphomas, mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, lymphoplasmacytic lymphoma, B-cell non-Hodgkin lymphoma, T cell non-Hodgkin lymphoma, natural killer cell lymphoma, cutaneous T cell lymphoma, Alibert-Bazin syndrome, Sezary syndrome, primary cutaneous anaplastic large-cell lymphoma, peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma (AITL), anaplastic large-cell lymphoma (ALCL), systemic ALCL, enteropathy-type T cell lymphoma (EATL), or hepatosplenic gamma / delta T cell lymphoma. In certain embodiments, the oral cancer is a squamous cell carcinoma, verrucous carcinoma, minor salivary gland carcinomas, lymphoma, benign oral cavity tumor, eosinophilic granuloma, fibroma, granular cell tumor, karatoacanthoma, leiomyoma, osteochondroma, lipoma, schwannoma, neurofibroma, papilloma, condyloma acuminatum, verruciform xanthoma, pyogenic granuloma, rhabdomyoma, odontogenic tumors, leukoplakia, erythroplakia, squamous cell lip cancer, basal cell lip cancer, mouth cancer, gum cancer, or tongue cancer. In certain embodiments, the ovarian cancer is a ovarian epithelial cancer, mucinous epithelial ovarian cancer, endometrioid epithelial ovarian cancer, clear cell epithelial ovarian cancer, undifferentiated epithelial ovarian cancer, ovarian low malignant potential tumors, primary peritoneal carcinoma, fallopian tube cancer, germ cell tumors, teratoma, dysgerminoma ovarian germ cell cancer, endodermal sinus tumor, sex cord-stromal tumors, sex cord-gonadal stromal tumor, ovarian stromal tumor, granulosa cell tumor, granulosa-theca tumor, Sertoli- Leydig tumor, ovarian sarcoma, ovarian carcinosarcoma, ovarian adenosarcoma, ovarian leiomyosarcoma, ovarian fibrosarcoma, Krukenberg tumor, or ovarian cyst. In certain embodiments, the pancreatic cancer is a pancreatic exocrine gland cancer, pancreatic endocrine61JBI6937065768.12701 / 207W01 gland cancer, or pancreatic adenocarcinoma, islet cell tumor, or neuroendocrine tumor. In some embodiments, the prostate cancer is a prostate adenocarcinoma, prostate sarcoma, transitional cell carcinoma, small cell carcinoma, or neuroendocrine tumor. In certain embodiments, the sinus cancer is a squamous cell carcinoma, mucosa cell carcinoma, adenoid cystic cell carcinoma, acinic cell carcinoma, sinonasal undifferentiated carcinoma, nasal cavity cancer, paranasal sinus cancer, maxillary sinus cancer, ethmoid sinus cancer, or nasopharynx cancer. In certain embodiments, the skin cancer is a basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, Kaposi sarcoma (KS), actinic keratosis, skin lymphoma, or keratoacanthoma. In certain embodiments, the soft tissue cancer is an angiosarcoma, dermatofibrosarcoma, epithelioid sarcoma, Ewing’s sarcoma, fibrosarcoma, gastrointestinal stromal tumors (GISTs), Kaposi sarcoma, leiomyosarcoma, liposarcoma, dedifferentiated liposarcoma (DL), myxoid / round cell liposarcoma (MRCL), well-differentiated liposarcoma (WDL), malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma (RMS), or synovial sarcoma. In certain embodiments, the spinal cancer is a spinal metastatic tumor. In some embodiments, the stomach cancer is a stomach adenocarcinoma, stomach lymphoma, gastrointestinal stromal tumors, carcinoid tumor, gastric carcinoid tumors, Type I ECL-cell carcinoid, Type II ECL-cell carcinoid, or Type III ECL-cell carcinoid. In certain embodiments, the testicular cancer is a seminoma, non-seminoma, embryonal carcinoma, yolk sac carcinoma, choriocarcinoma, teratoma, gonadal stromal tumor, leydig cell tumor, or sertoli cell tumor. In certain embodiments, the throat cancer is a squamous cell carcinoma, adenocarcinoma, sarcoma, laryngeal cancer, pharyngeal cancer, nasopharynx cancer, oropharynx cancer, hypopharynx cancer, laryngeal cancer, laryngeal squamous cell carcinoma, laryngeal adenocarcinoma, lymphoepithelioma, spindle cell carcinoma, verrucous cancer, undifferentiated carcinoma, or lymph node cancer. In certain embodiments, the thyroid cancer is a papillary carcinoma, follicular carcinoma, Hurthle cell carcinoma, medullary thyroid carcinoma, or anaplastic carcinoma. In certain embodiments, the uterine cancer is an endometrial cancer, endometrial adenocarcinoma, endometroid carcinoma, serous adenocarcinoma, adenosquamous carcinoma, uterine carcinosarcoma, uterine sarcoma, uterine leiomyosarcoma, endometrial stromal sarcoma, or undifferentiated sarcoma. In certain embodiments, the vaginal cancer is a squamous cell carcinoma, adenocarcinoma, melanoma, or sarcoma. In certain embodiments, the vulvar cancer is a squamous cell carcinoma or adenocarcinoma.62JBI6937065768.12701 / 207W01

[0189] In certain embodiments, the described anti-Vpi7 / anti-DLL3 bispecific antibody can be provided in a buffered composition for storage or use. Suitable buffers for the storage of the described anti-Vpi7 / anti-DLL3 bispecific antibody would serve to maintain the stability of the antibody or antibody fragment by minimizing deterioration while stored, not promoting aggregation of the antibody or antibody fragment, or minimizing adhesion to the storage vessel.

[0190] As used herein, the term “effective amount” refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject.

