D3 binding molecules and uses thereof

Abstract

The present disclosure provides D3 binding molecules, including anti-D3 antibodies, and uses thereof.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to International Patent Application No. PCT / CN2021 / 119011, filed September 17, 2021, the disclosure of which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This application incorporates by reference the sequence listing submitted herewith.

[0003] This application relates generally to Delta-like canonical Notch ligand 3 (D3) binding molecules, including anti-D3 antibodies, and uses thereof. [Background technology]

[0004] Delta-like canonical Notch ligand 3 (D3 or DLL3) is a type I transmembrane protein that belongs to the DSL family of Notch ligands. D3 is normally expressed only in intracellular membranes, especially in the Golgi apparatus. Additional Notch family ligands include Delta-like canonical Notch ligand 1 (D1), Delta-like canonical Notch ligand 4 (D4), Jagged canonical Notch ligand 1 (J1), and Jagged canonical Notch ligand 2 (J2). Other ligands, except for D3, can activate Notch signaling. D3 functions as an inhibitor of Notch signaling by preventing the binding of Notch to its ligands. D3 is highly expressed on the surface of lung tumor cells, including small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC). Although D3 is normally expressed only in intracellular membranes, it is a potential therapeutic tumor target for any tumor expressing D3, including SCLC and LCNEC.

[0005] Lung cancer is the most common cause of cancer death, with approximately 2 million cases diagnosed worldwide each year. Approximately 15% of all lung cancer cases are SCLC. SCLC is the most aggressive form of lung cancer, with very limited treatment options (surgery, chemotherapy, and radiation therapy). In 2019, the FDA approved atezolizumab (anti-PD-L1) as a first-line treatment for SCLC, but efficacy is limited to 2 months, highlighting the need for further therapeutic development.

[0006] There remains a need to identify new D3-targeting agents, including D3-binding molecules such as monoclonal antibodies for use as antibody conjugates, such as ADCs, or as bispecific antibodies, or for the development of CAR-T therapies. Summary of the Invention

[0007] The present disclosure relates to compounds, methods, compositions and products that provide D3 binding molecules with improved efficacy. The advantages provided by the present disclosure are broadly applicable to the fields of antibody therapy and diagnostics, and can be used in combination with other therapeutic agents, such as antibodies that react with various targets.

[0008] The present disclosure provides D3 binding molecules, such as monoclonal antibodies, capable of specifically binding to human D3 and cross-reactive with cynomolgus monkey and / or mouse D3. Such D3 binding molecules offer certain advantages over currently used and / or known drugs, compositions and / or methods in the art. These advantages include potential for internalization, improved therapeutic and pharmacological properties, improved specificity, improved safety profile, reduced immunogenicity, and other advantageous properties such as improved ease of preparation or reduced product cost, and higher stability, especially when compared to drug candidates already known in the art.

[0009] In the present disclosure, we have developed D3 binding molecules, such as monoclonal antibodies against D3, that can be used to treat tumors that overexpress D3.

[0010] The present disclosure provides D3 binding molecules, nucleic acid molecules encoding the same, expression vectors and host cells used to express the D3 binding molecules, and methods for using the D3 binding molecules. The D3 binding molecules of the present disclosure provide a potent agent for treating multiple cancers (including lung cancer) through the regulation of human immune function.

[0011] In some embodiments, the disclosure provides D3 binding molecules comprising at least one immunoglobulin single variable domain (e.g., a VHH domain) that specifically binds to D3, such as human D3, cynomolgus monkey D3, and / or mouse DLL-3. In some embodiments, the single variable domain comprises CDR1, CDR2, and CDR3, where: CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, 4, 7 or 10; CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 11; CDR3 comprises the amino acid sequence set forth in SEQ ID NO:3, 6 or 9.

[0012] In some embodiments, the single variable domain disclosed herein comprises: (A) CDR1 set forth in SEQ ID NO: 1, CDR2 set forth in SEQ ID NO: 2, and CDR3 set forth in SEQ ID NO: 3; (B) CDR1 set forth in SEQ ID NO: 4, CDR2 set forth in SEQ ID NO: 5, and CDR3 set forth in SEQ ID NO: 6; (C) CDR1 set forth in SEQ ID NO: 7, CDR2 set forth in SEQ ID NO: 8, and CDR3 set forth in SEQ ID NO: 9, or (D) Includes CDR1 set forth in SEQ ID NO: 10, CDR2 set forth in SEQ ID NO: 11, and CDR3 set forth in SEQ ID NO: 6.

[0013] In some embodiments, the single variable domain disclosed herein comprises: (A) CDR1 set forth in SEQ ID NO: 27, CDR2 set forth in SEQ ID NO: 28 or 56, and CDR3 set forth in SEQ ID NO: 29; (B) CDR1 set forth in SEQ ID NO: 38, CDR2 set forth in SEQ ID NO: 39, and CDR3 set forth in SEQ ID NO: 40, or (C) comprising CDR1 set forth in SEQ ID NO: 49, CDR2 set forth in SEQ ID NO: 50, and CDR3 set forth in SEQ ID NO: 51; However, CDR numbering follows the Contact numbering system.

[0014] In some embodiments, the single variable domain disclosed herein comprises: (A) an amino acid sequence set forth in any one of SEQ ID NOs: 12 to 18 and 55; (B) an amino acid sequence that is at least 85%, 90%, or 95% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 12-18 and 55, while maintaining a specific binding affinity for D3 (e.g., substantially, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%), or (C) An amino acid sequence that has one or more (e.g., 1, 2 or 3) amino acid additions, deletions and / or substitutions compared to the amino acid sequence set forth in any one of SEQ ID NOs: 12 to 18 and 55, while maintaining (e.g., substantially, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) the specific binding affinity for D3.

[0015] In some embodiments, the D3 binding molecules disclosed herein comprise one or more substitutions, additions and / or deletions of amino acids within the framework regions of the single variable domain (e.g., VHH), such as FRW1, FRW2, FRW3 and / or FRW4. In some embodiments, FRW1 at the N-terminus and / or FRW4 at the C-terminus of the single variable domain are truncated, e.g., by no more than 5, 4, 3, 2 or 1 amino acid.

[0016] In some embodiments, the single variable domain (eg, VHH) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 12-18 and 55.

[0017] In some embodiments, the D3 binding molecule disclosed herein further comprises one or more human IgG constant domains, such as one or more human IgG1, IgG2, IgG3, or IgG4 constant domains. In some embodiments, the IgG constant domain is a human IgG1 constant domain or a variant thereof. An example of an amino acid sequence of an IgG1 constant domain is as set forth in SEQ ID NO: 19. In some embodiments, the D3 binding molecule comprises one or more variants of a human IgG1 constant domain, such as an IgG1 Fc with L234A / L235A substitutions according to EU numbering.

[0018] In some embodiments, the D3 binding molecules disclosed herein have one or more of the following properties: (a) binds to human D3, cyno D3, and / or mouse D3 with nM-grade EC50 as measured by ELISA or FACS; (b) It showed dose-dependent internalization in cells expressing human D3. (c) It binds to human D3 with a KD of 0.1 nM or less as measured by SPR.

[0019] In some embodiments, the D3 binding molecules disclosed herein are chimeric, humanized, or fully human antibodies. In some embodiments, the D3 binding molecules are dimers.

[0020] In some embodiments, a D3 binding molecule disclosed herein comprises a single variable domain set forth in any one of SEQ ID NOs: 12-18 and 55, and an IgG constant domain set forth in SEQ ID NO:19.

[0021] In some embodiments, the disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding a D3 binding molecule disclosed herein, e.g., a D3 binding molecule comprising a single variable domain (e.g., a VHH).

[0022] In some embodiments, the disclosure provides a vector comprising the nucleic acid molecule disclosed herein.

[0023] In some embodiments, the disclosure provides a host cell comprising an expression vector or a nucleic acid molecule disclosed herein.

[0024] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a D3 binding molecule disclosed herein and a pharma- ceutically acceptable carrier.

[0025] In some embodiments, the present disclosure provides a method for preparing a D3 binding molecule, the method comprising expressing the D3 binding molecule in a host cell disclosed herein and isolating the D3 binding molecule from the host cell.

[0026] In some embodiments, the present disclosure provides a method for modulating a D3-associated immune response in a subject, comprising administering to the subject a D3 binding molecule disclosed herein, thereby modulating the subject's D3-associated immune response.

[0027] In some embodiments, the present disclosure provides a method for treating or preventing a D3-positive or D3-overexpressing cancer in a subject, comprising administering to the subject an effective amount of a D3 binding molecule or pharmaceutical composition disclosed herein. In some embodiments, the cancer is lung cancer, including, for example, SCLC and LCNEC.

[0028] In some embodiments, the present disclosure provides for the use of a D3 binding molecule disclosed herein in the manufacture of a medicament for diagnosing, treating or preventing a D3-positive cancer.

[0029] In some embodiments, the present disclosure provides a D3 binding molecule as disclosed herein for use in the diagnosis, treatment or prevention of a D3-positive cancer.

[0030] In some aspects, the present disclosure relates to kits or devices and associated methods that use the D3 binding molecules disclosed herein, or the pharmaceutical compositions disclosed herein.

[0031] The above is a summary and therefore necessarily contains simplifications, generalizations, and omissions of details. Thus, those skilled in the art will appreciate that this summary is merely illustrative and is not intended to be limiting in any way. Other aspects, features, and advantages of the binding molecules, methods, compositions, and / or devices and / or other subject matter described herein will become apparent in the teachings set forth herein. The summary is provided to introduce in a simplified form a set of concepts that are further described below in the Detailed Description of the Invention. This summary is not intended to identify key features or important features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. [Brief description of the drawings]

[0032] [Figure 1] Exemplary binding results of each antibody to immobilized WT115-hPro1.ECD.his as measured by ELISA are shown. [Diagram 2] Panels a and b show exemplary binding results of each antibody to WT115-293F.hPro1.2E5 cells as measured by FACS. [Diagram 3] Panels a and b show exemplary binding results of each antibody to WT115-Flpin293.cPro1.pool cells as measured by FACS. [Figure 4a] Exemplary binding results of each antibody to immobilized WT115-MBP-mPro1.ECD.hFc as measured by ELISA are shown. [Figure 4b]Exemplary binding results of each antibody to immobilized WT115-MBP-mPro1.ECD.hFc as measured by ELISA are shown. [Diagram 5] Panels a and b show exemplary results of internalization of each antibody by WT115-293F.hPro1.2E5 cells as measured by FACS. [Figure 6a] Exemplary epitope binning results for each antibody against immobilized WT115-hPro1.ECD.his as measured by ELISA. Binning with WT1156-P3R2-1C2-uIgG1. [Figure 6b] Exemplary epitope binning results for each antibody against immobilized WT115-hPro1.ECD.his as measured by ELISA. Binning with WT1156-P3R2-1H6-uIgG1. [Figure 6c] Exemplary epitope binning results for each antibody against immobilized WT115-hPro1.ECD.his as measured by ELISA. Binning with WT1156-P8R2-1H1-uIgG1. [Figure 6d] Exemplary epitope binning results for each antibody against immobilized WT115-hPro1.ECD.his as measured by ELISA are shown. Binning with WT115-BMK1. [Figure 7a] Exemplary ELISA binding results of each antibody to soluble WT115-hPro1.ECD.his or truncated protein by ELISA are shown. [Figure 7b] The results of ELISA binding of each antibody to immobilized WT115-hPro1.ECD.his or truncated protein by ELISA are shown. [Figure 7c] A diagram of the truncated protein is shown. [Figure 8] Exemplary inter-family binding results of each antibody with human D1 and human D4 as measured by ELISA are shown. [Figure 9]1 shows the results of an exemplary serum stability study of WT1156-P3R2-1C2-z109-uIgG1. [Figure 10] 1 shows an alignment of exemplary immunoglobulin single variable domains WT1156-P3R2-1C2 (1C2), WT1156-P3R2-1C2-z102 (1C2-z102), WT1156-P3R2-1C2-z109 (1C2-z109), WT1156-P3R2-1C9 (1C9), WT1156-P3R2-1H6 (1H6), WT1156-P3R2-1H6-z100 (1H6-z100), and WT1156-P8R2-1H1 (1H1). The boundaries of each CDR are indicated by the numbering of Kabat, AbM, Chothia, Contact, and IMGT. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Although the present disclosure can be implemented in many different forms, certain exemplary embodiments of the present disclosure are disclosed herein to illustrate the principles of the present disclosure. It should be emphasized that the present disclosure is not limited to the specific embodiments shown. Any section headings used herein are for organizational purposes only and should not be interpreted as limiting the subject matter of the invention described.

[0034] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include plural referents and plural terms shall include singular referents. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to "a protein" includes a plurality of proteins, a reference to "a cell" includes a mixture of cells, and so forth. In this application, the use of "or" means "and / or" unless otherwise indicated. Furthermore, the use of the term "comprises," as well as other forms such as "comprises" and "includes," is not limiting. Furthermore, the ranges provided in the specification and the appended claims include the endpoints and all points between the endpoints.

[0035] Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. For example, Abbas et al., Cellular and Molecular Immunology, 6th ed., WBSaunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular See Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art.

[0036] definition For a better understanding of the present disclosure, definitions and explanations of relevant terms are provided below.

[0037] The terms "antibody" (e.g., anti-D3 antibody) and "antigen-binding molecule" (e.g., D3 binding molecule) are used interchangeably in the broadest sense and encompass all forms of antibodies that exhibit the desired biological or binding activity. The terms include, but are not limited to, humanized antibodies, fully human antibodies, chimeric antibodies, and single domain antibodies (sdAbs, which usually contain only one chain similar to a heavy chain), and fragments of any of the above, such as antibodies containing at least one VHH domain, so long as they exhibit the desired antigen-binding activity. Conventional antibodies contain heavy and light chains. Heavy chains can be classified as μ, δ, γ, α, and ε, which define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The heavy chains are made up of a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain consists of one or more constant regions, e.g., three constant regions (C H 1. C H 2, and C H 3). The light chain can include a light chain variable region (V L ) and the light chain constant region (C L ) V H and V L The region can be further divided into regions of hypervariability (called complementarity determining regions (CDRs)) interspersed with regions that are relatively conserved (called framework regions (FRWs)). H and V L can comprise three CDRs (complementarity determining regions) and four FRs (framework regions) in the following order from N-terminus to C-terminus: FRW1, CDR1, FRW2, CDR2, FRW3, CDR3, FRW4. Can also be of different antibody isotypes, for example IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0038] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is often defined as extending from an amino acid residue at position Cys226 (according to the EU numbering system), or Pro230 (according to the EU numbering system), to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain.

[0039] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor: BCR), and the like. Effector functions such as these generally require that the Fc region be combined with a binding region or domain (e.g., an antibody variable region or domain, including a VHH domain), and can be assessed using a variety of assays as disclosed herein.

[0040] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature, and which has not been manipulated, modified and / or altered by man (e.g., isolated, purified, selected, or combined with other sequences, such as variable region sequences). Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0041] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion), preferably one or more amino acid substitution(s). In some embodiments, a variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or the Fc region of a parent polypeptide, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions in the native sequence Fc region or in the Fc region of the parent polypeptide. A variant Fc region may have at least about 80% homology, or at least about 90% homology, e.g., at least about 95% homology, with a native sequence Fc region and / or the Fc region of a parent polypeptide. A variant Fc region described herein may have a loss of effector function (e.g., a silent Fc).

[0042] Antibodies as described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including, e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0043] The terms "immunoglobulin single variable domain" or "single variable domain" or "VHH domain" or "VHH" or "heavy chain only antibody variable domain" may be used interchangeably herein and refer to a single chain antigen binding domain that can bind to an antigen or epitope independently of a different variable domain. VHH domains (e.g., variable domains of heavy chain antibodies) represent the smallest known antigen binding units generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13):3490-8. Epub 2007 Jun 15(2007)). VHH domains may be human domains, but also include single domains from other species such as rodent, nurse shark, and camelid VHH domains. Camelid VHHs are immunoglobulin single variable domain polypeptides from species such as camel, llama, alpaca, dromedary, and guanaco that produce heavy chain antibodies that are naturally devoid of light chains. Such VHH domains may be humanized according to standard techniques available in the art and are considered "single domain antibodies". As used herein, VHH includes camelid VHH domains and humanized VHH domains.

