D3 binding molecules and uses thereof

Abstract

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

Description

[0001] Related Applications

[0002] This application is a divisional application of the Chinese invention patent application filed on September 16, 2022, with invention name “D3 binding molecules and their uses” and application number 202280072803.0.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the priority benefit of international patent application No. PCT / CN2021 / 119011 filed on September 17, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0005] Sequence Listing

[0006] This application incorporates by reference the Sequence Listing filed with this application. Technical Field

[0007] The present application generally relates to delta-like canonical Notch ligand 3 (D3) binding molecules, including anti-D3 antibodies and uses thereof. Background Art

[0008] Delta-like canonical Notch ligand 3 (D3 or DLL3) is a type I transmembrane protein that belongs to the DSL family of Notch ligands. It is usually expressed only on intracellular membranes, especially on the Golgi apparatus. Other 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). In addition to D3, other ligands can activate Notch signaling. D3 acts as an inhibitor of Notch signaling by interfering with the binding between Notch and 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 usually expressed only on intracellular membranes, it is a potential therapeutic tumor target for any tumor that expresses D3, including SCLC and LCNEC.

[0009] Lung cancer is the most common cause of cancer death, with approximately 2 million people diagnosed with lung cancer each year worldwide; approximately 15% of lung cancer cases are SCLC, 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) for first-line treatment of SCLC, and the limited 2-month benefit highlights the need to develop other therapies.

[0010] There remains a need to find new D3-targeting agents, including D3-binding molecules, such as monoclonal antibodies, for use as antibody conjugates (such as ADCs) or as bispecific antibodies and for the development of CAR-T therapies. Summary of the invention

[0011] The present disclosure relates to compounds, methods, compositions and articles of manufacture that provide D3 binding molecules with improved efficacy. The benefits provided by the present disclosure are broadly applicable to the field of antibody therapy and diagnosis, and can be used in combination with other therapeutic agents such as antibodies reactive with a variety of targets.

[0012] The present disclosure provides D3 binding molecules, such as monoclonal antibodies, that can specifically bind to human D3 and cross-react with cynomolgus monkey and / or mouse D3. Such D3 binding molecules provide certain advantages over currently used and / or known agents, compositions and / or methods in the art. These advantages include internalization efficacy, improved therapeutic and pharmacological properties, increased specificity, improved safety profiles, reduced immunogenicity, and other favorable properties, such as improved ease of preparation or reduced cost of goods, greater stability, especially compared to drug candidates known in the art.

[0013] In the present disclosure, D3 binding molecules, such as monoclonal antibodies, directed against D3 have been developed, which can be used to treat D3 overexpressing tumors.

[0014] The present disclosure provides D3 binding molecules, nucleic acid molecules encoding the same, expression vectors and host cells for expressing the D3 binding molecules, and methods of using the D3 binding molecules. The D3 binding molecules of the present disclosure provide effective agents for treating various cancers (including lung cancer) by regulating human immune function.

[0015] In some embodiments, the present disclosure provides a D3 binding molecule comprising at least one immunoglobulin single variable domain (e.g., 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, and wherein:

[0016] CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, 4, 7 or 10;

[0017] CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, 5, 8 or 11; and

[0018] CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, 6 or 9.

[0019] In some embodiments, the single variable domain disclosed herein comprises:

[0020] (A) CDR1 shown in SEQ ID NO: 1; CDR2 shown in SEQ ID NO: 2; and CDR3 shown in SEQ ID NO: 3;

[0021] (B) CDR1 shown in SEQ ID NO:4; CDR2 shown in SEQ ID NO:5; and CDR3 shown in SEQ ID NO:6;

[0022] (C) CDR1 shown in SEQ ID NO:7; CDR2 shown in SEQ ID NO:8; and CDR3 shown in SEQ ID NO:9; or

[0023] (D) CDR1 shown in SEQ ID NO:10; CDR2 shown in SEQ ID NO:11; and CDR3 shown in SEQ ID NO:6.

[0024] In some embodiments, the single variable domain disclosed herein comprises:

[0025] (A) CDR1 shown in SEQ ID NO:27; CDR2 shown in SEQ ID NO:28 or 56; and CDR3 shown in SEQ ID NO:29;

[0026] (B) CDR1 shown in SEQ ID NO:38; CDR2 shown in SEQ ID NO:39; and CDR3 shown in SEQ ID NO:40; or

[0027] (C) CDR1 shown in SEQ ID NO:49; CDR2 shown in SEQ ID NO:50; and CDR3 shown in SEQ ID NO:51;

[0028] The CDR number is based on the Contact numbering system.