[0191] In certain embodiments, an effective amount refers to the amount of therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition to be treated or a symptom associated therewith; (ii) reduce the duration of the disease, disorder or condition to be treated, or a symptom associated therewith; (iii) prevent the progression of the disease, disorder or condition to be treated, or a symptom associated therewith; (iv) cause regression of the disease, disorder or condition to be treated, or a symptom associated therewith; (v) prevent the development or onset of the disease, disorder or condition to be treated, or a symptom associated therewith; (vi) prevent the recurrence of the disease, disorder or condition to be treated, or a symptom associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (viii) reduce hospitalization length of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (ix) increase the survival of a subject with the disease, disorder or condition to be treated, or a symptom associated therewith; (xi) inhibit or reduce the disease, disorder or condition to be treated, or a symptom associated therewith in a subject; and / or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0192] The effective amount or dosage can vary according to various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, e.g., age, body weight, health), whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.

[0193] In certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described63JBI6937065768.12701 / 207W01 herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.

[0194] As used herein, the terms “treat,” “treating,” and “treatment” are all intended to refer to an amelioration or reversal of at least one measurable physical parameter related to a cancer, which is not necessarily discernible in the subject, but can be discernible in the subject. The terms “treat,” “treating,” and “treatment,” can also refer to causing regression, preventing the progression, or at least slowing down the progression of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder, or condition, such as a tumor or more preferably a cancer. In a particular embodiment, “treat,” “treating,” and “treatment” refer to prevention of the recurrence of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an increase in the survival of a subject having the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to elimination of the disease, disorder, or condition in the subject.

[0195] In certain embodiments, the presently disclosed antibodies or compositions thereof can be used in combination with a supplemental therapy.

[0196] As used herein, the term “in combination,” in the context of the administration of two or more therapies to a subject, refers to the use of more than one therapy. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. For example, a first therapy (e.g., a composition described herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject.Non-limiting Exemplary Embodiments

[0197] In certain non-limiting embodiments, the present disclosure is directed to antibodies or antigen-binding fragments thereof binding DLL3. In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises a spFv. In certain embodiments, the anti-64JBI6937065768.12701 / 207W01DLL3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0198] In certain non-limiting embodiments, the present disclosure is directed to antibodies or antigen-binding fragments thereof binding DLL3. In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises a spFv. In certain embodiments, the anti- DLL3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region in a VH-VL orientation. In certain embodiments, the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, and the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0199] In certain non-limiting embodiments, the present disclosure is directed to antibodies or antigen-binding fragments thereof binding DLL3. In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises a spFv. In certain embodiments, the anti- DLL3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 33; and the light chain variable region comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 34.

[0200] In certain non-limiting embodiments, the present disclosure is directed to antibodies or antigen-binding fragments thereof binding DLL3. In certain embodiments, the anti-DLL365JBI6937065768.12701 / 207W01 antibody or antigen-binding fragment thereof comprises a spFv. In certain embodiments, the anti- DLL3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region in a VH-VL orientation. In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 33; and the light chain variable region comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 34.

[0201] In certain non-limiting embodiments, the present disclosure is directed to antibodies or antigen-binding fragments thereof binding DLL3. In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises a spFv. In certain embodiments, the anti- DLL3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region in a VL-VH orientation. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 33; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 34.

[0202] In certain non-limiting embodiments, the present disclosure is directed to antibodies or antigen-binding fragments thereof binding DLL3. In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises a spFv. In certain embodiments, the anti- DLL3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region in a VH-VL orientation. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 33; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 34.

[0203] In certain non-limiting embodiments, the present disclosure is directed to antibodies or antigen-binding fragments thereof binding DLL3. In certain embodiments, the anti-DLL3 antibody or antigen-binding fragment thereof comprises a spFv. In certain embodiments, the spFv comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 37.

[0204] In certain non-limiting embodiments, the present disclosure is directed to antibodies or antigen-binding fragments thereof binding DLL3. In certain embodiments, the anti-DLL366JBI6937065768.12701 / 207W01 antibody or antigen-binding fragment thereof comprises a spFv. In certain embodiments, the spFv comprises the amino acid sequence set forth in SEQ ID NO: 37.

[0205] In certain non-limiting embodiments, the present disclosure is directed to bispecific antibodies binding Vpi7 and DLL3. In certain embodiments, the bispecific antibody comprises a Fab binding Vpi7 and an spFv binding DLL3. In certain embodiments, the Fab comprises a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the spFv comprises a heavy chain variable region comprising an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 33; and a light chain variable region comprising an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 34.

[0206] In certain non-limiting embodiments, the present disclosure is directed to bispecific antibodies binding Vpi7 and DLL3. In certain embodiments, the bispecific antibody comprises a Fab binding Vpi7 and an spFv binding DLL3. In certain embodiments, the Fab comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the spFv comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34.

[0207] In certain non-limiting embodiments, the present disclosure is directed to bispecific antibodies binding Vpi7 and DLL3. In certain embodiments, the bispecific antibody comprises a first heavy chain (HC1), a first light chain (LC1), and a second heavy chain (HC2), wherein HC1 is associated with LC1 to form a binding site for Vpi7 and HC2 comprises an spFv binding DLL3. In certain embodiments, the HC1 comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the LC1 comprises67JBI6937065768.12701 / 207W01 an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the HC2 comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 37.

[0208] In certain non-limiting embodiments, the present disclosure is directed to bispecific antibodies binding Vpi7 and DLL3. In certain embodiments, the bispecific antibody comprises a first heavy chain (HC1), a first light chain (LC1), and a second heavy chain (HC2), wherein HC1 is associated with LC1 to form a binding site for Vpl7 and HC2 comprises an spFv binding DLL3. In certain embodiments, the HC1 comprises the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the LC 1 comprises the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the HC2 comprises the amino acid sequence set forth in SEQ ID NO: 37.EXAMPLES

[0209] The following examples are based on the premise that T cells demonstrate potent antitumor functions. These cells express TCR-haplotype-Vpi7 and majority of these cells exhibit efficient cytotoxicity of tumor target cells. This ability is then harnessed using bispecific antibodies constructed such that one arm binds to the Vpi7 structure and the other arm binds to an antigen expressed by the cancer cells. Thus, the bispecific antibody bridges the effector and target cells together resulting in cancer cell killing.Example 1: Molecule Design, Sequence, and Structure o f the Antibodies Disclosed Herein

[0210] Summary. The disclosed bispecific antibodies were specifically developed for the treatment of advanced tumors where DLL3 is known to be highly expressed on the cell surface. Specifically, they are immunoglobulin (Ig) G1 bispecific antibodies that simultaneously binds to the variable domain of the beta subunit of the T cell receptor, Vpi7 (contributed by gene segment, TRBV19; Uniprot ID: A0A075B6N1) on T lymphocytes (T cells) and to delta-like protein 3, DLL3 (Uniprot ID: Q9NYJ7) on tumor cells.