[0044] The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, llama or alpaca, have been introduced into human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0045] As used herein, the term "Ka" refers to the on-rate of a particular antibody-antigen interaction, and as used herein, the term "Kd" refers to the off-rate of a particular antibody-antigen interaction. The Kd value of an antibody can be determined using methods well established in the art. As used herein, the term "KD" refers to the dissociation constant of a particular antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (e.g., Kd / Ka) and expressed as a molar concentration (M). A preferred method for determining the Kd of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore® system.

[0046] As used herein, the terms "specific binding" or "specifically binds" refer to a non-random binding reaction between two molecules, such as, for example, between an antibody and an antigen.

[0047] As used herein, the term "high affinity" refers to an affinity of 1×10 to a target antigen. -7 M or less, preferably 5×10 -8 M or less, and even more preferably 1×10 -8 M or less, and even more preferably 5×10 -9 M or less, and even more preferably 1×10 -9 M refers to a D3 binding molecule, such as an antibody, having a KD of less than or equal to M.

[0048] As used herein, "EC 50 The term "median effective concentration," also referred to as "median effective concentration," refers to the concentration of a drug, antibody, or toxin that induces a response halfway between the baseline and maximum after a particular exposure time. In the context of this disclosure, EC 50 is expressed in units of "nM".

[0049] As used herein, the term "epitope" or "antigenic determinant" refers to a portion of an antigen to which an immunoglobulin or antibody specifically binds. An "epitope" is also referred to as an "antigenic determinant." An epitope or antigenic determinant generally comprises a chemically active surface group of molecules, such as amino acids, carbohydrates, or sugar side chains, and generally has a specific three-dimensional structure and specific charge characteristics. For example, an epitope generally comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique three-dimensional conformation that may be "linear" or "conformational." See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). In a linear epitope, all of the interaction sites between the protein and the interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. In conformational epitopes, the interaction sites are located across amino acid residues that are separated from each other in the protein. Antibodies can be screened according to their competitiveness for binding to the same epitope by conventional techniques well known to those skilled in the art. For example, competitive or cross-competitive experiments can be carried out to obtain antibodies that compete or cross-compete with each other for binding to antigen. A high-throughput method for obtaining antibodies that bind to the same epitope based on their cross-competition is described in International Patent Application WO03 / 48731.

[0050] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to D3 protein is substantially free of antibodies that specifically bind to antigens other than D3 protein). However, an isolated antibody that specifically binds to human D3 protein may have cross-reactivity to other antigens, such as D3 proteins from other species. Additionally, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0051] The term "vector" as used herein refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector allows for the expression of a protein encoded by a polynucleotide inserted therein, the vector is called an expression vector. A vector can carry genetic material elements that are expressed in a host cell by transformation, transduction, or transfection into the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda or M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (such as SV40). A vector may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0052] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as Drosophila S2 cells or Sf9 cells, and animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0053] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined by aligning and comparing the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest of the molecules compared. In these calculations, gaps in the alignment (if any) are preferably resolved by a specific mathematical model or computer program (e.g., "algorithm"). Methods that can be used to calculate identity between aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAM J. Applied Math. 48:1073.

[0054] As used herein, the term "immunogenicity" refers to the ability to stimulate the production of specific antibodies or sensitized lymphocytes in an organism. Immunogenicity refers not only to the property of an antigen to stimulate specific immune cells to activate, proliferate, and differentiate, ultimately producing immune effector substances such as antibodies and sensitized lymphocytes, but also to a specific immune response that can produce antibodies or sensitized T lymphocytes in the immune system of an organism after stimulating the organism with the antigen. Immunogenicity is one of the important properties of an antigen. Whether an antigen can successfully induce the production of an immune response in a host depends on several factors, such as the characteristics of the antigen, the reactivity of the host, and the immunization means.

[0055] As used herein, the term "transfection" or "transfecting" refers to a process by which nucleic acid is introduced into eukaryotic cells, particularly mammalian cells. Transfection protocols and techniques include, but are not limited to, lipid transfection, and chemical and physical methods such as electroporation. Many transfection methods are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0056] As used herein, the term "SPR" or "surface plasmon resonance" refers to and includes an optical phenomenon that allows for the analysis of real-time biospecific interactions by detecting changes in protein concentration in a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further explanation, see the Examples and Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0057] The term "fluorescence activated cell sorting" or "FACS" as used herein refers to a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescence properties of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting". Retrieved 2017-11-09.). Instruments for performing FACS are well known to those skilled in the art and are generally commercially available. Examples of such instruments include the FACS Star Plus, FACScan, and FACSort instruments sold by Becton Dickinson (Foster City, Calif.), Epics C sold by Coulter Epics Division (Hialeah, Fla.), and MoFlo sold by Cytomation (Colorado Springs, Colo.).

[0058] The term "subject" includes any human or non-human animal, preferably a human.

[0059] As used herein, the term "D3-associated condition" or "D3-related condition" refers to any condition that is caused by, exacerbated by, or otherwise correlates with an increase or decrease (generally an increase) in expression or activity of D3 (e.g., human D3).

[0060] As used herein, the term "cancer" refers to any tumor or any malignant cell growth or proliferation, whether primary or metastasis mediated, including solid tumors and non-solid tumors such as leukemia.

[0061] As used herein in connection with the treatment of a condition, the terms "treatment," "treat," or "treated" generally refer to a treatment or therapy, whether human or animal, in which some desired therapeutic effect is achieved, such as, for example, inhibition of progression of a condition, including slowing the rate of progression, stopping the rate of progression, regressing the condition, improving the condition, and curing the condition. Preventative treatment (e.g., prevention, prevention) is also included. In the case of cancer, "treat" can refer to attenuating or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or a combination thereof. In the case of tumors, "treatment" includes removal of all or part of the tumor, inhibition or delay of tumor growth and metastasis, prevention or delay of tumor development, or a specific combination thereof.

[0062] As used herein, the term "therapeutically effective amount" refers to the amount of active compound, or material, composition or dosage form containing active compound, that is effective in producing some desired therapeutic effect commensurate with a reasonable benefit / risk ratio when administered according to a desired treatment regimen.For example, the "therapeutically effective amount" of a D3 binding molecule refers to an amount or concentration that is effective in treating a D3-related disease or condition in humans.

[0063] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced.

[0064] As used herein, the term "pharmaceutical acceptable" means that the vehicle, diluent, excipient and / or salts thereof are chemically and / or physically compatible with the other ingredients in the formulation and physiologically compatible with the recipient.

[0065] As used herein, the term "pharmaceutical acceptable carrier and / or excipient" refers to a carrier, stabilizer, and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, as is well known in the art (e.g., Remington's Pharmaceutical Sciences.Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including, but not limited to, pH adjusters, surfactants, adjuvants, or ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers, surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween®-80, and ionic strength enhancers include, but are not limited to, sodium chloride. The carrier, excipient, or stabilizer is non-toxic to cells or mammals exposed thereto at the dosages and concentrations used. In many cases, the carrier is a pH-buffered aqueous solution. Examples of carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The term "carrier" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, or vehicle with which the therapeutic is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, as needed. The composition may have the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, and the like. Oral compositions, including formulations, can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable carriers are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa. A composition containing a pharmaceutical compound can include, for example, a prophylactically or therapeutically effective amount of a D3 binding agent (e.g., an anti-D3 antibody) in isolated or purified form, together with a suitable amount of carrier to provide the form for proper administration to a subject (e.g., a patient). The formulation should be suitable for the mode of administration.

[0066] As used herein, the term "adjuvant" refers to a non-specific immune enhancing agent that can enhance the immune response to an antigen or change the type of immune response in an organism when administered to an organism together with or prior to an antigen. There are various adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0067] D3 binding molecule In some embodiments, the present disclosure provides a D3 binding molecule. In the broad sense, a D3 binding molecule may include any molecule that specifically binds to D3. In some contexts, a "D3 binding molecule" may include a "D3 antagonist" and an "anti-D3 antibody." A "D3 antagonist" refers to any compound or biomolecule that blocks D3 activity. An "anti-D3 antibody" includes, but is not limited to, a chimeric antibody, a humanized antibody, a human antibody, or a single domain antibody. A D3 binding molecule is not limited to a polypeptide or a protein, but may include other moieties such as nucleotides, hybrids, glucans, and combinations thereof. As exemplified herein, a D3 binding molecule may be an anti-D3 antibody or an anti-D3 fusion protein.

[0068] In some embodiments, the D3 binding molecule disclosed herein comprises at least one VHH that specifically binds to D3. Furthermore, the D3 binding molecule may be a single domain antibody that comprises one VHH. For example, a single domain antibody can selectively bind to a specific antigen (e.g., D3). In some embodiments, the D3 binding molecule comprises a VHH fused to an immunoglobulin Fc region, for example, an Fc region of an IgG (e.g., IgG4 or IgG1). In some embodiments, the Fc region is an Fc region of a human IgG1. Fusing a VHH to an Fc region may allow for more efficient recruitment of effector functions. Also, fusing a VHH to an Fc region is believed to aid in the formation of a dimer of the D3 binding molecule, which may help extend the half-life of the D3 binding molecule in vivo.

[0069] As is well known in the art, VHH molecules derived from camelid antibodies have one of the smallest intact antigen-binding domains known (approximately 15 kDa, i.e. 10 times smaller than conventional IgG) and are therefore well suited for delivery to dense tissues and for accessing the limited space between macromolecules.

[0070] The VHHs disclosed herein can be produced by the skilled artisan according to methods well known in the art or any future methods, for example, VHHs can be obtained, for example, by immunizing camels and obtaining hybridomas therefrom, or by cloning a library of VHHs using molecular biology techniques well known in the art, followed by selection using phage display.

[0071] For example, VHHs can be obtained by immunization of a llama or alpaca with the desired antigen and subsequent isolation of mRNA encoding the heavy chain antibody. Genetic libraries of single domain antibodies containing millions of clones are generated by reverse transcription and polymerase chain reaction. Screening techniques such as phage display and ribosome display are useful in identifying clones that bind to the antigen. One technique is phage display, where a library of (e.g., human) antibodies is synthesized on phage, the library is screened with the desired antigen or an antibody-binding portion thereof, and phages that bind to the antigen are isolated and immunoreactive fragments can be obtained therefrom. Methods for preparing and screening such libraries are well known in the art, and kits for generating phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, Cat. No. 27-9400-01, and Stratagene SurfZAP™ Phage Display Kit, Cat. No. 240612). There are other methods and reagents available for generating and screening antibody display libraries (see, for example, Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991)).

[0072] When promising clones are identified, their DNA sequences are optimized, for example, by affinity maturation or humanization, which makes it possible to prevent human immunological responses to the antibody.

[0073] Thus, VHHs can be produced by: (1) isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization" of a naturally occurring VHH domain (described below) or by expression of nucleic acids encoding such a humanized VHH domain; (4) by "camelization" of a naturally occurring VH domain derived from any animal species, particularly a mammalian species such as human, or by expression of nucleic acids encoding such a camelized VH domain; (5) by "camelization" of "domain antibodies" or "Dabs" as described by Ward et al., supra. " or by expression of a nucleic acid encoding such a camelized VH domain, (6) using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences, (7) by preparing a nucleic acid encoding a VHH using nucleic acid synthesis techniques and then expressing the nucleic acid so obtained, (8) by subjecting the heavy chain antibody or VHH to affinity maturation, mutagenesis (e.g. random or site-directed mutagenesis) and / or any other technique for increasing the affinity and / or specificity of the VHH, and / or (9) by any combination of the above. Suitable methods and techniques for doing the above will be apparent to the skilled artisan based on the disclosure herein and include, for example, the methods and techniques described in more detail herein.

[0074] Single domain antibodies are usually generated by PCR cloning repertoires of variable domains from blood, lymph node, or spleen cDNA obtained from immunized animals into phage display vectors. Antigen-specific single domain antibodies are generally selected by panning phage libraries on immobilized antigen (e.g., antigen coated on the plastic surface of a test tube, biotinylated antigen immobilized on streptavidin beads, or membrane proteins expressed on cell surfaces). The affinity of sdAbs can be increased by mimicking this strategy in vitro, for example, by site-directed mutagenesis of the CDR regions and performing additional rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentration) (Wesolowski et al., Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol (2009) 198:157-174).

[0075] Methods for preparing VHHs that specifically bind to antigens or epitopes are described in references such as, for example, R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645, 17 June, 2009, and WO94 / 04678.

[0076] In some embodiments, the VHH may be truncated at the N-terminus or C-terminus to comprise only partial FRW1 and / or FRW4, or to lack one or both of these framework regions, so long as the VHH substantially maintains its antigen binding and specificity (e.g., substantially maintains, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

[0077] The present disclosure also provides D3 binding molecules with masking and / or cleavable moieties, in which one or more of the D3 binding domains of the D3 binding molecule are masked (e.g., by a masking moiety) and / or activatable (e.g., by a cleavable moiety). Techniques for masking D3 binding molecules (e.g., antibodies) are well known in the art and include SAFE body masking technology (see, e.g., US2019 / 0241886) and Probody masking technology (see, e.g., US2015 / 0079088). Such techniques can be used to generate masked and / or activatable D3 binding molecules (e.g., antibodies). Such masked and / or activatable D3 binding molecules (e.g., antibodies) are useful for preparing conjugates, including immunoconjugates, antibody drug conjugates (ADCs), masked ADCs, and activatable antibody drug conjugates (AADCs), that include any one of the D3 binding molecules (e.g., antibodies) of the present disclosure, including those that are directly or indirectly conjugated to another substance, such as a drug. For example, the D3 binding molecules of the present disclosure can be covalently attached to one or more agents, such as drugs, by synthetic linkers.

[0078] Optionally, the D3 binding molecule is linked or conjugated (directly or indirectly) to a moiety having an effector function, such as cytotoxic activity (e.g., a chemotherapeutic moiety or a radioisotope) or immune recruitment activity. The moiety to be linked or conjugated (directly or indirectly) includes a cytotoxic drug (e.g., a toxin such as auristatin) or a non-cytotoxic drug (e.g., a signal transduction regulator such as a kinase, or a masking moiety that masks one or more binding domains of the D3 binding molecule, or a cleavable moiety that allows the D3 binding molecule to be activated by unmasking one or more binding domains of the D3 binding molecule in the tumor microenvironment in the form of a masked complex by cleaving the cleavable moiety). Moieties that promote immune recruitment may include other antigen binding agents, such as viral proteins that selectively bind to cells of the innate immune system. Alternatively, or in addition, the D3 binding molecule is optionally linked or conjugated (directly or indirectly) to a moiety that facilitates isolation from a mixture (e.g., a tag) or a moiety that has reporter activity (e.g., a detection label or reporter protein). It will be appreciated that the features of D3 binding molecules described herein extend to polypeptides that include D3 binding molecule fragments.

[0079] In some embodiments, the D3 binding molecules described herein may be linked or conjugated (directly or indirectly) to a polypeptide, which may result in the generation of an activatable antibody. In some embodiments, the D3 binding molecules are linked or conjugated (directly or indirectly) to a substance. In some embodiments, the substance is a drug, which provides an ADC or AADC when the antibody of the ADC contains a masking moiety and a cleavable moiety.

[0080] In some embodiments, the D3 binding molecules described herein are conjugated or recombinantly linked (directly or indirectly) to a therapeutic agent (e.g., a cytotoxic agent) or a diagnostic or detectable agent. Conjugated or recombinantly linked antibodies, including masked or activatable conjugates, may be useful, for example, in the treatment or prevention of a disease, disorder or condition, such as cancer or tumor.