[0211] To mitigate any potential interactions with Fey receptors (FcyRs) and complement protein Clq, the bispecific antibodies disclosed herein contain mutations of L234A, L235A, and D265S (EU numbering scheme) (Edelman et al. 1968; Kabat 1991; Wilkinson et al. 2021) in the constant region (Fc) (see Table 5). These residues play a critical role in mediating interactions68JBI6937065768.12701 / 207W01 with FcyRs on immune cells and Clq in the complement pathway. Thus, these mutations were strategically designed to reduce the affinity for Fey receptors and complement protein Clq (Wilkinson et al. 2021) of the bispecific antibodies disclosed herein. These mutations were also designed and engineered to serve specific and complementary purposes to enhance functionality of the antibodies disclosed herein. These modifications resulted in a significant reduction in Fc- mediated effector functions, minimized immune activation through antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), and resulted in enhanced therapeutic precision of the bispecific antibodies disclosed herein. Moreover, these modifications proved through experimentation to prevent non-specific immune cell engagement, which could otherwise compromise safety and therapeutic specificity. Furthermore, these mutations were designed to ensure that the primary mode of action of the antibodies disclosed herein was the redirection of Vpi7+ T cells to DLL3 -expressing tumor cells without being confounded by unintended Fc-mediated interactions.

[0212] As described below, the bispecific antibodies disclosed herein were optimized for heterodimerization and employ the “knobs-into-holes” engineering strategy to ensure selective pairing of heavy chains, critical for bispecific functionality.

[0213] As described below, the bispecific antibodies disclosed herein comprise an anti-Vpi7 Fab region on HC1 and an anti-DLL3 single-chain variable fragment (scFv) with a “stapled” linker on HC2 (referred as spFc). In addition to the "hole" mutations in HC1, mutation of H435R, Y436F were added to disrupt protein A binding (Zwolak et al. 2017).

[0214] As described below, HC2 of the bispecific antibodies disclosed herein includes an anti- DLL3 single-chain fragment variable in “VL-VH” orientation, featuring a “stapled” linker (spFv). The spFv consists of a linker, SGGSGGCPPCGGSGG, having a central “C1PPC2” motif wherein Cl forms a disulfide bond with (in the case of the spFv in the “light-heavy” orientation) an engineered cysteine at position 43 (Chothia numbering scheme) (Chothia and Lesk 1987) in the variable light domain and C2 forms a disulfide bond with an engineered cysteine at position 100 in the variable heavy domain (Boucher et al. 2023). This provides additional structural reinforcement to the scFv, minimizing flexibility and preventing misfolding or aggregation.

[0215] Detailed information on all mutations in Fc, using EU and sequence-based numbering, is provided in Table 5.69JBI6937065768.12701 / 207W01

[0216] In summary, the structural and functional designs disclosed herein were engineered into the bispecific antibodies disclosed herein to allow for precise targeting of DLL3 -expressing tumor cells through the engagement of V 17+ T cells.

[0217] Results. The presently disclosed bispecific antibodies simultaneously binds to the variable domain of the beta subunit of the T cell receptor, TRBV19 or V017 (Uniprot ID: A0A075B6N1) on T lymphocytes (T cells) and to delta-like protein 3, DLL3 (Uniprot ID: Q9NYJ7) on tumor cells. The bispecific antibodies disclosed herein include mutations of L234A, L235A, and D265S (“AAS”, EU numbering (7, 2)) in the constant region (Fc) to abolish interaction with Fc receptors, and heterodimerization is enhanced using the knobs-into-holes mutations (Ridgway et al., Protein Eng 9, 617-621 (1996); Atwell et al., J Mol Biol 2 Q, 26-35 (1997)). The bispecific antibodies disclosed herein included an anti-TRBV19 Fab region on heavy chain (HC) 1. HC1 comprises the “hole” mutations: T366S, L368A, Y407V, and additionally mutation of H435R, Y436F to disrupt protein A binding. HC2 features an anti-DLL3 single-chain fragment variable featuring a “stapled” linker (spFv). The spFv comprises a linker having a central “C1PPC2” motif wherein Ci forms a disulfide bond with (in the case of the spFv in the “lightheavy” orientation) an engineered cysteine at position 43 (Chothia numbering) in the variable light domain and C2 forms a disulfide bond with an engineered cysteine at position 100 in the variable heavy domain. Heavy chain 2 comprises the “knob” mutation: T366W.

[0218] The bispecific antibody disclosed by sequences in Table 4 was designated as “bispecific J8” and was designed to target DLL3 for T cell redirection targeting T cells expressing the TRBV19 T cell receptor. The bispecific J8 was developed for the treatment of DLL3 expressing tumors, including but not limited to where DLL3 is expressed on the cell surface or is accessible from the cell surface.