[0081] Diagnosis and detection can be accomplished, for example, by combining the D3 binding molecule with a detectable substance, such as an enzyme, including, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; a prosthetic group, including, but not limited to, streptavidin / biotin or avidin / biotin; a fluorescent substance, including, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; a luminescent substance, including, but not limited to, luminol; a bioluminescent substance, including, but not limited to, luciferase, luciferin, or aequorin; or a chemiluminescent material, including, but not limited to, acridinium-based compounds or HALOTAG. iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga and 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140L Positron emission tomography (PET) imaging can be performed by combining positron-emitting metals with a variety of positron emission tomography techniques; and non-radioactive paramagnetic metal ions; radioactive materials including, but not limited to, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, or 117Sn; positron-emitting metals using various positron emission tomography techniques; and non-radioactive paramagnetic metal ions.

[0082] Conjugates of antibodies and agents (including when the agent is a drug for the preparation of an ADC or AADC) can be prepared using a variety of bifunctional protein coupling agents, such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate). The present disclosure further contemplates that conjugates of antibodies and agents (including when the agent is a drug for the preparation of an ADC or AADC) can be prepared using any suitable method disclosed in the art (e.g., Bioconjugate Techniques (Hermanson ed., 2d ed. 2008)).

[0083] Traditional conjugation strategies between antibodies and substances (including when the substance is a drug for preparing an ADC or AADC) are based on random conjugation chemistry involving the ε-amino group of Lys residues or the thiol group of Cys residues, resulting in heterogeneous conjugates. Recently developed techniques allow site-specific conjugation to antibodies, resulting in uniform loading and avoiding subpopulations of conjugates with altered antigen binding or pharmacokinetics. These techniques include engineering "thiomab" containing cysteine ​​substitutions at positions on the heavy and light chains that provide reactive thiol groups and do not disrupt immunoglobulin folding and assembly or alter antigen binding (see, e.g., Junutula et al., 2008, J. Immunol.Meth. 332: 41-52; and Junutula et al., 2008, Nature Biotechnol.26:925-32). Alternatively, selenocysteine ​​can be cotranslationally inserted into the antibody sequence by recoding the stop codon UGA from the stop to the insertion of selenocysteine, allowing site-specific covalent attachment at the nucleophilic selenol group of selenocysteine ​​in the presence of other natural amino acids (e.g., Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105:12451-56; and Hofer et al., 2009, Biochemistry 48(50):12047-57).

[0084] The D3 binding molecules described herein may be monospecific, bispecific, trispecific, or of higher order multispecificity. Such substances may include antibodies. Multispecific antibodies, such as bispecific antibodies, are monoclonal antibodies that have binding specificities for at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., bispecific antibodies to D3 with a first binding domain to a first epitope of D3 and a second binding domain to a second epitope of D3). In some embodiments, multispecific (e.g., bispecific) antibodies can be constructed based on the sequences of the antibodies described herein. In some embodiments, the multispecific antibodies described herein are bispecific antibodies. In some embodiments, the bispecific antibodies are murine, chimeric, human, or humanized antibodies. In some embodiments, one of the binding specificities of the bispecific antibody is for D3 and the other is for any other target (e.g., antigen). In some embodiments, a multispecific (e.g., bispecific) antibody can comprise multiple target (e.g., antigen) binding domains, where different binding domains are specific for different targets (e.g., a first binding domain that binds to D3 and a second binding domain that binds to another target (e.g., antigen), such as an immune checkpoint regulator (e.g., a negative checkpoint regulator). In some embodiments, a multispecific (e.g., bispecific) antibody molecule can bind to multiple (e.g., two or more) epitopes on the same target (e.g., antigen).In some embodiments, one of the binding specificities is for D3 and the other is for a cytotoxic T-lymphocyte antigen-4 (CTLA-4), CD80, CD86, programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), lymphocyte activation gene 3 (LAG-3, also known as CD223), galectin 3, B- and T-lymphocyte attenuator (BTLA), T-cell membrane protein 3 (TIM3), galectin 9 (GAL9), B7-H1, B7-H3, B7-H4, Ig and against one or more of T cell immunoreceptor with ITIM domain (TIGIT / Vstm3 / WUCAM / VSIG9), V domain Ig suppressor of T cell activation (VISTA), glucocorticoid-induced tumor necrosis factor receptor-related (GITR) protein, herpes virus entry mediator (HVEM), OX40, CD27, CD28, CD137, CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052, and CGEN-15092.

[0085] Methods for producing multispecific antibodies are well known in the art, for example, by co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (see, for example, Milstein and Cuello, 1983, Nature 305:537-40). For further details on the production of multispecific antibodies (e.g., bispecific antibodies), see, for example, Bispecific Antibodies (Kontermann ed., 2011).

[0086] The present disclosure provides a humanized antibody that binds to D3. There are various methods known in the art for humanizing non-human antibodies. For example, a humanized antibody may have one or more amino acid residues that are introduced from a non-human source. These non-human amino acid residues are often called "import" residues, and are usually derived from "import" variable domains. Humanized antibodies that bind to D3 can be made using techniques well known to those skilled in the art (e.g., Zhang et al., Molecular Immunology, 42(12): 1445-1451, 2005; Hwang et al., Methods, 36(1): 35-42, 2005; Dall'Acqua et al., Methods, 36(1): 43-60, 2005; Clark, Immunology Today, 21(8): 397-402, 2000, and U.S. Patent Nos. 6,180,370, 6,054,927, 5,869,619, 5,861,155, 5,712,120, and 4,816,567).

[0087] D3 binding molecules may be referred to as anti-D3 antibodies in the following sections.

[0088] Anti-D3 antibodies with functional properties For example, the antibodies of the present disclosure, including antibodies comprising at least one VHH domain, are characterized by specific functional features or properties of the antibody. In some embodiments, the RLR agonist has the following properties: (a) binds to human D3, cyno D3, and mouse D3 with nM-grade EC50s as measured by ELISA or FACS; (b) Dose-dependent internalization in human cells engineered to express D3; (c) It binds to the ECD of human D3 with a KD of 0.1 nM or less as measured by SPR.

[0089] The antibodies of the present disclosure bind to cell surface D3 with high affinity. The binding of the antibodies of the present disclosure to D3 can be assessed using one or more techniques well established in the art, such as ELISA. The binding specificity of the antibodies of the present disclosure can also be determined by monitoring the binding of the antibodies to cells expressing D3 protein, for example, by flow cytometry. For example, the antibodies can be tested by flow cytometry assays (e.g., FACS) in which the antibodies are reacted with cell lines expressing human D3, such as CHO cells and 293 cells transfected to express D3 on the cell surface. Additionally or alternatively, the binding of the antibodies, including binding kinetics (e.g., Kd value), can be tested in BIAcore binding assays. Still other suitable binding assays include, for example, ELISA assays using recombinant D3 protein. For example, the antibodies of the present disclosure bind to cell surface D3 (e.g., ECD of human D3) protein at 1×10 -7 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1×10 -10 K below M D Combine with.

[0090] In some embodiments, an antibody of the disclosure has an EC50 of about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM or less for cynomolgus monkey or mouse D3 as measured by FACS. 50 Combine with.

[0091] Anti-D3 antibodies containing VHH CDRs In some embodiments, the anti-D3 antibodies disclosed herein comprise at least one immunoglobulin single variable domain (e.g., VHH), wherein the VHH comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, 4, 7, or 10, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, or 11, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3, 6, or 9. In some embodiments, the CDR numbering is according to a combination of Kabat and AbM numbering.

[0092] The extent of the framework regions and each CDR can be precisely identified using methods well known in the art, such as, for example, the Kabat definition, the Chothia definition, the AbM definition, the Contact definition, the IMGT definition (all of which are well known in the art), and any combination thereof. See, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; Edelman et al., Proc Natl Acad Sci US A. 1969 May, 63(1):78-85; and Martin and Allen, in "Handbook of Therapeutic Antibodies", chapter 5, 2007. See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. Correspondence or alignment between numbering according to different definitions can be found, for example, at www.imgt.org / (see also Giudicelli V et al. IMGT (International ImMunoGeneTics Database) Nucleic Acids Res. (1997) 25:206-11; and Lefranc MP et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol. (2003) 27:55-77).

[0093] As will be appreciated by those skilled in the art, the exact numbering and arrangement of the complementarity determining regions (CDRs) may vary depending on the numbering system. However, it should be understood that the disclosure of the variable heavy chain, variable light chain, and / or VHH sequence includes the disclosure of the relevant (unique) CDRs. Thus, the disclosure of each variable region is the disclosure of each CDR (e.g., CDR1, CDR2, and CDR3). Two antibodies with the same VH, VL, or VHH CDRs mean that their CDRs are the same as determined by the same approach (e.g., Kabat, AbM, Chothia, Contact, and IMGT numbering approaches well known in the art).

[0094] Variable regions and CDRs in an antibody sequence can be identified according to general rules developed in the art (e.g., Kabat, AbM, Chothia, Contact, and IMGT numbering systems) or by aligning the sequence against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described and accessible at the "Abysis" website (www.bioinf.org.uk / abs) (maintained by A. C. Martin in the Department of Biochemistry & Molecular Biology University College London, London, England) and the VBASE2 website (www.vbase2.org), as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). Preferably, the sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Data Bank (PDB), and structure data from the PDB. See Dr. Andrew C. Martin's book chapter Protein Sequence and Structure Analysis of Antibody Variable Domains. Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13:978-3540413547, also available at the website bioinforg.uk / abs).The Abysis database website further includes general rules that have been developed to identify CDRs that can be used in accordance with the teachings herein. Figure 10 shows an alignment of exemplary immunoglobulin single variable domains, with the boundaries of each CDR indicated by the Kabat, AbM, Chothia, Contact, and IMGT numbering.

[0095] In some embodiments, the D3 binding molecules disclosed herein comprise at least one immunoglobulin single variable domain (e.g., a VHH), wherein the VHH comprises FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4, where CDR1 has the amino acid sequence set forth in SEQ ID NO: 1, 4, 7 or 10, CDR2 has the amino acid sequence set forth in SEQ ID NO: 2, 5, 8 or 11, and CDR3 has the amino acid sequence set forth in SEQ ID NO: 3, 6 or 9. In some embodiments, the FRW1 and FRW4 at the N-terminus and C-terminus of the VHH comprised in the D3 binding molecule may be truncated to comprise only partial FRW1 and / or FRW4, or to lack one or both of these framework regions, so long as the VHH substantially maintains antigen binding and specificity.

[0096] In some embodiments, provided herein are anti-D3 antibodies (such as anti-D3 single domain antibodies) that comprise one, two, or all three CDRs of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, provided are anti-D3 antibodies (such as anti-D3 single domain antibodies) that comprise one, two, or all three CDRs of the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, provided are anti-D3 antibodies (such as anti-D3 single domain antibodies) that comprise one, two, or all three CDRs of the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, provided are anti-D3 antibodies (such as anti-D3 single domain antibodies) that comprise one, two, or all three CDRs of the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, provided are anti-D3 antibodies (such as anti-D3 single domain antibodies) that comprise one, two, or all three CDRs of the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, provided are anti-D3 antibodies (such as anti-D3 single domain antibodies) that comprise one, two, or all three CDRs of the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, an anti-D3 antibody (such as an anti-D3 single domain antibody) is provided that comprises one, two, or all three CDRs of the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, an anti-D3 antibody (such as an anti-D3 single domain antibody) is provided that comprises one, two, or all three CDRs of the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the anti-D3 single domain antibody is camelid. In some embodiments, the anti-D3 antibody (such as an anti-D3 single domain antibody) is humanized. In some embodiments, the anti-D3 antibody (such as an anti-D3 single domain antibody) comprises an acceptor human framework, for example, a human immunoglobulin framework or a human consensus framework.

[0097] In some embodiments, an anti-D3 antibody (such as a single domain antibody) comprises a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 12. In some embodiments, an anti-D3 antibody (such as a single domain antibody) comprises a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 12. In some embodiments, an anti-D3 antibody (such as a single domain antibody) comprises a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 12. In some embodiments, an anti-D3 antibody (such as a single domain antibody) comprises a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 set forth in SEQ ID NO: 12. In some embodiments, an anti-D3 antibody (such as a single domain antibody) comprises a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 set forth in SEQ ID NO: 12. In some embodiments, an anti-D3 antibody (such as a single domain antibody) comprises a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 set forth in SEQ ID NO: 12. In some embodiments, the anti-D3 single domain antibody (such as a single domain antibody) has CDR1, CDR2, and CDR3 with the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 12. The CDR sequences can be determined by well-known numbering systems. In some embodiments, the CDRs are according to the IMGT numbering. In some embodiments, the CDRs are according to the Kabat numbering. In other embodiments, the CDRs are according to the Chothia numbering. In other embodiments, the CDRs are according to the Contact numbering. In some embodiments, the CDRs are according to the AbM numbering. In some embodiments, the anti-D3 single domain antibody is a camelid. In some embodiments, the anti-D3 antibody (such as an anti-D3 single domain antibody) is humanized. In some embodiments, the anti-D3 antibody (such as an anti-D3 single domain antibody) comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0098] In some embodiments, the single domain antibody has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 13. In some embodiments, the single domain antibody has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 13. In other embodiments, the single domain antibody has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 13. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having the amino acid sequence of CDR1 and CDR2 set forth in SEQ ID NO: 13. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having the amino acid sequence of CDR1 and CDR3 set forth in SEQ ID NO: 13. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having the amino acid sequence of CDR2 and CDR3 set forth in SEQ ID NO: 13. In some embodiments, the single domain antibody has a CDR1, a CDR2, and a CDR3 having the amino acid sequence of CDR1, a CDR2, and a CDR3 set forth in SEQ ID NO: 13. The CDR sequences can be determined by well-known numbering systems. In some embodiments, the CDRs follow the IMGT numbering. In some embodiments, the CDRs follow Kabat numbering. In other embodiments, the CDRs follow Chothia numbering. In other embodiments, the CDRs follow Contact numbering. In some embodiments, the CDRs follow AbM numbering. In some embodiments, the anti-D3 single domain antibody is camelid. In some embodiments, the anti-D3 single domain antibody is humanized. In some embodiments, the anti-D3 single domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0099] In some embodiments, the single domain antibody has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 14. In some embodiments, the single domain antibody has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 14. In other embodiments, the single domain antibody has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 14. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having the amino acid sequence of CDR1 and CDR2 set forth in SEQ ID NO: 14. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having the amino acid sequence of CDR1 and CDR3 set forth in SEQ ID NO: 14. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having the amino acid sequence of CDR2 and CDR3 set forth in SEQ ID NO: 14. In some embodiments, the single domain antibody has a CDR1, a CDR2, and a CDR3 having the amino acid sequence of CDR1, a CDR2, and a CDR3 set forth in SEQ ID NO: 14. The CDR sequences can be determined by well-known numbering systems. In some embodiments, the CDRs follow the IMGT numbering. In some embodiments, the CDRs follow Kabat numbering. In other embodiments, the CDRs follow Chothia numbering. In other embodiments, the CDRs follow Contact numbering. In some embodiments, the CDRs follow AbM numbering. In some embodiments, the anti-D3 single domain antibody is camelid. In some embodiments, the anti-D3 single domain antibody is humanized. In some embodiments, the anti-D3 single domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0100] In some embodiments, the single domain antibody has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 15. In some embodiments, the single domain antibody has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 15. In other embodiments, the single domain antibody has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 15. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having the amino acid sequence of CDR1 and CDR2 set forth in SEQ ID NO: 15. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having the amino acid sequence of CDR1 and CDR3 set forth in SEQ ID NO: 15. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having the amino acid sequence of CDR2 and CDR3 set forth in SEQ ID NO: 15. In some embodiments, the single domain antibody has a CDR1, a CDR2, and a CDR3 having the amino acid sequence of CDR1, a CDR2, and a CDR3 set forth in SEQ ID NO: 15. The CDR sequences can be determined by well-known numbering systems. In some embodiments, the CDRs are according to the IMGT numbering. In some embodiments, the CDRs follow Kabat numbering. In other embodiments, the CDRs follow Chothia numbering. In other embodiments, the CDRs follow Contact numbering. In some embodiments, the CDRs follow AbM numbering. In some embodiments, the anti-D3 single domain antibody is camelid. In some embodiments, the anti-D3 single domain antibody is humanized. In some embodiments, the anti-D3 single domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0101] In some embodiments, the single domain antibody has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 16. In some embodiments, the single domain antibody has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 16. In other embodiments, the single domain antibody has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 16. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having the amino acid sequence of CDR1 and CDR2 set forth in SEQ ID NO: 16. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having the amino acid sequence of CDR1 and CDR3 set forth in SEQ ID NO: 16. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having the amino acid sequence of CDR2 and CDR3 set forth in SEQ ID NO: 16. In some embodiments, the single domain antibody has a CDR1, a CDR2, and a CDR3 having the amino acid sequence of CDR1, a CDR2, and a CDR3 set forth in SEQ ID NO: 16. The CDR sequences can be determined by well-known numbering systems. In some embodiments, the CDRs are according to the IMGT numbering. In some embodiments, the CDRs follow Kabat numbering. In other embodiments, the CDRs follow Chothia numbering. In other embodiments, the CDRs follow Contact numbering. In some embodiments, the CDRs follow AbM numbering. In some embodiments, the anti-D3 single domain antibody is camelid. In some embodiments, the anti-D3 single domain antibody is humanized. In some embodiments, the anti-D3 single domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0102] In some embodiments, the single domain antibody has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 17. In some embodiments, the single domain antibody has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 17. In other embodiments, the single domain antibody has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 17. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having the amino acid sequence of CDR1 and CDR2 set forth in SEQ ID NO: 17. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having the amino acid sequence of CDR1 and CDR3 set forth in SEQ ID NO: 17. In some embodiments, the single domain antibody has a CDR1, a CDR2, and a CDR3 having the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 17. In some embodiments, the single domain antibody has a CDR1, a CDR2, and a CDR3 having the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 17. The CDR sequences can be determined by well-known numbering systems. In some embodiments, the CDRs are according to the IMGT numbering. In some embodiments, the CDRs follow Kabat numbering. In other embodiments, the CDRs follow Chothia numbering. In other embodiments, the CDRs follow Contact numbering. In some embodiments, the CDRs follow AbM numbering. In some embodiments, the anti-D3 single domain antibody is camelid. In some embodiments, the anti-D3 single domain antibody is humanized. In some embodiments, the anti-D3 single domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0103] In some embodiments, the single domain antibody has a CDR1 having the amino acid sequence of CDR1 set forth in SEQ ID NO: 18. In some embodiments, the single domain antibody has a CDR2 having the amino acid sequence of CDR2 set forth in SEQ ID NO: 18. In other embodiments, the single domain antibody has a CDR3 having the amino acid sequence of CDR3 set forth in SEQ ID NO: 18. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having the amino acid sequence of CDR1 and CDR2 set forth in SEQ ID NO: 18. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having the amino acid sequence of CDR1 and CDR3 set forth in SEQ ID NO: 18. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having the amino acid sequence of CDR2 and CDR3 set forth in SEQ ID NO: 18. In some embodiments, the single domain antibody has a CDR1, a CDR2, and a CDR3 having the amino acid sequence of CDR1, a CDR2, and a CDR3 set forth in SEQ ID NO: 18. The CDR sequences can be determined by well-known numbering systems. In some embodiments, the CDRs are according to the IMGT numbering. In some embodiments, the CDRs follow Kabat numbering. In other embodiments, the CDRs follow Chothia numbering. In other embodiments, the CDRs follow Contact numbering. In some embodiments, the CDRs follow AbM numbering. In some embodiments, the anti-D3 single domain antibody is camelid. In some embodiments, the anti-D3 single domain antibody is humanized. In some embodiments, the anti-D3 single domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0104] In some embodiments, provided herein is a single domain antibody that binds to D3, comprising the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which (i) CDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:53, or SEQ ID NO:54, and (ii) CDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:55, or SEQ ID NO:56. , SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:56, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:47, SEQ ID NO:50, or SEQ ID NO:52, and / or (iii) CDR3 comprises the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:48, or SEQ ID NO:51. In some embodiments, the anti-D3 single domain antibody is Camelidae. In some embodiments, the anti-D3 single domain antibody is humanized. In some embodiments, the anti-D3 single domain antibody comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