[0219] The bispecific J8 was generated by co-expression of the anti-TRBV19 hole heavy chain 1, its cognate light chain (LC), and the anti-DLL3 knob heavy chain 2. The anti-TRBV19 variable region was de novo sequenced by mass spectrometry. The de novo-sequenced mouse anti- TRBV19 v-region was humanized using the procedure described previously (Fransson et al., JMol Z> / o / 398, 214-231 (2010)). Briefly, mouse germlines were identified as IGHV3-6*01 and lGKVl- 110*01. The closest human germlines were, for VL: IGKV1-39*O1 and IGKV2-30*01 and for VH: IGHV3-23*01 and IGHV4-61*01. For the heavy chain variable domain, IGHV4-61 *01 was preferred due to its lack of low risk deamidation motifs in framework region 3. For the light chain70JBI6937065768.12701 / 207W01 variable domain, IGKV1 -39*01 was selected. The humanized variable region was further modified by mutation of N33S in the light chain CDR1 variable domain to remove a deamidation risk. The final resulting v-region was included in the bispecific J8.

[0220] The parent anti-DLL3 variable region was discovered by immunizing transgenic mice with recombinant human DLL3 protein. The parent anti-DLL3 variable region contained sequence liabilities. Specifically, in the variable heavy domain contained a germline mutation at N27, which is normally tyrosine in the IGHV1 -46*03 germline. However, since this residue was near the CDR, it was mutated to an amino acid with a similar sidechain, glutamine (N27Q). Additionally, Metl05 in the joining region was mutated to Thr (M105T). The light chain v-region was mutated back to Gly (A99G). Together, mutation of N27Q and M105T in the variable heavy domain and A99G in the variable light domain gave rise to the final variable region featured in the bispecific J8. This modified variable region was formatted as a stapled single-chain fragment variable (spFv) in the final bispecific J8.

[0221] The amino acid sequences for the bispecific J8 heavy chains and light chain were confirmed by peptide mapping and mass spectrometry. Specifically, the Heavy Chain 1 of the bispecific J8 is set forth in SEQ ID NO: 23, the Light Chain 1 of the bispecific J8 is set forth in SEQ ID NO: 24 and the Heavy Chain 2 of the bispecific J8 is set forth in SEQ ID NO: 37.Table 471JBI6937065768.12701 / 207W01

[0222] The complementarity-determining regions (CDRs), defined according to ABM numbering, in each chain are bolded and underlined. The Glu residue at position 1 of the heavy chain 1, Asp residue at position 1 of the light chain 1, and Asp residue at position 1 of the heavy chain 2 constitute the N-termini of the mature chain. Both heavy chains of the bispecific J8 IgGl- AAS have the following point mutations: L234A, L235A, and D265S. Heavy chain 1 features the “hole” mutations: T366S, L368A, Y407V and the RF mutations: H435R, Y436F, while heavy chain 2 features the “knob” mutation: T366W. The knobs-into-holes mutations promote heterodimerization of the Fc (Von Kreudenstein et al., MAbs 5, 646-654 (2013)). The “RF” mutations disrupt binding to protein A. The mutations for silencing (AAS), the knobs-into-holes mutations, RF mutations, and the engineered cysteines are underlined in the sequences above. Additional details on the constant region mutations of the bispecific J8 can be found in Table 5 below.Table 5

[0223] The bispecific antibody sequences were further analyzed for potential immunogenicity using the T-regulatory (Treg) adjusted scores from the EpiVax Epimatrix in silico immunogenicity prediction program (Table 6) (De Groot et al., Clinical immunology 131, 189-201 (2009)). EpiVax program computationally calculated the binding potential to the most common HLA molecules within each of the “supertypes”. The report provided results that were representative of > 90 % of human populations worldwide without the necessity of testing each haplotype individually. Epivax score was calculated by aggregating the EpiMatrix scores of all predicted T-cell epitopes72JBI6937065768.12701 / 207W01 contained within a given protein sequence and adjusting for expected T-cell epitope content and protein length. Although based on extensive in silica analysis, the EpiVax Epimatrix score has limited experimental ability to predict immunogenicity in patients.Table 6

[0224] Overall, the anti-DLL3 v-region had an acceptable EpiVax score for its VL and for its VH, while the anti-TRBV19 arm had an ideal Epivax score.

[0225] Anti-DLL3 / anti-Vpi7 antibodies with the same binding specificities as antibody J8 have been shown to mediate cytotoxicity against DLL3 target cell lines in PBMCs and to proliferate V[317+ T cells in whole PBMC cytotoxicity assays. WO202130657 (Example 5.4, incorporated by reference herein).JBI6937065768.12701 / 207W01Example 2: PK profile ofJ8 in NSG mice

[0226] A study was designed to evaluate the pharmacokinetics of J8 at 3mg / kg and 15mg / kg following a single dose i.v administration in NOD-SCID Gamma (NSG) mice bearing subcutaneous SHP-77 xenografts.

[0227] Experimental procedures.

[0228] SHP-77 cell stocks were thawed in water bath at 37°C, and immediately suspended in warm complete growth media (RPMI1640 with 10% FBS and 1% Anti-anti). The SHP-77 cell suspension was then centrifuged at 800rpm for 5minutes and the resulting cell pellet was then resuspended in complete culture medium. The cells were expanded, counted, and were then seeded in new flasks with complete media and placed in an incubator at 37°C and 5% CO2. The subculturing was repeated to get the required cell number. On the day of cell injection, the flasks with -70-80% confluence were subjected to above procedures and the cells with >90% viability cells were re-suspended with an appropriate volume of RPMI1640 serum free media to achieve the final cell concentration (5xl06cells / 200pL / animal with 50% Matrigel) and kept on ice for injection into animals.

[0229] Female NSG mice (6-8 weeks old) housed in Individually Ventilated Cages (IVCs) were used for the study. The cell injection was performed in a laminar flow hood under sterile conditions. Five million (5xl06) of SHP-77 cells in 200pL of serum free medium were implanted subcutaneously (s.c) in the right flank region of each animal under isoflurane anesthesia (0.4- 0.81iter / min oxygen with 2% isoflurane). Following injection, animals were returned to their respective cages and the implanted area was monitored for tumor growth. Once the tumor attained palpable stage, out of 62 mice, 48 mice with optimum tumor range (73-103mm3) were selected and randomized into 6 treatment groups (G1-G2: n=16 mice / group; G3-G6: n=4 mice / group), keeping almost similar starting mean tumor volume for each group.

[0230] Human PBMCs were isolated using the Ficoll gradient method and frozen in aliquots in liquid nitrogen. The PBMC vials were thawed at 37°C in water bath and treated with TCR V 17- APC antibody for Ih at 4°C in dark. lOmL of sorting buffer (made by mixing 500mL Auto MACS Rinsing solution and 25mL MACS BSA Stock solution) was added after Ih and cells were centrifuged at 400rcf for 5min at 4°C. The supernatant was discarded, and wash was repeated. The cells were re-suspended in lOOpL of sorting buffer (lOOpL for 106cells). 20pL of anti-APC beads were added for up to 106cells. Beads were scaled up according to the cell numbers. Cells were74JBI6937065768.12701 / 207W01 incubated for 15min in the dark at 4°C. 1 OmL of sorting buffer was added and centrifuged for 5min at 400rcf. MACS separator and the LS columns were prepared during centrifugation. Columns were rinsed with 3mL of the sorting buffer and flow through was discarded. Cell pellet was resuspended in 600pL of the sorting buffer (500pL for 107cells). Cell suspension was applied to LS column with a micropipette. Flow through was collected into a 50mL conical tube (this was the negative fraction). Each column was washed 7times with 4mL sorting buffer. Flow through was collected into the same 15mL conical tube. Column was removed from MACS separator and placed on top of a clean 15mL tube to collect cells that adhered to the columns (positive fraction of VP17 T cells). 5mL of the sorting buffer was pipetted onto the column and the magnetically labeled cells were immediately flushed out by firmly pushing the plunger into the column. 3mL of sort buffer was added onto the column and cells were flushed again. Both the fractions were spun at 400rcf for 5min at 4°C. Pellet was re-suspended in 10% RPMI media and cells were counted. ImL media per 1.5xl06cells was added. It was made sure that the culture volumes should not exceed 55mL per T175 flask.

[0231] Anti-Biotin MACS iBead Particles were prepared (for activation of Vpi7 T cells). lOOpL CD2-Biotin, lOOpL CD3-Biotin and lOOpL CD28 Biotin were pipetted into a sealable 2mL tube and mixed. Anti-Biotin MACS iBeads were vortexed and 500pL was added to antibody mix. 200pL Bead buffer (DPBS+0.5%FBS+2mM EDTA) was added to make total volume of ImL and incubated for 2h at 4°C with shaking. Anti -Biotin MACS iBead Particles were re-suspended thoroughly and 25pL (2.5* 106loaded Anti-Biotin MACS iBead Particles) per 5* 106cells were transferred in 10% FBS-RPMI (without IL-2). Cells were incubated at 37°C in CO2 incubator for 3 days.

[0232] After three days of culture, the cells were counted, and the concentration of culture was adjusted to 0.75-1.5 xlO6cells / mL with 10% FBS-RPMI supplemented with IL-2 (65ng / mL). The cultures were split and maintained in IL-2 containing media.

[0233] Results. As shown in Figure 3, tumor growth decrease was observed in 18 treated groups with Vpi7 cell engrafted group when compared to without Vpi7 cell engrafted group at same dose levels. A full description of the experimental design for the experiment shown in Figure 3 is provided in Table 7.75JBI6937065768.12701 / 207W01Table 7

[0234] In this efficacy study, pharmacokinetics following a single dose of J8 (3 & 15mg / kg) was evaluated in NSG mice bearing SHP-77 tumors. Animals bearing SHP-77 tumors were randomly divided into six groups (G1-G2: n=16 mice / group and G3-G6: n=4 mice / group) with a mean tumor volume of ~86mm3per group (tumor ranged from 73- 107mm3). On the day of randomization, Human Vpi7 T cells (10 xlO6cells / animal) were injected via i.p. route in G1-G4. After 24h (day 1), the dosing was initiated. First, IVIG (lOmg / O.lmL / animal; single dose) and Fc block (0.2mg / 0.1mL / animal; single dose) were injected i.p. to the animals from groups 1-4. Then, after 30minutes of IVIG and Fc block administration, J8 was injected i.v. as per the respective dosage. For testing the plasma pharmacokinetic concentrations of J8, Ni-NTA ELISA plates bound with His-tagged recombinant-DLL3 protein (DL3W35) were used to capture J8 in plasma. Mice injected with Vpi7 T cells + IVIG + Fc and 3mg / kg J8 (G1 and G3) and 15mg / kg J8 (G2 and G4) exhibited generally dose dependent plasma concentrations, which decreased overtime. The plasma concentration analysis of J8 at 3mg / kg and 15mg / kg in NSG mice bearing SHP-77 xenografts showed dose and time dependent concentrations in the presence of Vpi7 T cells. Additionally, plasma concentration of J8 at 3mg / kg and 15mg / kg in the absence of Vpi7 T cells was reversed.

[0235] Example 3: Cytotoxicity ofJ8

[0236] Methods to Assess DLL3 Binding. The DLL3 expression level was measured on the surface of GFP-labelled endogenously DLL3 expressing SHP-77, NCLH82 and NCLH1436 cell lines. The target cell line cultures were harvested and seeded at a density of 0.1 * 106cells per well in 96 well plates. Live / dead stain (Violet Fluorescent dye) as lpL / l >< 106cells in PBS was added76JBI6937065768.12701 / 207W01 and incubated at 4°C for 20min. Samples were washed once with FACS buffer (PBS+2%FBS). lOOnM of J8 and DLL3 Bivalent IgG (DL3B757) were added at a volume of lOOpL per well and incubated at 37°C for 40min. Samples were washed twice with FACS buffer. Secondary antibody [AF647 AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, Fey fragment specific] was added at a dilution of 1 :500 with FACS buffer and incubated at 4°C for 30min. Samples were washed twice with FACS buffer. Quantum APC MESF beads (Idrop) were added to the wells separately for receptor level analysis. Data was acquired on Flow cytometer.