[0105] In some embodiments, CDR1 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 1, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 2, and CDR3 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 20 according to the IMGT numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 21 according to the IMGT numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22 according to the IMGT numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 23 according to the Kabat numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 2 according to the Kabat numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 according to the Kabat numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 24 according to the Chothia numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 25 according to the Chothia numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 26 according to the Chothia numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:27 according to the Contact numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO:28 or 56 according to the Contact numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:29 according to the Contact numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:1 according to the AbM numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO:30 according to the AbM numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:3 according to the AbM numbering. In some embodiments, the anti-D3 single domain antibody is a camelid. In some embodiments, the anti-D3 single domain antibody is humanized. In some embodiments, the anti-D3 single domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0106] In some embodiments, CDR1 comprises the exemplary amino acid sequence set forth in SEQ ID NO:4, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO:5, and CDR3 comprises the exemplary amino acid sequence set forth in SEQ ID NO:6. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:31 according to the IMGT numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO:32 according to the IMGT numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:33 according to the IMGT numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:34 according to the Kabat numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO:5 according to the Kabat numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:6 according to the Kabat numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:35 according to the Chothia numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO:36 according to the Chothia numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:37 according to the Chothia numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 38 according to the Contact numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 39 according to the Contact numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40 according to the Contact numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 4 according to the AbM numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 41 according to the AbM numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6 according to the AbM numbering. In some embodiments, the anti-D3 single domain antibody is camelid. In some embodiments, the anti-D3 single domain antibody is humanized. In some embodiments, the anti-D3 single domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0107] In some embodiments, CDR1 comprises the exemplary amino acid sequence set forth in SEQ ID NO:7, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO:8, and CDR3 comprises the exemplary amino acid sequence set forth in SEQ ID NO:9. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:42 according to the IMGT numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO:43 according to the IMGT numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:44 according to the IMGT numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:45 according to the Kabat numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO:8 according to the Kabat numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:9 according to the Kabat numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:46 according to the Chothia numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO:47 according to the Chothia numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO:48 according to the Chothia numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 49 according to the Contact numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 50 according to the Contact numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 51 according to the Contact numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 7 according to the AbM numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 52 according to the AbM numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 9 according to the AbM numbering. In some embodiments, the anti-D3 single domain antibody is camelid. In some embodiments, the anti-D3 single domain antibody is humanized. In some embodiments, the anti-D3 single domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0108] In some embodiments, CDR1 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 10, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 11, and CDR3 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 53 according to the IMGT numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 32 according to the IMGT numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 33 according to the IMGT numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 34 according to the Kabat numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 11 according to the Kabat numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6 according to the Kabat numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 54 according to the Chothia numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 36 according to the Chothia numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 37 according to the Chothia numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 38 according to the Contact numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 39 according to the Contact numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40 according to the Contact numbering. In some embodiments, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 10 according to the AbM numbering, CDR2 comprises the exemplary amino acid sequence set forth in SEQ ID NO: 41 according to the AbM numbering, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6 according to the AbM numbering. In some embodiments, the anti-D3 single domain antibody is camelid. In some embodiments, the anti-D3 single domain antibody is humanized. In some embodiments, the anti-D3 single domain antibody comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0109] In some embodiments, the single domain antibody further comprises one or more framework regions of WT1156-P3R2-1C2, WT1156-P3R2-1C9, WT1156-P8R2-1H1, WT1156-P3R2-1H6, WT1156-P3R2-1C2-z102, WT1156-P3R2-1C2-z109, and / or WT1156-P3R2-1H6-z100. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence set forth in SEQ ID NO: 12. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence set forth in SEQ ID NO: 13. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence set forth in SEQ ID NO: 14. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence set forth in SEQ ID NO: 15. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence set forth in SEQ ID NO: 16. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence set forth in SEQ ID NO: 17. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence set forth in SEQ ID NO: 18. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence set forth in SEQ ID NO: 55.

[0110] In some embodiments, the single domain antibodies provided herein are humanized single domain antibodies.

[0111] Framework region as described herein is determined based on the boundaries of CDR numbering system.In other words, when CDR is determined by IMGT, Kabat, Cothia, Contact or AbM, framework region is the amino acid residue that surrounds the CDR in variable region from N-terminus to C-terminus in the format of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residues N-terminal to the amino acid residues of CDR1 as defined, for example, by the IMGT numbering system, the Kabat numbering system, the Chotia numbering system, the Contact numbering system, or the AbM numbering system; FR2 is defined as the amino acid residues between the amino acid residues of CDR1 and CDR2 as defined, for example, by the IMGT numbering system, the Kabat numbering system, the Chotia numbering system, the Contact numbering system, or the AbM numbering system; FR3 is defined as the amino acid residues between the amino acid residues of CDR2 and CDR3 as defined, for example, by the IMGT numbering system, the Kabat numbering system, the Chotia numbering system, the Contact numbering system, or the AbM numbering system; and FR4 is defined as the amino acid residues C-terminal to the amino acid residues of CDR3 as defined, for example, by the IMGT numbering system, the Kabat numbering system, the Chotia numbering system, the Contact numbering system, or the AbM numbering system.

[0112] In some embodiments, an isolated anti-D3 single domain antibody is provided, comprising a VHH domain having an amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, a polypeptide is provided, comprising an amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, an isolated anti-D3 single domain antibody is provided, comprising a VHH domain having an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, a polypeptide is provided, comprising an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, an isolated anti-D3 single domain antibody is provided, comprising a VHH domain having an amino acid sequence as set forth in SEQ ID NO: 14. In some embodiments, a polypeptide is provided, comprising an amino acid sequence as set forth in SEQ ID NO: 14. In some embodiments, an isolated anti-D3 single domain antibody is provided, comprising a VHH domain having an amino acid sequence as set forth in SEQ ID NO: 15. In some embodiments, a polypeptide is provided, comprising an amino acid sequence as set forth in SEQ ID NO: 15. In some embodiments, an isolated anti-D3 single domain antibody is provided, comprising a VHH domain having an amino acid sequence as set forth in SEQ ID NO: 16. In some embodiments, a polypeptide is provided, comprising an amino acid sequence as set forth in SEQ ID NO: 16. In some embodiments, an isolated anti-D3 single domain antibody is provided, comprising a VHH domain having an amino acid sequence as set forth in SEQ ID NO: 17. In some embodiments, a polypeptide is provided, comprising an amino acid sequence as set forth in SEQ ID NO: 17. In some embodiments, an isolated anti-D3 single domain antibody is provided, comprising a VHH domain having the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a polypeptide is provided, comprising the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, an isolated anti-D3 single domain antibody is provided, comprising a VHH domain having the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, a polypeptide is provided, comprising the amino acid sequence set forth in SEQ ID NO: 55.

[0113] Anti-D3 antibody containing VHH sequence In some embodiments, the anti-D3 antibody comprises at least one immunoglobulin single variable domain (e.g., VHH), wherein the VHH is (A) an amino acid sequence set forth in any one of SEQ ID NOs: 12 to 18 and 55; (B) an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 12-18 and 55; or (C) Comprises or consists of an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid additions, deletions and / or substitutions compared to any one of SEQ ID NOs: 12 to 18 and 55.

[0114] Percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as implemented in the ALIGN program (version 2.0) using a PAM120 weighted residue table, gap length penalty = 12, gap penalty = 4. Additionally, percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol 48:444-453 (1970)) as implemented in the GAP program of the GCG software package (available at http: / / www.gcg.com) using either a Blossum62 matrix or a PAM250 matrix, and gap weights = 16, 14, 12, 10, 8, 6 or 4, and length weights = 1, 2, 3, 4, 5 or 6.

[0115] Additionally or alternatively, the protein (antibody) sequences of the present disclosure can be further used as a "query sequence" to search public databases to identify, for example, related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the antibody molecules of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al, (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0116] In some embodiments, the amino acid sequence of the VHH may be at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 12-18, and 55.

[0117] In some further embodiments, the anti-D3 antibody may include conservative substitutions or modifications of amino acids in the variable and / or constant regions. It is understood in the art that certain conservative sequence modifications can be made that do not eliminate antigen binding. See, e.g., Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864- 26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O' Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0118] As mentioned above, the term "conservative substitution" as used herein refers to an amino acid substitution that does not adversely affect or change the essential properties of the protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques well known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced with another amino acid residue having a similar side chain to the corresponding amino acid residue, for example, a physically or functionally similar residue (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the corresponding amino acid residue is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997), which are incorporated by reference).

[0119] In some embodiments, the anti-D3 antibody comprises at least one VHH, and the VHH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 12 to 18. In some embodiments, the anti-D3 antibody comprises a VHH having an amino acid sequence set forth in any one of SEQ ID NOs: 12 to 18.

[0120] In some embodiments, the anti-D3 antibody is a chimeric antibody comprising a VHH fused to a human IgG1 or IgG4 Fc region, wherein the VHH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 12-18 and 55. In some embodiments, the anti-D3 antibody is a chimeric antibody comprising a VHH and a human IgG1 Fc region. Such antibodies are exemplified herein as "WT1156-P3R2-1C2-uIgG1", "WT1156-P3R2-1H6-uIgG1", "WT1156-P8R2-1H1-uIgG1", and "WT1156-P3R2-1C9-uIgG1". In some further embodiments, the anti-D3 antibody is a humanized antibody comprising a VHH and a human IgG1 Fc region. Such antibodies are exemplified herein as "WT1156-P3R2-1C2-z102-uIgG1," "WT1156-P3R2-1C2-z109-uIgG1," and "WT1156-P3R2-1H6-z100-uIgG1."

[0121] In some embodiments, at least one addition, deletion, and / or substitution of an amino acid in the VHH region is not present in any of the CDR sequences, but is present in a framework (FRW) sequence. For example, the above-mentioned antibodies or antigen-binding portions thereof may comprise one or more substitutions of amino acids within the framework sequences of the VHH region, such as FRW1, FRW2, FRW3, and / or FRW4.

[0122] In some embodiments, the antibodies or antigen-binding portions thereof provided herein comprise any suitable framework region (FRW) sequence, so long as the antigen-binding domain is capable of specifically binding to D3.

[0123] As noted above, an antibody or antigen-binding portion thereof can include one or more amino acid modifications in the heavy and / or light chain variable regions, including where the modifications are conservative substitutions. It is understood in the art that certain conservative sequence modifications can be made that do not eliminate antigen binding. See, e.g., Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864- 26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O' Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0124] In some embodiments, the antibody or antigen-binding portion thereof comprises a VHH domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 12 to 18 and 55, and an Fc region comprising the amino acid sequence set forth in SEQ ID NO: 19.

[0125] The antigen-binding domain of the D3 binding molecule is not limited to the VHH format, and may be, for example, but not limited to, Fab, Fab', F(ab') 2 The antigen-binding domain can take a variety of other forms, such as a single-chain antibody, an Fv fragment, a single-chain antibody molecule (scFv), etc. In some embodiments, the antigen-binding domain is an Fv fragment, which has the VH and VL domains in separate chains held together by tight non-covalent interactions.

[0126] Fc region containing IgG constant domains The anti-D3 antibodies and antigen-binding fragments provided herein further comprise an Fc region comprising one or more human IgG constant domains. The human IgG constant domain may be a human IgG1, IgG2, IgG3 or IgG4 constant domain, preferably a human IgG1 constant domain. An example of an amino acid sequence of an Fc region comprising a human IgG1 constant domain is set forth in SEQ ID NO: 19. In some embodiments, the Fc region is a human IgG1 Fc region, such as a wild-type Fc region or an Fc variant comprising one or more amino acid modifications (e.g., Leu234Ala / Leu235Ala or LALA) that alter antibody-dependent cellular cytotoxicity (ADCC) or other effector function.

[0127] In some embodiments, the Fc modification comprises a LALA mutation, e.g., L234A and L235A mutations, according to EU numbering as in Kabat et al. The Kabat numbering system is often used when referring to residues in the variable domain (approximately residues 1-107 in the light chain and residues 1-113 in the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU numbering as in Kabat" or "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise specified herein, designation of a residue number in the constant domain of an antibody refers to the numbering of the residues according to the EU numbering system.