[0237] Methods for Cytotoxicity. PBMCs, frozen and stored as described in Example 2, were thawed from liquid nitrogen and counted. Tumor target cells were seeded at 5,000 cells per well per lOOpL of 10% FBS-media in 96 well plates. J8, tarlatamab and Vpi7xNull antibodies were prepared at 4X concentration, and each antibody was added at a starting DRC concentration of lOOnM, 3-fold dilutions and 10 to 11 dilutions (50pL per well). PBMCs were thawed from liquid nitrogen stocks and analyzed by flow cytometry for the Vpi7 T cell and CD3 T cell percentage. The number of PBMCs for the required E:T was calculated according to the percentages of Vpi7 T cells and CD3 T cell and 50pL per well were added. In the control wells, the volume was adjusted with complete media to match the treated wells. Plates were transferred to the IncuCyte machine and incubated at 37°C and 5% CO2. Plates were checked for condensation using single scan to make sure there were no blurred images. The IncuCyte scan (live cell imaging) program was scheduled with interval of 6-12h for a period of 5 or more days. The green integrated intensities (GII) were exported from the IncuCyte, and percent lysis was calculated using the following formula: Percent Lysis= (GII for ET Control- GII for Sample value) / GII for ET Control) *100.

[0238] Method for Assessing If 17 T Cell Activation. J8, tarlatamab and Vpi7xNull antibodies were prepared at 4X concentration, and each antibody was added at final concentrations of lOOnM, 33.33nM and 11.1 InM. The plates were collected at 24h, 48h and 72h timepoints and centrifuged at 1200rpm for 5min. The supernatants (150pL) were collected and stored in -80°C for cytokine analysis. lOOpL of PBS was added to the cell pellets in the plates and mixed thoroughly. The suspension was transferred to 96-well V bottom plates and centrifuged at 1400rpm for 5min. lOOpL of live dead stain (IpL per l >< 106cells in PBS) was added to the wells. The plates were incubated at 4°C for 20min. lOOpL of the FACS buffer was added to the wells and the centrifuged at 1400rpm for 5min. Antibodies against CD25, CD69 and CD71 activation markers were added in the next step. 2pL of each antibody per well in lOOpL of antibody cocktail was added to wells.77JBI6937065768.12701 / 207W01The plates were incubated at 4°C for 30min. lOOpL of FACS buffer was added to the wells and the plate centrifuged at 1400rpm for 5min. This washing step was repeated. The pellet was resuspended in 50pL of FACS buffer. 25pL of cell count precision beads were added to the wells, mixed thoroughly and acquired in the flow cytometer.

[0239] Results. Live cell imaging (IncuCyte) based cytotoxicity assays were performed with PBMCs from 4 healthy human donors (DI, D2, D3 and D4) as effector cells with SHP-77 / GFP, NCLH82 / GFP and NCLH1436 / GFP target cell lines. Additionally, HCC1833 / GFP and G361 / GFP cell lines were evaluated in the IncuCyte based cytotoxicity assay with J8, resulting in no cytotoxicity and low cytotoxicity (about 60% killing), respectively (data not shown). DMS53 / GFP cells were very slow growing. Therefore, these cells were not pursued further.

[0240] J8 showed potent cytotoxicity with SHP-77 / GFP (Figures 4A-4E), NCI-H82 (Figures 5A-5E), and NCI-H1436 (Figures 6A-6E) cell lines with all four donor PBMCs at 1 : 1 E:T (Vpi7 T cell to Target cell ratio) on day-5 of the cytotoxicity assay. Tarlatamab showed lower ECso values at matching E:T of 1: 1 (CD3 T cell to Target cell ratio) with all three cell lines. Vpi7xNull control did not show any cytotoxicity with any of the cell lines or donors. The cytotoxicity by J8 and tarlatamab showed similar kinetic profile at top three highest concentration of lOOnM, 33.33nM and 11.1 InM in a kinetic analysis with all four donors with all three cells lines. A dose dependent reduction in cytotoxicity was seen when J8 and tarlatamab were tested at 0.5: 1 E:T with SHP- 77 / GFP, NCI-H82 / GFP and NCLH1436 / GFP Cell lines (Figure 7). Kinetic analysis at 0.5:1 E:T with all three cell lines together in a graph along with 1 : 1 E:T at top three highest concentration of lOOnM, 33.33nM and 11.1 InM of J8 is shown in Figure 8. Overall, there was comparable cytotoxicity with J8 and tarlatamab with all three cell lines. At 1 : 1 E:T, complete cytotoxicity was observed at around day-5 with both J8 and tarlatamab.

[0241] J8 showed potent activation of CD69, CD25 and CD71 marker on Vpi7 T cells at 1 : 1 E:T and at top three highest concentration of lOOnM, 33.33nM and l l.l lnM. CD69 showed early activation till day-3 followed by CD25 and CD71 markers continuously increasing until tested day-5. J8.

[0242] Summary. In the present study, J8 was evaluated for its cytotoxic potential on endogenously DLL3 expressing tumor cell lines. PBMCs from health donors were used as a source of effector immune cells (Vpi7 T cells and Pan T cells).78JBI6937065768.12701 / 207W01

[0243] J8 showed dose-dependent cytotoxicity when tested with multiple E:T (2: 1, 1 : 1 and 0.5: 1). A dose dependent response was seen with multiple concentrations ranging from lOOnM to 0.02nM, resulting in a sigmoidal curve with EC50 values in single digit nM range with SHP- 77 / GFP cells and sub-nM range with NCI-H82 / GFP and NCI-H1436 / GFP. Time-kinetic showed complete kill by day 5 for all the three cell lines tested. The maximum level of cytotoxicity was comparable to tarlatamab at matching ET and / or PBMC numbers (same number of PBMCs as used for Vpi7 to target ratio or E:T for J8).

[0244] J8 was able to activate CD69, CD25 and CD71 markers on Vpi7 T cells with all three cell lines tested. CD69, an early activation marker, showed early activation till day-3. CD25 and CD71 showed continuous increase in activation till the tested day-5.