[0128] Nucleic acid molecules encoding antibodies of the present disclosure In some embodiments, the present disclosure provides a nucleic acid molecule that encodes the D3 binding molecule disclosed herein, for example, comprises a nucleic acid sequence that encodes the single variable domain of the D3 binding molecule disclosed herein. The nucleic acid of the present disclosure can be obtained using standard molecular biology techniques.

[0129] A nucleic acid encoding a VHH region can be converted into a full-length heavy chain gene by operably linking the VHH-encoding nucleic acid to another nucleic acid encoding one or more heavy chain constant regions (e.g., CH1, CH2 and CH3). The sequences of human heavy chain constant region genes are well known in the art (see, e.g., Kabat et al. (1991), supra), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, e.g., an IgG1 constant region.

[0130] Once the nucleic acids encoding the VHH segments are obtained, these nucleic acids can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes into full-length antibody chain genes. In these manipulations, the nucleic acid encoding the VHH is operably linked to another nucleic acid encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used in this context is intended to mean that two or more nucleic acids are linked such that the amino acid sequences encoded by the two or more nucleic acids remain in frame.

[0131] In some embodiments, the present disclosure relates to a nucleic acid molecule comprising a nucleic acid sequence encoding a single variable domain (eg, a VHH) of a D3 binding molecule disclosed herein.

[0132] In some embodiments, the nucleic acid molecule is (A) a nucleic acid sequence encoding a VHH region set forth in any one of SEQ ID NOs: 12 to 18 and 55; (B) a nucleic acid sequence having at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of (A); and (C) comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences that hybridize to the complementary strand of the nucleic acid sequence of (A) under high stringency conditions.

[0133] In some embodiments, provided herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an anti-D3 single domain antibody comprising the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, provided herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an anti-D3 single domain antibody comprising the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, provided herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an anti-D3 single domain antibody comprising the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, provided herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an anti-D3 single domain antibody comprising the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, provided herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an anti-D3 single domain antibody comprising the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, provided herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an anti-D3 single domain antibody comprising the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, provided herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an anti-D3 single domain antibody comprising the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, provided herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an anti-D3 single domain antibody comprising the amino acid sequence set forth in SEQ ID NO: 55.

[0134] In some embodiments, the percent identity results from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0135] Exemplary high stringency conditions include hybridization at 45°C in 5xSSPE and 45% formamide, and a final wash at 65°C in 0.1xSSC. It is understood in the art that conditions of equivalent stringency can be obtained by varying temperature and buffer or salt concentration, as described in Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3-6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length of the probe and the percentage of guanosine / cytosine (GC) base pairing. Hybridization conditions can be calculated as described in Sambrook, et al. (Eds.), Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47-9.51.

[0136] host cell The host cells disclosed in this disclosure may be any cell suitable for expressing the antibodies of this disclosure, including, for example, yeast, bacterial, plant and mammalian cells. Mammalian host cells for expressing the antibodies of this disclosure include Chinese hamster ovary cells (CHO cells) (e.g., dhfr CHO cells as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, used with the DHFR selection marker as described, for example, in RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159:601-621), 293F cells, NSO myeloma cells, COS cells and SP2 cells. In particular, another expression system for use with NSO myeloma cells is the GS gene expression system disclosed in WO87 / 04462, WO89 / 01036 and EP338,841.Also, SV40-transformed monkey kidney cell line CV1 (SCO-7, ATCC CRL 1651), human embryonic kidney cell line (293 cells or 293 cells subcloned in suspension culture for growth, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary (CHO) cells / DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216); mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary carcinoma (MMT 060562, ATCC CCL51), TRI cells (Mather et al., 1982, Annals NYAcad.Sci.383:44-68), MRC5 cells, FS4 cells; mouse myeloma cells such as NOS (e.g., RCB0213, 1992, Bio / Technology 10:169) and SP2 / 0 cells (e.g., SP2 / 0-Ag14 cells, ATCC CRL 1581); YB2 / 0 cells (e.g., YB2 / 3HL.P2.G11.16Ag.20 cells, ATCC CRL 1662); PER.C6 cells; and human hepatoma line (Hep G2). CHO cells are one of the cell lines that can be used herein, with CHO-K1, DUK-B11, CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics 12:555 (1986)), and Lec13 being exemplary host cell lines. In the case of CHO-K1, DUK-B11, DG44 or CHO-DP12 host cells, they can be modified to lack the ability to fucosylate expressed proteins.In some embodiments, the host cell herein is selected from a CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NSO cell, or a lymphocytic cell.

[0137] Suitable prokaryotes for this purpose include eubacteria such as gram-negative or gram-positive organisms, for example Enterobacteriaceae, for example Escherichia (e.g. E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Serratia, for example Serratia marcescans, and Shigella, as well as Bacillus, for example B. subtilis and B. licheniformis, Pseudomonas, for example P. aeruginosa, and Streptomyces.

[0138] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; as well as several other genera, species, and strains of filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger, are generally available and useful herein.

[0139] If a recombinant expression vector encoding an antibody is introduced into a mammalian host cell, the antibody is produced by culturing the host cells for a sufficient time that the antibody is expressed within the host cells or that the antibody is secreted into the medium in which the host cells are grown. The antibody can be recovered from the medium using standard protein purification methods.

[0140] Pharmaceutical Compositions In some aspects, the present disclosure provides a pharmaceutical composition comprising a D3 binding molecule disclosed herein, for example comprising a single variable domain (e.g., VHH) of a D3 binding molecule disclosed herein, and a pharmaceutically acceptable carrier. In some aspects, the present disclosure provides a pharmaceutical composition comprising a nucleic acid encoding a D3 binding molecule disclosed herein, for example comprising a single variable domain (e.g., VHH) of a D3 binding molecule disclosed herein, and a pharmaceutically acceptable carrier. In some aspects, the present disclosure provides a pharmaceutical composition comprising a cell expressing a D3 binding molecule disclosed herein, for example comprising a single variable domain (e.g., VHH) of a D3 binding molecule disclosed herein, and a pharmaceutically acceptable carrier.

[0141] Ingredients of the composition The pharmaceutical composition may optionally contain one or more additional components, including one or more pharmacoactive ingredients, such as another antibody or drug.The pharmaceutical composition of the present disclosure can also be administered in combination therapy with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, including when the anti-D3 antibody enhances immune response.Pharmaceutical acceptable carriers include, for example, pharmacologic acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, and various combinations of other ingredients known in the art.

[0142] Suitable components of the pharmaceutical composition can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers or stabilizers, such as sugars and cyclodextrins. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene and / or propyl gallate. As disclosed in the present disclosure, a composition can include an antibody or antigen-binding fragment of the present disclosure and further include one or more antioxidants, such as methionine, to prevent or reduce loss of binding affinity, thereby increasing the stability and shelf life of the antibody. Thus, in some embodiments, the present disclosure provides a composition comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants, such as methionine. The present disclosure further provides various methods of combining the antibody or antigen-binding fragment thereof with one or more antioxidants, such as methionine, to prevent oxidation of the antibody or antigen-binding fragment thereof and increase its shelf life and / or activity.

[0143] To further illustrate, pharma- ceutically acceptable carriers can include, for example, aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, or Dextrose and Lactated Ringer's Injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate buffer or citrate buffer; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone; emulsifying agents such as polysorbate 80 (TWEEN®-80); sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multi-dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffers, stabilizers, dissolution promoters, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0144] Administration, Formulation and Dosage The pharmaceutical compositions of the present disclosure can be administered to subjects in need of administration by various routes, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise implanted or inhaled. The compositions can be formulated as solid, semisolid, liquid, or gaseous formulations, including but not limited to tablets, capsules, powders, granules, ointments, liquids, suppositories, enemas, injections, inhalants, and aerosols. The appropriate formulation and administration route can be selected according to the intended use and treatment regimen.

[0145] Formulations suitable for enteral administration include hard or soft gelatin capsules, pills, tablets including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.

[0146] Suitable formulations for parenteral administration (e.g., by injection) include aqueous or non-aqueous isotonic pyrogen-free sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may further contain other pharma- ceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of isotonic carriers suitable for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Similarly, the particular administration regimen, including dosage, timing, and repetition, will depend on the particular individual and that individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, and the like).

[0147] The frequency of dosing can be determined and adjusted over the course of treatment and is based on the reduction in the number of proliferative or tumorigenic cells, the maintenance of such a reduction in tumor cell numbers, the reduction in tumor cell proliferation, or the delay in metastatic progression. In some embodiments, the dosage can be adjusted or reduced to manage potential side effects and / or toxicity. Alternatively, sustained release formulations of the subject therapeutic compositions may be appropriate.

[0148] Those skilled in the art will recognize that appropriate dosages may vary from patient to patient. Determining optimal dosages generally involves balancing the level of therapeutic benefit against risk or adverse side effects. In particular, the dosage level selected will depend on a variety of factors, including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds and / or substances used in combination, the species, sex, age, weight, condition, general health, and previous medical history of the patient. The amount of compound and route of administration are ultimately at the discretion of the physician, veterinarian, or clinician, but generally the dosage will be selected to provide a local concentration at the site of action that will produce the desired effect without causing significant adverse or deleterious side effects.

[0149] In general, the D3 binding molecules of the present disclosure can be administered in a variety of ranges. These ranges include about 5 μg / kg body weight to about 100 mg / kg body weight per dose, about 50 μg / kg body weight to about 5 mg / kg body weight per dose, about 100 μg / kg body weight to about 10 mg / kg body weight per dose, and any value within these ranges. Other ranges include about 100 μg / kg body weight to about 20 mg / kg body weight per dose, and about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the dosage is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, at least about 10 mg / kg body weight.

[0150] In any event, the antibodies or antigen-binding portions thereof of the present disclosure are preferably administered as needed to a subject in need thereof. The frequency of administration can be determined by one of skill in the art, such as the attending physician, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, and the general health of the subject being treated.

[0151] In some embodiments, the course of treatment involving the D3 binding molecule of the present disclosure includes multiple administrations of the selected pharmaceutical agent over a period of several weeks or months.For example, the D3 binding molecule of the present disclosure can be administered once daily, every 2 days, every 4 days, every week, every 10 days, every 2 weeks, every 3 weeks, every month, every 6 weeks, every 2 months, every 10 weeks, or every 3 months.In this regard, it will be appreciated that dosages can be modified and intervals can be adjusted based on patient response and clinical practice.

[0152] Dosages and regimens can also be empirically determined for a disclosed therapeutic composition in an individual who has received one or more administrations. For example, an individual can be administered incremental doses of a therapeutic composition prepared as described herein. In some embodiments, the dose can be gradually increased or decreased or attenuated based on empirically determined or observed side effects or toxicity, respectively. To assess the effectiveness of a selected composition, markers of a particular disease, disorder, or condition can be tracked, as previously described. In the case of cancer, this includes direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques, improvement as assessed by direct tumor biopsy and microscopic examination of tumor samples, indirect measurement of tumor markers (e.g., PSA for prostate cancer) or tumorigenic antigens, relief of pain or paralysis; improvement in speech, vision, breathing, or other disorders associated with the tumor, increased appetite, or improved quality of life as measured by accepted tests or increased survival. It will be apparent to one of skill in the art that dosages will vary depending on the individual, the type of neoplastic condition, the stage of the neoplastic condition, whether the neoplastic condition has begun to metastasize to other sites in the individual, and past and current treatments being administered.

[0153] Formulations suitable for parenteral administration (e.g., intravenous injection) can include the D3 binding molecules disclosed herein at a concentration of about 10 μg / ml to about 100 mg / ml. In some embodiments, the concentration of the D3 binding molecule (e.g., an antibody or antigen-binding portion thereof) includes 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, or 1 mg / ml. In some embodiments, the concentration of the D3 binding molecule (e.g., an antibody or antigen-binding portion thereof) includes 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 8 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml or 100 mg / ml.

[0154] Applications of this disclosure The antibodies, antibody compositions and methods of the present disclosure have many in vitro and in vivo utilities and applications, including, for example, detection of D3 or enhancement of immune response.For example, these molecules can be administered to cells in culture in vitro or ex vivo, or to human subjects, for example, in vivo, to enhance immunity in various situations.Immune response can be modulated, for example, enhanced, stimulated, or upregulated.

[0155] For example, the subject includes a human patient who needs to enhance immune response. The method is particularly suitable for treating a human patient with a disease that can be treated by enhancing immune response (e.g., T cell-mediated immune response). In some embodiments, the method is particularly suitable for treating cancer in vivo. To achieve antigen-specific immune enhancement, the anti-D3 antibody may be administered together with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a subject with a tumor or a subject with a virus). When the antibody against D3 is administered together with another agent, the two can be administered in any order or simultaneously.

[0156] The present disclosure further provides a method for detecting the presence of human D3 antigen in a sample or for measuring the amount of human D3 antigen, comprising contacting the sample and a control sample with a human monoclonal antibody or an antigen-binding portion thereof that specifically binds to human D3, for example, under conditions that allow the formation of a complex between the antibody or portion thereof and human D3. The formation of the complex is then detected, where a difference in the complex formation of the sample compared to the control sample indicates the presence of PCSK3 in the sample. Furthermore, the anti-D3 antibody of the present disclosure can be used to purify human D3 by immunoaffinity purification.

[0157] Treatment of diseases including cancer In some embodiments, the present disclosure provides a method of treating a disorder or disease in a mammal, comprising administering to a subject (e.g., a human) in need of treatment a therapeutically effective amount of an anti-D3 antibody or antigen-binding portion thereof disclosed herein. In some embodiments, the present disclosure provides an anti-D3 antibody or antigen-binding portion thereof disclosed herein for use in treating a disease or disorder. In some embodiments, the present disclosure provides a use of an anti-D3 antibody or antigen-binding portion thereof disclosed herein in the manufacture of a medicament for treating a disease or disorder. The disorder or disease may be cancer.

[0158] Various cancers in which D3 is implicated, whether malignant or benign, primary or secondary, can be treated or prevented by the methods provided by the present disclosure.Cancers can include, but are not limited to, lung cancer (including various subtypes such as small cell lung cancer and non-small cell lung cancer), adrenal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, gastric cancer, ovarian cancer, cervical cancer, uterine cancer, esophageal cancer, colorectal cancer, prostate cancer, pancreatic cancer, thyroid cancer, carcinoma, sarcoma, glioblastoma, and various head and neck tumors.Examples of cancers include, for example, small cell lung cancer, large cell neuroendocrine carcinoma, glioblastoma, Ewing's sarcoma, and cancers with a neuroendocrine phenotype.

[0159] The anti-D3 antibodies disclosed herein can be used to treat lung cancer, such as bronchogenic carcinoma, non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma, such as lung adenocarcinoma. The lung cancer can be refractory, recurrent, or resistant to platinum-based agents (e.g., carboplatin, cisplatin, oxaliplatin, topotecan) and / or taxanes (e.g., docetaxel, paclitaxel, larotaxel, or cabazitaxel).

[0160] The cancers treated by the anti-D3 antibodies disclosed herein may be large cell neuroendocrine carcinoma (LCNEC), medullary thyroid carcinoma, glioblastoma, neuroendocrine prostate carcinoma (NEPC), high-grade gastroenteropancreatic carcinoma (GEP) and malignant melanoma. The anti-D3 antibodies disclosed herein can be used to treat neuroendocrine tumors (both NETs and pNETs) occurring in the kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid carcinoma), and lung (small cell lung carcinoma and large cell neuroendocrine carcinoma).

[0161] As noted above, anti-D3 antibodies are particularly effective in treating lung cancer, including the following subtypes: small cell lung cancer and non-small cell lung cancer (e.g., squamous non-small cell lung cancer or squamous small cell lung cancer) and large cell neuroendocrine carcinoma.