[0245] J8 was able to proliferate Vpi7 T cells, increasing its percentage within CTV labeled effector cells and increasing the Vpi7 T cell absolute numbers.

[0246] J8 showed lower cytokine release levels when compared to tarlatmab in a cytotoxicity assay with matching PBMC numbers (same number of PBMCs as used for Vpi7 to target ratio or E:T for J8).Example 4. in vivo Efficacy of J8

[0247] The objective of the studies presented in this Example 4 was to evaluate and compare the anti-tumor efficacies of DLL3 -targeting bispecific antibodies in NSG mice bearing SHP-77 subcutaneous tumor xenografts. Briefly, 6-8 weeks old female NSG mice were injected subcutaneously with SHP-77 cells (~5xl06cells / 200pL / mouse with 50% matrigel) on the right flank region to develop SHP-77 tumors. Mice bearing SHP-77 tumors with a tumor range of il l 7mm3were randomized based on tumor volume and divided into eleven groups (n=8mice per group) with a mean tumor volume of ~88mm3per group. In addition to single dose control groups of vehicle, effector cells provided as either human T cells (hT; 20x106cells / animal) or human Vpi7 T cells (hvpi7; 10xl06 / animal) were administered based on the study group. The anti-tumor efficacy of J8 at three dose levels- Img / kg, 3mg / kg and 15mg / kg as a monotherapy, tarlatamab at 3mg / kg, and Vpi7 x null isotype control) at 15mg / kg in NSG mice bearing SHP-77 (human lung small cell lung cancer) xenograft tumors was evaluated.

[0248] J8 demonstrated greater anti-tumor efficacy in hvpi7 (10xl06 / animal) engrafted groups compared to hT cell engrafted groups. In hvpi7 T cell engrafted groups, J8 showed significant and complete tumor suppression at all three dose levels (Img / kg, 3mg / kg & 15mg / kg) compared79JBI6937065768.12701 / 207W01 to vehicle. The anti-cancer efficacy of tarlatamab treatment was nearly identical in the hT cell and hvpi7 cell engrafted groups. The isotype control Vpi7 x null, and J8 at both doses tested in hT cell engrafted groups (3mg / kg and 15mg / kg), did not show significant change in tumor growth compared to hT cells alone group.

[0249] The ex-vivo flow cytometric analysis of treated blood on day 8 and day 30 revealed higher CD25 expression in hvpi7 T cells engrafted groups than in hT cells engrafted groups. Further, PD1 exhaustion marker expression was detected on day 30 bleed hT cells and hvpi7 T cells for all the tested groups. Cells from donor- 1 hT cell and hvpi7 T cell injected mice showed more activation marker expression and less exhaustion marker expression in comparison to Donor 2 cells across all the groups.

[0250] Plasma PK of J8 showed dose dependent concentrations with Cmax achieved by day 10 after repeated administration on every third day and these concentrations were maintained until the dosing period.

[0251] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that the present disclosure is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present disclosure as defined by the present description.INCORPORATION BY REFERENCE

[0252] All publications, patents, and patent applications herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls.80JBI6937

Claims

1. 065768.12701 / 207W01CLAIMS1. A bispecific antibody comprising:(a) a Fab binding V 17 comprising a heavy chain variable region comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 8; and(b) an spFv binding DLL3 comprising a heavy chain variable region comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 33, and a light chain variable region an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 34.

2. The bispecific antibody of claim 31, wherein:(a) the Fab comprising a heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8; and(b) the spFv comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34.

3. A bispecific antibody comprising(a) a first heavy chain (HC1) comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 23,(b) a first light chain (LC1) comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 24, and(c) a second heavy chain (HC2) comprising an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 37; wherein HC1 is associated with LC1 to form a binding site for V 17, and HC2 comprises an spFv binding DLL3.

4. A bispecific antibody comprising(a) a first heavy chain (HC1) comprising the amino acid sequence set forth in SEQ IDNO: 23,(b) a first light chain (LC1) comprising the amino acid sequence set forth in SEQ ID NO: 24, and81JBI6937065768.12701 / 207W01(c) a second heavy chain (HC2) comprising the amino acid sequence set forth in SEQ ID NO: 37; wherein HC1 is associated with LC1 to form a binding site for V 17, and HC2 comprises an spFv binding DLL3.

5. A bispecific antibody comprising:(a) a first binding domain that binds Vpi7 comprising a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 53, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; and(b) a second binding domain that binds DLL3 comprising a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; wherein the first binding domain or the second binding domain comprises an spFv.

6. The bispecific antibody of claim 1, wherein the second binding domain comprises an spFv.

7. The bispecific antibody of claim 1 or 2, wherein the second binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33.

8. The bispecific antibody of any one of claims 1-3, wherein the second binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34.

9. The bispecific antibody of any one of claims 1-4, wherein the second binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34.

10. The bispecific antibody of any one of claims 1-5, wherein the second binding domain comprises the amino acid sequence set forth in SEQ ID NO: 36 or SEQ ID NO: 37.82JBI6937065768.12701 / 207W0111 . The bispecific antibody of any one of claims 1-6, wherein the second binding domain comprises the amino acid sequence set forth in SEQ ID NO: 37.

12. The bispecific antibody of any one of claims 1-7, wherein the first binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 9.

13. The bispecific antibody of any one of claims 1-8, wherein the first binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 10.

14. The bispecific antibody of any one of claims 1-9, wherein the first binding domain comprises(a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; or(b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10.

15. The bispecific antibody of claim 10, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8.

16. The bispecific antibody of any one of claims 1-11, wherein the first binding domain comprises the amino acid sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 20.

17. The bispecific antibody of claim 13, wherein the first binding domain comprises the amino acid sequence set forth in SEQ ID NO: 19.

18. The bispecific antibody of any one of claims 1-13, wherein the first binding domain comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23.