[0162] Stimulating the immune response In some aspects, the present disclosure also provides a method of enhancing (e.g., stimulating) the immune response of a subject by administering to the subject a D3 binding molecule, such as an anti-D3 antibody or antigen-binding portion thereof of the present disclosure. In some aspects, the present disclosure provides an anti-D3 antibody or antigen-binding portion thereof disclosed herein for use in enhancing (e.g., stimulating) the immune response of a subject. In some aspects, the present disclosure provides a use of an anti-D3 antibody or antigen-binding portion thereof disclosed herein in the manufacture of a medicament for enhancing (e.g., stimulating) the immune response of a subject. For example, in some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0163] The term "enhancing an immune response" or grammatical variations thereof means stimulating, eliciting, increasing, improving, or enhancing any response of the immune system of a mammal. The immune response may be a cellular response (e.g., cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (e.g., an antibody-mediated response), and may be a primary or secondary immune response. Examples of enhanced immune responses include increasing the number of CD4 + These include increased helper T cell activity and generation of cytolytic T cells. Enhancement of the immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, cytokine release (e.g., IL-2 production or IFN-γ production), tumor regression, survival of tumor-bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxic activity. For example, the methods of the present disclosure are useful for enhancing immune responses by mammals when compared to immune responses by untreated mammals or animals not treated using the methods disclosed herein.

[0164] D3 binding molecules can be used alone as monotherapy or in combination with chemotherapy, radiation therapy, targeted therapy, cellular immunotherapy, and the like.

[0165] Combined with chemotherapy The D3 binding molecule (eg, an anti-D3 antibody) can be used in combination with chemotherapy, including, for example, anti-cancer agents, cytotoxic agents, or chemotherapeutic agents.

[0166] The term "anti-cancer agent" or "anti-proliferative agent" refers to any agent that can be used to treat a cell proliferative disease, such as cancer, including, but not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, debulking agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormonal therapy, radiotherapy, and anti-metastatic and immunotherapeutic agents. As discussed above, it will be understood that in some embodiments, such anti-cancer agents may comprise a conjugate and may be conjugated to the disclosed anti-D3 antibodies prior to administration. For example, in some embodiments, a selected anti-cancer agent is conjugated to an unpaired cysteine ​​of an engineered antibody to provide an engineered conjugate (e.g., an antibody drug conjugate) as described herein. Thus, such engineered conjugates are expressly contemplated to be within the scope of the present disclosure. In some embodiments, the disclosed anti-cancer agents are administered in combination with an anti-D3 conjugate comprising a different therapeutic agent as described above.

[0167] As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells and reduces or inhibits the function of cells and / or causes destruction of cells. In some embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule or enzymatically active toxins from bacteria (e.g., diphtheria toxin, Pseudomonas aeruginosa endotoxins and exotoxins, Staphylococcus aureus enterotoxin A), fungi (e.g., alpha-sarcin, restrictocin), plants (e.g., abrin, ricin, modeccin, viscumin, pokeweed antiviral protein, saporin, gelonin, momorisin, trichosanthin, barley toxin, Aleurites fordii protein, dianthin protein, Phytolacca mericana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis, and the like. officinalis) inhibitors, gelonin, mitegellin, restrictocin, phenomycin, neomycin, and the trichothecenes) or animals (e.g., cytotoxic RNases such as extracellular pancreatic RNase, DNase I, including fragments and / or variants thereof).

[0168] For purposes of this disclosure, a "chemotherapeutic agent" includes compounds (e.g., cytotoxic or cytostatic agents) that non-specifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. Such chemicals often target intracellular processes necessary for cell growth or division, and are therefore particularly effective against cancerous cells, which generally grow and divide rapidly. For example, vincristine depolymerizes microtubules and inhibits cells from entering mitosis. In general, chemotherapeutic agents may include any chemical that inhibits or is designed to inhibit cancerous cells, or cells that may become cancerous or give rise to tumorigenic progeny (e.g., TICs). Such agents are often administered, and are often most effective, in combination with regimens such as CHOP or FOLFIRI.

[0169] Examples of anti-cancer agents that can be used in combination with the D3 binding molecules (e.g., anti-D3 antibodies) of the present disclosure (either as a component of a site-specific conjugate or unconjugated) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethylenimines and methylamelamines, acetogenins, camptothecins, bryostatins, kallistatins, CC-1065, cryptophycins, dolastatins, duocarmycins, eleutherobins, pancratistatins, sarcodictyins, spongiostatins, nitrogen mustards, antibiotics, enediyne antibiotics, dynemicins, bisphosphonates, esperamicins, chromoprotein ...bisphosphonates, esperamicins, bisphosphonates, esperamicins, bisphosphonates, esperamicins, bisphosphonates, esperamicins, bisphosphonates, esperamicins, bisphosphonates, esperamicins, bisphosphonates, esperamicins, bis chromophores, aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin® doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;Antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogues, purine analogues, androgens, antiadrenal drugs, folic acid supplements, e.g. folinic acid acid), aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diazicon, elfornithine, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK® polysaccharide complex (JHS Natural Products, Eugene, OR), razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, vinblastine; platinum; etoposide (VP- 16);Ifosfamide;Mitoxantrone;Vincristine;NAVELBINE(R) Vinorelbine;Novantrone;Teniposide;Edatrexate;Daunomycin;Aminopterin;Xeloda;Ibandronate;Irinotecan (Camptosar, CPT-11), topoisomerase inhibitor RFS2000;Difluoromethylornithine;Retinoids;Capecitabine;Combretastatin;Leucovorin;Included in this definition are oxaliplatin; inhibitors of PKC-α, Raf, H-Ras, EGFR and VEGF-A that reduce cell proliferation, as well as pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antiestrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit the enzyme aromatase (which regulates estrogen production in the adrenal glands), and antiandrogens; as well as antihormonal agents that act to regulate or inhibit hormone action on tumors, such as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes, such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; vinorelbine and esperamicin, as well as pharmaceutically acceptable salts, acids or derivatives of any of the above.;

[0170] Combined with radiation therapy The present disclosure also provides for the combination of D3 binding molecules with radiation therapy (e.g., any mechanism that induces localized DNA damage in tumor cells, such as gamma irradiation, X-rays, ultraviolet radiation, microwaves, electronic radiation, etc.). Combination therapy using directed delivery of radioisotopes to tumor cells is also contemplated, and the disclosed D3 binding molecules can be used in combination with targeted anticancer drugs or other targeting means. Generally, radiation therapy is administered in pulses over a period of about 1 to about 2 weeks. Radiation therapy may be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiation therapy may be administered as a single dose or as multiple sequential doses.

[0171] diagnosis The present disclosure provides in vitro and in vivo methods for detecting, diagnosing, or monitoring proliferative diseases, as well as methods for screening cells from patients to identify tumor cells, including tumorigenic cells. Such methods include identifying individuals with cancer for cancer treatment or monitoring cancer progression, comprising contacting a patient or a sample obtained from a patient (either in vivo or in vitro) with an anti-D3 antibody as described herein, and detecting the presence or absence, or level of association, of the antibody to the bound or free target molecule in the sample. In some embodiments, the anti-D3 antibody comprises a detectable label or reporter molecule as described herein.

[0172] In some embodiments, association of an anti-D3 antibody with particular cells in a sample can indicate that the sample may contain tumorigenic cells, thereby indicating that an individual with cancer may be effectively treated with an anti-D3 antibody described herein.

[0173] Samples can be analyzed by a number of assays, including, for example, radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblot assays, Western blot analysis, and flow cytometry assays. As is well known to those of skill in the art, compatible in vivo theragnostic or diagnostic assays can include art-recognized imaging or monitoring techniques, such as magnetic resonance imaging, computed tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, and the like.

[0174] Pharmaceutical Packs and Kits Pharmaceutical packs and kits are also provided that include one or more containers containing one or more doses of the D3 binding molecule. In some embodiments, unit doses are provided that include a predetermined amount of a composition that includes, for example, the D3 binding molecule with or without one or more additional agents. In some embodiments, such unit doses are provided in a single-use pre-filled syringe for injection. In some embodiments, the composition included in the unit dose can include saline, sucrose, etc., buffers such as phosphates, etc., and / or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition can be provided as a lyophilized powder that can be reconstituted by adding an appropriate liquid, such as sterile water or saline. In some embodiments, the composition includes one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. A label on or associated with the container(s) indicates that the enclosed composition is used to treat a selected neoplastic disease condition.

[0175] The present disclosure also provides a kit for preparing a single or multiple dose administration unit of the D3 binding molecule and optionally one or more anti-cancer drugs. The kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be formed from a variety of materials, such as glass or plastic, and can contain a pharma- ceutically effective amount of the disclosed D3 binding molecule, either in complex or uncomplexed form. In some embodiments, the container(s) can have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). Such a kit generally includes a pharma- ceutically acceptable formulation of the D3 binding molecule, either in complex or uncomplexed form, in a suitable container, and optionally, one or more anti-cancer drugs in the same or different containers. The kit may also include other pharma- ceutically acceptable formulations for either diagnosis or combination therapy. For example, such kits may include, in addition to the D3 binding molecules of the present disclosure, any one or more of a wide range of anti-cancer agents, such as chemotherapeutic or radiotherapeutic agents; anti-angiogenic agents; anti-metastatic agents; targeted anti-cancer agents; cytotoxic agents; and / or other anti-cancer agents.

[0176] For example, the kit may have a single container containing the D3 binding molecule with or without additional components, or may have separate containers for each desired agent. If combined therapeutic agents are provided for conjugation, a single solution may be premixed in molar equivalent combinations or with one component in greater amount than the other. Alternatively, the conjugate and any anticancer agent of the kit may be kept separate in separate containers prior to administration to a patient. The kit may include a second / third container means for containing a sterile pharma- ceutically acceptable buffer or other diluent, such as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution, and dextrose solution.

[0177] When each component of the kit is provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, such as a sterile aqueous solution or saline. However, the components of the kit may also be provided as a dry powder(s). When a reagent or component is provided as a dry powder, the powder can be reconstituted by adding a suitable solvent. It is envisioned that the solvent may be provided in a separate container.

[0178] As briefly mentioned above, the kits can also include a means for administering the D3 binding molecule and any components to a patient, such as one or more needles, IV bags or syringes, as well as eye droppers, pipettes, or other similar devices from which the formulations can be injected or introduced into an animal or applied to affected areas of the body. The kits of the present disclosure also typically include a means for containing the vials or the like and other components in close confinement for commercial sale, such as injection or blow molded plastic containers into which the desired vials and other devices can be placed and retained.

[0179] Sequence Listing Overview Attached to this application is a sequence listing that includes a number of amino acid sequences. Tables A-F below provide an overview of the included sequences. The exemplary antibodies are collectively referred to in this disclosure as "WBPT1156 antibodies."

[0180] [Table A] [Table B] [Table C] [Table D] [Table E] [Table F] EXAMPLES

[0181] The present disclosure, generally described above, will be more readily understood with reference to the following examples, which are for illustrative purposes only and are not intended to limit the present disclosure, and are not intended to represent that the following experiments are all or the only experiments performed.

[0182] Example 1 Preparation of antigens, benchmark antibodies and cell lines 1.1 Preparation of antigen DNA sequences encoding the extracellular domain (ECD) sequences of cynomolgus monkey D3 (Uniprot number A0A2K5WSR4) and mouse D3 (Uniprot number O88516) were synthesized at Sangon Biotech (Shanghai, China) and subcloned into modified pcDNA3.3 expression vectors with an MBP tag at the N-terminus and an AVI-His tag or human Fc tag at the C-terminus. Human D3 (Uniprot number Q9NYJ7) was purchased from AcroBiosystems (catalog DL3-H52H4).

[0183] A DNA sequence encoding a truncated isoform of human D3 (disclosed in WO2017 / 021349) was synthesized by Sangon Biotech (Shanghai, China) and subcloned into a modified pcDNA3.3 expression vector containing an MBP tag and an AVI-His tag at the C-terminus.

[0184] The purified expression vector was transfected into Expi293 cells (Invitrogen-A14527). The cells were cultured for 5 days, and the supernatant was collected for protein purification using Ni-NTA column (GE Healthcare, Cat. No. 175248) or Protein A column (GE Healthcare, Cat. No. 175438). The resulting mouse D3, cynomolgus D3, and truncated human D3 were analyzed by SDS-PAGE and SEC, and stored at -80°C.

[0185] Human D3 (ACRO DL3-H52H4) was designated WT115-hPro1.ECD.His. The resulting mouse D3 was designated WT115-MBP-mPro1.ECD.hFc. The human D3 protein is characterized by a Delta / Serrate / LAG-2 (DSL) domain, six epidermal growth factor (EGF)-like repeats (EGF domain), and a transmembrane domain. The truncated isoforms of human D3 are WT115-hPro1.V1.ECD.MBP.AVI.His (DSL domain + EGF1-6 domain + membrane proximal), WT115-hPro1.V2.ECD.MBP.AVI.His (EGF1-6 domain + membrane proximal), WT115-hPro1.V3.ECD.MBP.AVI.His (EGF2-6 domain + membrane proximal), and WT115-hPro1.V We named them WT115-hPro1.V4.ECD.MBP.AVI.His (EGF3-6 domains + membrane proximal), WT115-hPro1.V5.ECD.MBP.AVI.His (EGF4-6 domains + membrane proximal), WT115-hPro1.V6.ECD.MBP.AVI.His (EGF5-6 domains + membrane proximal), and WT115-hPro1.V7.ECD.MBP.AVI.His (EGF6 domain + membrane proximal). Diagrams of the truncated proteins are shown in Figure 7c.

[0186] 1.2 Construction of expression vector for BMK antibody Two anti-D3 antibodies were used as benchmark antibodies and are referred to herein as WT115-BMK1 and WT115-BMK2. DNA sequences encoding the variable regions of WT115-BMK1 (SEQ ID NO:212 and SEQ ID NO:213 in US2019 / 0046656) and WT115-BMK2 (SEQ ID NO:37 and SEQ ID NO:38 in WO2017 / 021349) were synthesized at Sangon Biotech (Shanghai, China) and subcloned into a modified pcDNA3.3 expression vector encoding the Fc region of human IgG1.

[0187] Plasmids containing the VH and VL genes were co-transfected into Expi293 cells. The cells were cultured for 5 days and the supernatant was collected for protein purification using a Protein A column (GE Healthcare, 175438). The resulting antibodies were analyzed by SDS-PAGE and SEC and stored at -80°C.

[0188] 1.3 Establishment of stable cell lines / cell pools 293F cells were transfected with an expression vector containing the gene encoding full-length human D3 (UniProt, Q9NYJ7-1) using Lipofectamine 2000. Flpin293 cells were transfected with an expression vector containing the gene encoding full-length cynomolgus D3. Cells were cultured in medium containing the appropriate selection marker. A stable cell line highly expressing human D3 (WT115-293F.hPro1.2E5) was selected after limiting dilution, and a stable cell pool highly expressing cynomolgus D3 (WT115.Flpin293.cPro1.pool) was selected with the appropriate selection antibiotic.

[0189] 1.4 Antibody biotinylation To perform NHS-PEO4-biotinylation, 1–10 mg / mL of antibody (IgG) was incubated with a 20-fold molar excess of NHS-PEO4-biotin reagent in a metal bath for 75 min at 25 °C or 2 h on ice. Excess biotin was then removed using a desalting spin column, and purified protein samples were collected from the flow-through solution. The level of biotin incorporation into proteins was measured by HABA assay. Biotinylated samples were diluted 10-fold with HABA / avidin solution, and the absorbance at A500 of the mixed solution was measured. The number of moles of biotin per mole of protein was calculated based on the A500 value.

[0190] Example 2 Generation of WBPT1156 antibody containing VHH 2.1 Preparation of anti-D3 VHH Anti-D3 VHHs were generated by camelid immunization and phage display technology. Briefly, alpacas (Vicugnapacos) were subcutaneously immunized with hFc-tagged human D3 ECD protein (ACRO, DL3-H5255). After immunization, peripheral blood was collected to construct a phage library displaying VHH fragments. After biopanning with the corresponding target ECD protein, positive VHH clones binding to D3 were selected.

[0191] 2.2 Sequencing of VHHs Positive E. coli clones selected by target-specific binding ELISA and FACS using E. coli supernatants were sent to Biosune (Shanghai, China) for nucleotide sequencing of VHH genes. Sequencing results were analyzed using CLC Main Workbench (Qiagen, Hilden, Germany). The sequences of the four unique positive VHH clones were WT1156-P3R2-1C2, WT1156-P3R2-1C9, WT1156-P3R2-1H6, and WT1156-P8R2-1H1, as shown in Tables A and B.

[0192] 2.3 Preparation of human Fc fusion antibodies containing VHH Four unique positive VHH clones were converted into VHH-Fc(hIgG1) fusion antibodies. Briefly, VHH genes were PCR amplified from pET-bac vectors using VHH-specific cloning primers containing appropriate restriction sites and cloned by fusing into modified human hIgG1 expression pcDNA3.3 vector to generate the corresponding clones of VHH-Fc(hIgG1) chimeric antibodies. The vectors were transiently transfected into 293F or Expi293 cells to express the antibodies. The cell culture supernatants containing the antibodies were collected and purified using protein A chromatography. The generated antibodies were named "WT1156-P3R2-1C2-uIgG1", "WT1156-P3R2-1C9-uIgG1", "WT1156-P3R2-1H6-uIgG1" and "WT1156-P8R2-1H1-uIgG1", respectively. The resulting antibodies were analyzed by SDS-PAGE and HPLC-SEC and stored at -80°C.

[0193] 2.4 Humanization Humanization of VHHs was performed by a "best-fit" approach. Briefly, humanized VHH sequences were generated by blasting against the amino acid sequences of the VHH framework regions in the human germline V gene database and replacing the human CDR sequences of the top hits with the CDR sequences of the VHHs using Kabat's CDR definitions. Then, key residues in the framework that play important roles in the affinity or developability of the antibody were backmutated to the parent residues, either alone or in combination. Each variant was codon-optimized for mammalian expression and synthesized by GENEWIZ (SuZhou, China). The designed VHH variants and parent VHH proteins were cloned into a human IgG1 expression vector to generate human IgG1 constructs. The antibodies were produced in HEK293 cells and purified using protein A chromatography. The variants with the desired affinities were finally selected as humanization leads.

[0194] The sequences of three unique humanized D3 antibodies, WT1156-P3R2-1C2-z102-uIgG1, WT1156-P3R2-1C2-z109-uIgG1, and WT1156-P3R2-1H6-z100-uIgG1, are also shown in Tables A and B.

[0195] Example 3 Characterization of D3-binding antibodies 3.1 Human D3 binding by ELISA Plates were pre-coated with WT115-hPro1.ECD.His at 1 μg / mL, 100 μL per well, overnight at 4° C. Antigen was diluted from stock solution in coating buffer (0.02 M Na 2 CO 3 and 0.18M NaHCO 3 , pH 9.2). The next day, the plate was washed once with 1x PBST (PBS containing 0.05% Tween®-20) and blocked by adding 200 μL of 1x PBS / 2% BSA. The antibody was serially diluted in blocking buffer (5-fold serial dilutions from 20 nM to 0.00128 nM). After 1 h of blocking, the plate was washed three times using 1x PBST, and then the antibody was added to the plate and incubated for 1 h at ambient temperature. Binding of the antibody to the immobilized human D3 was detected by an HRP-labeled secondary antibody (Bethyl, A80-304P) diluted to a concentration of 1:10000 in 1x PBS / 2% BSA. After incubation, the plate was washed six times using 1x PBST. Color was developed by dispensing 100 μL of TMB substrate and the reaction was stopped by adding 100 μL of 2 M HCl. The absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer. Anti-human D3 antibodies WT115-BMK1 and WT115-BMK2 were used as positive controls. A human IgG1 isotype antibody was used as a negative control. All samples were tested in duplicate.

[0196] As shown in FIG. 1 and Table 1, WT1156-P3R2-1C2-uIgG1, WT1156-P3R2-1C9-uIgG1, WT1156-P3R2-1H6-uIgG1, and WT1156-P8R2-1H1-uIgG1 can bind equally strongly to immobilized human D3 compared to WT115-BMK1 and WT115-BMK2. The EC50 of the WT1156 antibody is in the range of 0.013 nM to 0.026 nM. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.0094 nM and 0.011 nM, respectively.

[0197] [Table 1]

[0198] 3.2 Human D3 binding by FACS WT115-293F.hPro1.2E5 cells (1 x 10 5 Cells / well) were incubated with various concentrations of antibodies (5-fold serial dilutions from 200 nM to 0.0128 nM) for 1 h at 4 °C. After washing with 1x PBS / 1% BSA, R-PE-labeled goat anti-human IgG (1:150, Jackson ImmunoResearch, 109-115-098) was added as a secondary antibody and incubated with the cells for 1 h at 4 °C in the dark. Anti-human D3 antibodies WT115-BMK1 and WT115-BMK2 were used as positive controls. Human IgG1 isotype antibody was used as a negative control. Cells were washed and resuspended in 4% paraformaldehyde. The MFI of the cells was measured by flow cytometer and analyzed by FlowJo.

[0199] As shown in FIG. 2a and Table 1, WT1156-P3R2-1C2-uIgG1, WT1156-P3R2-1C9-uIgG1, WT1156-P3R2-1H6-uIgG1, and WT1156-P8R2-1H1-uIgG1 can bind to human D3-expressing cells as well as WT115-BMK2. The EC50 of the WT1156 antibody ranges from 0.15 nM to 0.52 nM. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.019 nM and 0.54 nM, respectively.

[0200] As shown in Figure 2b, WT1156-P3R2-1C2-z102-uIgG1 and WT1156-P3R2-1C2-z109-uIgG1 can strongly bind to human D3-expressing cells with EC50 = 0.088 nM and 0.14 nM, respectively. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.03 nM and 0.52 nM, respectively. The data of WT1156-P3R2-1C2-z109-uIgG and the two BMK antibodies are also summarized in Table 2.

[0201] [Table 2] 3.3 Cynomolgus D3 binding by FACS WT115-Flpin293.cPro1.Pooled cells (1 × 10 5Cells / well) were incubated with various concentrations of antibodies (4-fold serial dilutions from 10 nM to 0.00061 nM) for 1 h at 4 °C. After washing with 1x PBS / 1% BSA, Alexa Fluor 647-labeled goat anti-human IgG (1:150, Jackson ImmunoResearch, 109-605-098) was added as a secondary antibody and incubated with the cells for 1 h at 4 °C in the dark. Anti-human D3 antibodies WT115-BMK1 and WT115-BMK2 were used as positive controls. Human IgG1 isotype antibody was used as a negative control. Cells were washed with 1x PBS / 1% BSA, resuspended in 4% paraformaldehyde, and incubated with the cells for 0.5 h at 4 °C in the dark. Then, the buffer was exchanged for 1x PBS / 1% BSA, and the cells were filtered. The MFI of the cells was measured by flow cytometer and analyzed by FlowJo.

[0202] As shown in FIG. 3a and Table 1, WT1156-P3R2-1C2-uIgG1, WT1156-P3R2-1C9-uIgG1, WT1156-P3R2-1H6-uIgG1, and WT1156-P8R2-1H1-uIgG1 can bind to cynomolgus D3 expressing cells as well as WT115-BMK1 and WT115-BMK2. The EC50 of the WT1156 antibody is in the range of 0.12 nM to 0.44 nM. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.019 nM and 0.20 nM, respectively.

[0203] As shown in Figure 3b, WT1156-P3R2-1C2-z102-uIgG1, WT1156-P3R2-1C2-z109-uIgG1 and WT1156-P3R2-1H6-z100-uIgG1 can strongly bind to human D3 expressing cells with EC50 = 0.083nM, 0.096nM and 0.42nM, respectively. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.019nM and 0.20nM, respectively. The data of WT1156-P3R2-1C2-z109-uIgG and the two BMKs are also summarized in Table 2.

[0204] 3.4 Mouse D3 binding by ELISA Plates were pre-coated overnight at 4 °C with WT115-MBP-mPro1.ECD.hFc at 1 μg / mL, 100 μL per well. Antigen was diluted in coating buffer from a stock solution. The next day, plates were washed once with 1x PBST and blocking was performed by adding 200 μL of 1x PBS / 2% BSA. Antibodies were serially diluted in blocking buffer (5-fold serial dilutions from 20 nM to 0.000256 nM). After 1 h of blocking, plates were washed 3 times using 1x PBST, then antibodies were added to the plates and incubated for 1 h at ambient temperature. Anti-human D3 antibodies WT115-BMK1-biotin and WT115-BMK2-biotin were used as positive controls. WT114-BMK1-biotin antibody was used as negative control. Binding of the antibodies to the immobilized mouse D3 was detected by HRP-labeled secondary antibody (Invitrogen, SNN1004) diluted at a concentration of 1:30000 in 1x PBS / 2% BSA. After incubation, the plates were washed six times with 1x PBST. Color was developed by dispensing 100 μL of TMB substrate, and the reaction was stopped by adding 100 μL of 2M HCl. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer. All samples were tested in duplicate.

[0205] As shown in Figure 4a, WT1156-P3R2-1C2-uIgG1 can bind to mouse D3 as strongly as WT115-BMK1. The EC50 of WT1156-P3R2-1C2-uIgG1 is 0.0092nM. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.0039nM and 0.014nM, respectively.

[0206] As shown in Figure 4b, WT1156-P3R2-1C2-z102-uIgG1 and WT1156-P3R2-1C2-z109-uIgG1 can strongly bind to mouse D3 protein with EC50 = 0.0067nM and 0.0075nM, respectively. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.0039nM and 0.014nM, respectively. The data of WT1156-P3R2-1C2-z109-uIgG and the two BMKs are also summarized in Table 2.

[0207] 3.5 Internalization WT115-293F.hPro1.2E5 cells (4 × 10 4 Cells / well) were seeded in a 96-well plate, and the medium was removed from the plate after centrifugation. A 1x final maximum concentration of primary antibody (5-fold serial dilutions from 40nM to 0.00256nM, or 5-fold serial dilutions from 200nM to 0.0128nM) was prepared, and pHrodo (amine reactive, Thermo Fisher, P36011)-labeled secondary antibody (Affinipure F(ab')2 fragment goat anti-human IgG, Jackson ImmunoResearch, 109-006-098, ratio = molecular 1:1) dilutions were added to the plate with cell culture medium and incubated at 37°C for 5 hours. Anti-human D3 antibodies WT115-BMK1 and WT115-BMK2 were used as positive controls. Human IgG1 isotype antibody was used as negative control. After incubation, cells were stained with reagents (nuclei - Hoechst33342, 1000ng / ml, cytoplasm - Calcein AM, 1:2000 dilution in DPBS) and plates were incubated for 15 min at 37°C. Finally, cells were photographed with Operatta CLS and antibody endocytosis was analyzed by the parameter "number of spots per cell".

[0208] As shown in FIG. 5a and Table 1, WT1156-P3R2-1C2-uIgG1, WT1156-P3R2-1C9-uIgG1, WT1156-P3R2-1H6-uIgG1, and WT1156-P8R2-1H1-uIgG1 showed dose-dependent internalization ability equivalent to that of WT115-BMK1 and WT115-BMK2 in human D3-expressing cells. The EC50 of the WT1156 antibody ranges from 0.46 nM to 0.95 nM. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.19 nM and 0.58 nM, respectively.

[0209] As shown in Fig. 5b and Table 2, WT1156-P3R2-1C2-z109-uIgG1 showed dose-dependent internalization ability in human D3-expressing cells, with an EC50 of 12.2 nM. The EC50s of WT115-BMK1 and WT115-BMK2 were 3.47 nM and 14.4 nM, respectively.

[0210] 3.6 Kinetic binding affinity of anti-D3 antibody The binding affinity of anti-D3 antibodies to the ECD protein of human D3 was detected by SPR assay using Biacore T200. Each antibody tested was captured on an anti-human IgGFc antibody immobilized CM5 sensor chip (GE). Different concentrations of WT115-hPro1.ECD.His were injected over the sensor chip at a flow rate of 30 μl / min for a binding phase of 180 s, followed by a dissociation phase of 3600 s. The chip was regenerated with 10 mM glycine (pH 1.5) after each binding cycle.

[0211] The experimental data for human D3 was fitted by the steady-state affinity model, as shown in Table 3. The experimental data for WT-115-BMK1 for human D3 was fitted by the heterologous ligand model. The other experimental data was fitted by the 1:1 model using Langmuir analysis. The sensorgrams of the blank surface and buffer channel were subtracted from the test sensorgrams. The molar concentration of the analyte was calculated using a molecular weight of 34 KDa. The affinity of the test antibodies for human D3 is shown in Table 3.

[0212] [Table 3] 3.7 Epitope binning by competitive ELISA Plates were pre-coated with WT115-hPro1.ECD.His at 1 μg / mL, 100 μL per well, overnight at 4°C. Antigen was diluted from stock solution in coating buffer (0.02 M Na2CO3 and 0.18 M NaHCO3, pH 9.2). The next day, plates were washed once with 1×PBST and blocked using 200 μL of 1×PBS / 2% BSA. VHH antibodies were serially diluted in blocking buffer (5-fold serial dilutions from 10 nM to 0.00013 nM) and premixed with a fixed concentration of total IgG antibody (0.02 nM). After 1 h of blocking, plates were washed 3 times using 1×PBST, then the VHH antibody / total IgG antibody mixture was added to the plate and incubated for 1 h at ambient temperature. Binding of total IgG antibodies to immobilized human D3 was detected by HRP-labeled secondary antibody (Bethyl, A80-304P) diluted at a concentration of 1:10000 in 1x PBS / 2% BSA. After incubation, the plates were washed six times with 1x PBST. Color was developed by dispensing 100 μL of TMB substrate, and the reaction was stopped by adding 100 μL of 2M HCl. Absorbance was read at 450 nM and 540 nM using a microplate spectrophotometer. All samples were tested in duplicate.

[0213] As shown in Figure 6a, WT1156-P3R2-1C2.uIgG1 does not compete with the other three WBPT1156 antibodies for binding to human D3-ECD protein when tested by ELISA. Also, as shown in Figure 6b, WT1156-P3R2-1H6-uIgG1 can compete with WT1156-P8R2-1H1-uIgG1 and WT1156-P3R2-1C9-uIgG1 for binding to human D3-ECD protein. Figure 6c shows that WT1156-P8R2-1H1-uIgG1 can compete with WT1156-P3R2-1C9-uIgG1. As shown in Figure 6d, WBPT1156 4 antibodies do not compete with WT115-BMK1.

[0214] 3.8 Binding by ELISA using truncated D3 protein The binding epitope of the WT1156 antibody was tested by ELISA using truncated D3 protein as described in 1.1 and Figure 7c. The ELISA test was performed by pre-coating the plate with the antibody or antigen. The results are shown in Figures 7a and 7b, respectively.

[0215] To perform ELISA with precoated antibodies, ELISA plates were precoated overnight at 4°C with 2 μg / mL of antibody at 100 μL per well. Antibodies were diluted from stock solutions in coating buffer (0.02 M Na2CO3 and 0.18 M NaHCO3, pH 9.2). The next day, plates were washed once with 1×PBST and blocked using 200 μL of 1×PBS / 2% BSA. Fixed concentrations of full-length DLL ECD protein (WT115-hPro1.ECD.His) (3 μg / mL) or truncated D3 protein (3 μg / mL or 6 μg / mL) diluted in blocking buffer were then added. After 1 h of blocking, plates were washed 3 times using 1×PBST, and then antigen was added to the plates and incubated for 1 h at ambient temperature. Binding of the antibodies to the immobilized WBPT1156 was detected by HRP-labeled secondary antibody (GenScript, A00612) diluted at a concentration of 1:2000 in 1x PBS / 2% BSA. After incubation, the plates were washed six times with 1x PBST. Color was developed by dispensing 100 μL of TMB substrate. The color reaction was stopped with 2M HCl and absorbance was read at 450 nM and 540 nM using a microplate spectrophotometer. All samples were tested in duplicate. The results are shown in Figure 7a.