19. The bispecific antibody of any one of claims 1-14, wherein the first binding domain comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 24.

20. The bispecific antibody of claim 14 or 15, wherein the first binding domain comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 24.

21. The bispecific antibody of any one of claims 2-16, wherein the first binding domain is a half antibody.83JBI6937065768.12701 / 207W0122. The bispecific antibody of claim 1, wherein the first binding domain comprises an spFv.

23. The bispecific antibody of claim 18, wherein the first binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17.

24. The bispecific antibody of claim 18 or 19, wherein the first binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18.

25. The bispecific antibody of any one of claims 18-20, wherein the first binding domain comprises(a) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12;(b) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14;(c) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16; or(d) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18.

26. The bispecific antibody of claim 21, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 11 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12.

27. The bispecific antibody of claim 21, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16.

28. The bispecific antibody of any one of claims 18-26, wherein the first binding domain comprises the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 22.84JBI6937065768.12701 / 207W0129. The bispecific antibody of any one of claims 18-24, wherein the second binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31.

30. The bispecific antibody of any one of claims 18-25, wherein the second binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32.

31. The bispecific antibody of any one of claims 18-26, wherein the second binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32.

32. The bispecific antibody of any one of claims 18-27, wherein the second binding domain comprises the amino acid sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 38.

33. A bispecific antibody comprising:(a) a Fab binding Vpi7 comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 24; and(b) an spFv binding DLL3 comprising the amino acid sequence set forth in SEQ ID NO: 36.

34. A bispecific antibody comprising:(a) a Fab binding V017 comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 24; and(b) a heavy chain binding DLL3 comprising the amino acid sequence set forth in SEQ ID NO: 37.

35. A nucleic acid encoding the bispecific antibody of any one of claims 1-34.

36. A vector comprising the nucleic acid of claim 35.

37. A host cell comprising the nucleic acid of claim 35 or the vector of claim 36.

38. A composition comprising the bispecific antibody of any one of claims 1-34, the nucleic acid of claim 35, the vector of claim 36, or the host cell of claim 37.

39. The composition of claim 38, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.85JBI6937065768.12701 / 207W0140. A method for treating a cancer comprising a cell expressing DLL3 in a subject in need thereof, the method comprising administering an effective amount of the bispecific antibody of any one of claims 1-34 or the composition of claim 38 or 39.

41. The method of claim 40, wherein the cancer is adrenal cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational trophoblastic, head and neck cancer, Hodgkin lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, neuroendocrine tumor, nonHodgkin lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma spinal cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer endometrial cancer, vaginal cancer, or vulvar cancer.

42. The method of claim 41, wherein the lung cancer is small cell lung cancer, small cell carcinoma, combined small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, large-cell undifferentiated carcinoma, pulmonary nodule, metastatic lung cancer, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland-type lung carcinoma, lung carcinoid, mesothelioma, sarcomatoid carcinoma of the lung, or malignant granular cell lung tumor.

43. The method of claim 41 or 42, wherein the lung cancer is small cell lung cancer.

44. A method for eliminating a cancer cell expressing DLL3 in a subject in need thereof, the method comprising administering an effective amount of the bispecific antibody of any one of claims 1-34 or the composition of claims 38 or 39.

45. A method of inhibiting the growth or proliferation of cancer cells expressing DLL3 in a subject in need thereof, the method comprising administering an effective amount of the bispecific antibody of any one of claims 1-34 or the composition of claims 38 or 39.

46. A method of promoting T cell mediated killing of cancer cells expressing DLL3 in a subject in need thereof, the method comprising administering an effective amount of the bi specific antibody of any one of claims 1-34 or the composition of claims 38 or 39.

47. A method of directing a Vpi7-expressing T cell to a cancer cell expressing DLL3 in a subject in need thereof, the method comprising administering an effective amount of the bispecific antibody of any one of claims 1-34 or the composition of claims 38 or 39.86JBI6937065768.12701 / 207W0148. The method of claim 47, wherein the T cell is a CD8 T cell or a CD4 T cell.

49. The method of any one of claims 44-48, wherein the cancer cell is a cell of an adrenal cancer, an anal cancer, an appendix cancer, a bile duct cancer, a bladder cancer, a bone cancer, a brain cancer, a breast cancer, a cervical cancer, a colorectal cancer, an esophageal cancer, a gallbladder cancer, a gestational trophoblastic, a head and neck cancer, a Hodgkin lymphoma, an intestinal cancer, a kidney cancer, a leukemia, a liver cancer, a lung cancer, a melanoma, a mesothelioma, a multiple myeloma, a neuroendocrine tumor, a non-Hodgkin lymphoma, an oral cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a sinus cancer, a skin cancer, a soft tissue sarcoma spinal cancer, a stomach cancer, a testicular cancer, a throat cancer, a thyroid cancer, a uterine cancer endometrial cancer, a vaginal cancer, or a vulvar cancer.

50. The method of claim 49, wherein the lung cancer is small cell lung cancer, small cell carcinoma, combined small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, large-cell undifferentiated carcinoma, pulmonary nodule, metastatic lung cancer, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland-type lung carcinoma, lung carcinoid, mesothelioma, sarcomatoid carcinoma of the lung, or malignant granular cell lung tumor.

51. The method of claim 49 or 50, wherein the lung cancer is small cell lung cancer.

52. The bispecific antibody of any one of claims 1-34 or the composition of claim 38 or 39 for use in treating a cancer comprising a cell expressing DLL3, eliminating a cancer cell expressing DLL3, inhibiting the growth or proliferation of cancer cells expressing DLL3, and / or promoting T cell mediated killing of cancer cells expressing DLL3.

53. Use of the bispecific antibody of any one of claims 1-34 for the preparation of a medication for treating a cancer comprising a cell expressing DLL3, eliminating a cancer cell expressing DLL3, inhibiting the growth or proliferation of cancer cells expressing DLL3, and / or promoting T cell mediated killing of cancer cells expressing DLL3.87JBI6937

Images