[0216] To perform ELISA with precoated antigen, ELISA plates were precoated with 100 μL per well of WT115-hPro1.ECD.His (2 μg / mL) or truncated D3 protein (2 μg / mL or 5 μg / mL) overnight at 4 °C. Antigens were diluted from stock solutions in coating buffer (0.02 M Na2CO3 and 0.18 M NaHCO3, pH 9.2). The next day, plates were washed once with 1×PBST and blocked with 200 μL of 1×PBS / 2% BSA. A fixed concentration of antibody (2 μg / mL) was diluted in blocking buffer. After 1 h of blocking, plates were washed 3 times with 1×PBST, then antibodies were added to the plates and incubated for 1 h at ambient temperature. Binding of the antibodies to the immobilized human D3 was detected by HRP-labeled secondary antibody (Bethyl, A80-304P) diluted at a concentration of 1:10000 in 1x PBS / 2% BSA. After incubation, the plates were washed six times with 1x PBST. Color was developed by dispensing 100 μL of TMB substrate and the reaction was stopped by adding 100 μL of 2M HCl. Absorbance was read at 450 nM and 540 nM using a microplate spectrophotometer. All samples were tested in duplicate. The results are shown in Figure 7b.

[0217] As shown in Figures 7a and 7b, WT1156-P3R2-1C2-uIgG1 binds to WT115-hPro1.V1.ECD.MBP.AVI.His, WT115-hPro1.V2.ECD.MBP.AVI.His, and partially binds to WT115-hPro1.V3.ECD.MBP.AVI.His, but not to other isoforms, indicating that its binding epitope is located in EGF1-2. WT1156-P3R2-1C9-uIgG1 and WT1156-P3R2-1H6-uIgG1 bind to WT115-hPro1.ECD.His, but not to the truncated D3 isoform, indicating that the binding epitopes of these two antibodies are located in the N-terminus. When tested in an ELISA with pre-coated antibodies, WT1156-P8R2-1H1-uIgG1 shows binding to WT115-hPro1.ECD.His but not to the truncated D3 isoform (Figure 7a). When tested in an ELISA with pre-coated antigen, WT1156-P8R2-1H1-uIgG1 shows binding to WT115-hPro1.V1.ECD.MBP.AVI.His and WT115-hPro1.V2.ECD.MBP.AVI.His but not to other isoforms, as shown in Figure 7b, indicating that its binding epitope is likely located at the N-terminus-DSL-EGF-1. ELISA binding results show that WT115-BMK1 binds to the DSL domain and WT115-BMK2 binds to the EGF-3 domain, which is consistent with the results shown in US2019 / 0046656 and WO2017 / 021349, respectively.

[0218] 3.9 Interfamily binding of anti-human D3 antibodies Plates were pre-coated overnight at 4° C. with WT115-hPro1.ECD.His, WT115-hPro2.ECD.His (human D1, SinoBiological, Catalog No.: 11635-H08H) or WT115-hPro3.ECD.His (human D4, SinoBiological, Catalog No.: 10171-H08H) at 1 μg / mL, 100 μL per well. Antigens were diluted from stock solutions in coating buffer (0.02 M Na2CO3 and 0.18 M NaHCO3, pH 9.2). The next day, plates were washed once with 1×PBST (PBS with 0.05% Tween®-20) and blocked by adding 200 μL per well of 1×PBS / 2% BSA. During blocking, BMK and WT1156 antibodies were diluted to 10 nM in blocking buffer, and WT115-cAb (WT115-cAb1 is anti-D1 antibody purchased from SinoBiological, Catalog No.: 11635-MM07, WT115-cAb2 is anti-D4 antibody purchased from SinoBiological, Catalog No.: 10171-MM15) was diluted 1000-fold. After 1 h of blocking, the plate was washed three times using 1× PBST, and then each diluted antibody was added to the plate and incubated at ambient temperature for 1 h. Binding of each antibody to immobilized human D3 was detected by goat anti-human IgG-Fc fragment cross-absorbed antibody HRP (Bethyl, A80-304P) and mouse IgG-Fc fragment cross-absorbed antibody HRP (Bethyl, A90-231P) diluted 1:10000 in 1× PBS / 2% BSA. After incubation, the plates were washed six times with 1x PBST. Color was developed by dispensing 100 μL of TMB substrate and the reaction was stopped by adding 100 μL of 2M HCl. Absorbance was read at 450 nM and 540 nM using a microplate spectrophotometer. WT115-cAb1 and cAb2 were used as positive controls for anti-human D1 and D4, respectively. A human IgG1 isotype antibody was used as a negative control. All samples were tested in duplicate.

[0219] As shown in FIG. 8, the WBPT1156 antibody does not bind to either human D1 or human D4.

[0220] 3.10 Human serum stability Human serum was freshly isolated from healthy donors by centrifugation. Samples were diluted with serum to bring the serum volume to more than 90% of the total volume. Five aliquots of samples were incubated at 37° C. Samples were then collected on days 0, 1, 4, 7, and 14, respectively, and flash frozen together until analysis.

[0221] The stability of the samples was tested by binding to human D3 using ELISA. Briefly, plates were pre-coated with 100 μL / well of 1 μg / mL WT115-hPro1.ECD.His overnight at 4°C. The next day, plates were washed once with 1×PBST (PBS containing 0.05% Tween®-20) and blocked by adding 200 μL per well of 1×PBS / 2% BSA. During blocking, test antibodies were added to the plates at various concentrations (4-fold serial dilutions from 3 nM to 0.00018 nM). The plates were incubated for 1 h at room temperature. Binding of each antibody to immobilized human D3 was detected by goat anti-human IgG-Fc fragment cross-absorbed antibody HRP (Bethyl, A80-304P) and mouse IgG-Fc fragment cross-absorbed antibody HRP (Bethyl, A90-231P) diluted 1:5000 in 1×PBS / 2% BSA. After incubation, the plates were washed six times with 1x PBST. Color was developed by dispensing 100 μL of TMB substrate and the reaction was stopped by adding 100 μL of 2M HCl. Absorbance was read at 450 nM and 540 nM using a microplate spectrophotometer. A human IgG1 isotype antibody was used as a negative control. All samples were tested in duplicate.

[0222] As shown in FIG. 9, the binding profile of WT1156-P3R2-1C2-z109-uIgG1 to human D3 protein did not change even after incubation in human serum at 37° C. for up to 14 days.

[0223] Those skilled in the art will further appreciate that the present disclosure may be embodied in other specific forms without departing from its spirit or central attributes. It should be understood that the above description of the present disclosure discloses only exemplary embodiments thereof, and other variations are contemplated as being within the scope of the present disclosure. Thus, the present disclosure is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims as indicating the scope and content of the present disclosure.

Claims

1. A D3 binding molecule comprising an immunoglobulin single variable domain, wherein the single variable domain comprises CDR1, CDR2, and CDR3 set forth in a VHH comprising the amino acid sequence of SEQ ID NO: 17, 55, 18, 12, 13, 14, 15, or 16.

2. The D3 binding molecule of claim 1, wherein the CDR1, CDR2, and CDR3 are identified according to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the Contact numbering system, the IMGT numbering system, or any combination thereof.

3. A D3 binding molecule as described in claim 1, wherein (i) the CDR1 comprises the amino acid sequence of SEQ ID NO: 1, 20, 23, 24, or 27, (ii) the CDR2 comprises the amino acid sequence of SEQ ID NO: 2, 21, 28, 56, or 30, or AYY, and (iii) the CDR3 comprises the amino acid sequence of SEQ ID NO: 3, 22, 26, or 29. (1) the CDR1 comprises the amino acid sequence of SEQ ID NO: 1, the CDR2 comprises the amino acid sequence of SEQ ID NO: 2, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 3; (2) the CDR1 comprises the amino acid sequence of SEQ ID NO: 20, the CDR2 comprises the amino acid sequence of SEQ ID NO: 21, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 22; (3) the CDR1 comprises the amino acid sequence of SEQ ID NO: 23, the CDR2 comprises the amino acid sequence of SEQ ID NO: 2, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 3; (4) the CDR1 comprises the amino acid sequence of SEQ ID NO: 24, the CDR2 comprises the amino acid sequence of AYY, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 26; (5) the CDR1 comprises the amino acid sequence of SEQ ID NO: 27, the CDR2 comprises the amino acid sequence of SEQ ID NO: 28, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 29; (6) the CDR1 comprises the amino acid sequence of SEQ ID NO: 27, the CDR2 comprises the amino acid sequence of SEQ ID NO: 56, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 29; or (7) The CDR1 comprises the amino acid sequence of SEQ ID NO: 1, the CDR2 comprises the amino acid sequence of SEQ ID NO: 30, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 3; The D3 binding molecule of claim 3.

5. A D3 binding molecule as described in claim 1, wherein (i) the CDR1 comprises the amino acid sequence of SEQ ID NO: 7, 42, 45, 46, or 49, (ii) the CDR2 comprises the amino acid sequence of SEQ ID NO: 8, 43, 47, 50, or 52, and (iii) the CDR3 comprises the amino acid sequence of SEQ ID NO: 9, 44, 48, or 51. (1) the CDR1 comprises the amino acid sequence of SEQ ID NO: 7, the CDR2 comprises the amino acid sequence of SEQ ID NO: 8, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 9; (2) the CDR1 comprises the amino acid sequence of SEQ ID NO: 42, the CDR2 comprises the amino acid sequence of SEQ ID NO: 43, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 44; (3) the CDR1 comprises the amino acid sequence of SEQ ID NO: 45, the CDR2 comprises the amino acid sequence of SEQ ID NO: 8, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 9; (4) the CDR1 comprises the amino acid sequence of SEQ ID NO: 46, the CDR2 comprises the amino acid sequence of SEQ ID NO: 47, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 48; (5) the CDR1 comprises the amino acid sequence of SEQ ID NO: 49, the CDR2 comprises the amino acid sequence of SEQ ID NO: 50, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 51; or (6) The CDR1 comprises the amino acid sequence of SEQ ID NO: 7, the CDR2 comprises the amino acid sequence of SEQ ID NO: 52, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 9; The D3 binding molecule of claim 5.

7. A D3 binding molecule as described in claim 1, wherein (i) the CDR1 comprises the amino acid sequence of SEQ ID NO: 4, 31, 34, 35, or 38, (ii) the CDR2 comprises the amino acid sequence of SEQ ID NO: 5, 32, 36, 39, or 41, and (iii) the CDR3 comprises the amino acid sequence of SEQ ID NO: 6, 33, 37, or 40. (1) the CDR1 comprises the amino acid sequence of SEQ ID NO: 4, the CDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 6; (2) the CDR1 comprises the amino acid sequence of SEQ ID NO: 31, the CDR2 comprises the amino acid sequence of SEQ ID NO: 32, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 33; (3) the CDR1 comprises the amino acid sequence of SEQ ID NO: 34, the CDR2 comprises the amino acid sequence of SEQ ID NO: 5, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 6; (4) the CDR1 comprises the amino acid sequence of SEQ ID NO: 35, the CDR2 comprises the amino acid sequence of SEQ ID NO: 36, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 37; (5) the CDR1 comprises the amino acid sequence of SEQ ID NO: 38, the CDR2 comprises the amino acid sequence of SEQ ID NO: 39, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 40; or (6) The CDR1 comprises the amino acid sequence of SEQ ID NO: 4, the CDR2 comprises the amino acid sequence of SEQ ID NO: 41, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 6; The D3 binding molecule of claim 7.

9. A D3 binding molecule as described in claim 1, wherein (i) the CDR1 comprises the amino acid sequence of SEQ ID NO: 10, 53, 34, 54, or 38, (ii) the CDR2 comprises the amino acid sequence of SEQ ID NO: 11, 32, 36, 39, or 41, and (iii) the CDR3 comprises the amino acid sequence of SEQ ID NO: 6, 33, 37, or 40. (1) the CDR1 comprises the amino acid sequence of SEQ ID NO: 10, the CDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 6; (2) the CDR1 comprises the amino acid sequence of SEQ ID NO: 53, the CDR2 comprises the amino acid sequence of SEQ ID NO: 32, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 33; (3) the CDR1 comprises the amino acid sequence of SEQ ID NO: 34, the CDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 6; (4) the CDR1 comprises the amino acid sequence of SEQ ID NO: 54, the CDR2 comprises the amino acid sequence of SEQ ID NO: 36, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 37; (5) the CDR1 comprises the amino acid sequence of SEQ ID NO: 38, the CDR2 comprises the amino acid sequence of SEQ ID NO: 39, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 40; or (6) The CDR1 comprises the amino acid sequence of SEQ ID NO: 10, the CDR2 comprises the amino acid sequence of SEQ ID NO: 41, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 6; The D3 binding molecule of claim 9.

11. The D3 binding molecule of claim 1, further comprising framework region 1 (FRW1), framework region 2 (FRW2), framework region 3 (FRW3), and / or framework region 4 (FRW4).

12. A D3 binding molecule as described in claim 1, further comprising FRW1, FRW2, FRW3, and / or FRW4 described in a VHH comprising the amino acid sequence of SEQ ID NO: 17, 55, 18, 12, 13, 14, 15, or 16.

13. the single variable domain (A) the amino acid sequence of SEQ ID NO: 17, 55, 18, 12, 13, 14, 15, or 16; (B) an amino acid sequence that is at least 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 17, 55, 18, 12, 13, 14, 15, or 16, while retaining specific binding affinity for D3; or (C) an amino acid sequence having one or more amino acid additions, deletions, and / or substitutions compared to the amino acid sequence of SEQ ID NO: 17, 55, 18, 12, 13, 14, 15, or 16; The D3 binding molecule of claim 1, comprising:

14. The D3 binding molecule of claim 1, wherein the single variable domain comprises the amino acid sequence of SEQ ID NO: 17, 55, 18, 12, 13, 14, 15, or 16.

15. The D3 binding molecule of claim 1, further comprising a human IgG constant domain.

16. 16. The D3 binding molecule of claim 15, wherein the human IgG constant domain is a human IgG1, IgG2, IgG3 or IgG4 constant domain, such as a human IgG1 constant domain or a variant thereof.

17. The following properties: (a) binds to human D3, cynomolgus monkey D3, and / or mouse D3 with an nM-grade EC50 as measured by ELISA or FACS; (b) exhibiting dose-dependent internalization in cells expressing human D3; (c) binds to human D3 with a K of 0.1 nM or less as measured by SPR; The D3 binding molecule of claim 1, having one or more of the following:

18. The D3 binding molecule of claim 1, which is a chimeric antibody, a humanized antibody, or a fully human antibody.

19. The D3 binding molecule of claim 1, comprising a single variable domain comprising the amino acid sequence of SEQ ID NO: 17, 55, 18, 12, 13, 14, 15, or 16, and an IgG constant domain comprising the amino acid sequence of SEQ ID NO:

19.

20. The D3 binding molecule of claim 1, which is a dimer.

21. A fusion protein comprising a D3 binding molecule as defined in any one of claims 1 to 20 fused to a heterologous peptide, such as an antigen binding domain that targets a different antigen.

22. A nucleic acid molecule comprising a nucleic acid sequence encoding a single variable domain of a D3 binding molecule as defined in any one of claims 1 to 20.

23. A vector comprising the nucleic acid molecule of claim 22.

24. A host cell comprising the vector of claim 23.

25. A pharmaceutical composition comprising at least one D3 binding molecule as defined in any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

26. A method for producing a D3-binding molecule, comprising: expressing the D3-binding molecule in a host cell according to claim 24; - isolating the D3 binding molecule from the host cell.

27. A method for modulating a D3-associated immune response in a subject, comprising administering to the subject a D3 binding molecule as defined in any one of claims 1 to 20, thereby modulating the immune response in the subject.

28. 21. A method for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of a D3 binding molecule as defined in any one of claims 1 to 20, wherein the cancer is D3-positive or overexpresses D3.

29. 29. The method of claim 28, wherein the cancer is lung cancer or neuroendocrine cancer.

30. 30. The method of claim 29, wherein the cancer is small cell lung cancer (SCLC) or large cell neuroendocrine carcinoma (LCNEC).

31. An in vitro method for diagnosing D3-positive cancer in a subject, comprising contacting a sample obtained from the subject with a D3 binding molecule as defined in any one of claims 1 to 20, and detecting the presence or absence, or level of association, of the D3 binding molecule with a bound or free target molecule in the sample.

32. A kit for treating or diagnosing cancer, comprising a container containing a D3 binding molecule as defined in any one of claims 1 to 20.

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