[0029] In some embodiments, the single variable domain disclosed herein comprises:

[0030] (A) the amino acid sequence shown in any one of SEQ ID NOs: 12-18 and 55;

[0031] (B) an amino acid sequence that is at least 85%, 90% or 95% identical to the amino acid sequence shown in any one of SEQ ID NOs: 12-18 and 55, but still retains specific binding affinity for D3 (e.g., substantially retains, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%); or

[0032] (C) an amino acid sequence in which one or more (e.g., 1, 2 or 3) amino acids are added, deleted and / or substituted compared to the amino acid sequence shown in any one of SEQ ID NOs: 12-18 and 55, but the specific binding affinity for D3 is still maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

[0033] In some embodiments, the D3 binding molecules disclosed herein include one or more amino acid substitutions, additions and / or deletions in the framework regions, e.g., FRW1, FRW2, FRW3 and / or FRW4, of a single variable domain (e.g., VHH). In some embodiments, the N-terminal FRW1 and / or the C-terminal FRW4 of the single variable domain are truncated, e.g., truncated by no more than 5, 4, 3, 2, or 1 amino acids.

[0034] In some embodiments, the single variable domain (e.g., VHH) comprises the amino acid sequence set forth in any one of SEQ ID NOs: 12-18 and 55.

[0035] In some embodiments, the D3 binding molecules disclosed herein further comprise 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 the amino acid sequence of an IgG1 constant domain is shown in SEQ ID NO: 19. In some embodiments, the D3 binding molecules comprise one or more variants of human IgG1 constant domains, such as an IgG1 Fc with L234A / L235A substitutions, according to EU numbering.

[0036] In some embodiments, the D3 binding molecules disclosed herein have one or more of the following properties:

[0037] (a) binds to human D3, cynomolgus D3 and / or mouse D3 with an EC50 in nM as measured by ELISA or FACS;

[0038] (b) showed dose-dependent internalization potency in human D3-expressing cells; and

[0039] (c) Binds to human D3 with a KD of no more than 0.1 nM, as measured by SPR.

[0040] In some embodiments, the D3 binding molecules disclosed herein are chimeric antibodies, humanized antibodies, or fully human antibodies. In some embodiments, the D3 binding molecules are dimers.

[0041] In some embodiments, the D3 binding molecules disclosed herein comprise the single variable domain set forth in any one of SEQ ID NOs: 12-18 and 55 and the IgG constant domain set forth in SEQ ID NO:19.

[0042] In some embodiments, the disclosure provides nucleic acid molecules comprising a nucleic acid sequence encoding a D3 binding molecule disclosed herein, such as a D3 binding molecule comprising a single variable domain (eg, VHH).

[0043] In some embodiments, the present disclosure provides vectors comprising the nucleic acid molecules disclosed herein.

[0044] In some embodiments, the present disclosure provides host cells comprising an expression vector or nucleic acid molecule disclosed herein.

[0045] In some embodiments, the present disclosure provides pharmaceutical compositions comprising a D3 binding molecule disclosed herein and a pharmaceutically acceptable carrier.

[0046] In some embodiments, the present disclosure provides methods 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.

[0047] In some embodiments, the present disclosure provides methods of modulating a D3-related immune response in a subject, comprising administering to the subject a D3-binding molecule disclosed herein such that the D3-related immune response in the subject is modulated.

[0048] In some embodiments, the disclosure provides methods 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.

[0049] In some embodiments, the disclosure provides use of a D3-binding molecule disclosed herein in the manufacture of a medicament for diagnosing, treating, or preventing a D3-positive cancer.

[0050] In some embodiments, the disclosure provides a D3-binding molecule disclosed herein for use in diagnosing, treating, or preventing a D3-positive cancer.

[0051] In some aspects, the disclosure relates to kits or devices for using the D3-binding molecules disclosed herein or the pharmaceutical compositions disclosed herein, and related methods.

[0052] The foregoing is an overview and therefore necessarily contains simplifications, generalizations, and omitted details; therefore, those skilled in the art will appreciate that the overview is illustrative only 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 themes described herein will become apparent in the teachings set forth herein. An overview is provided to introduce the concept selections further described below in the detailed description in a simplified form. The overview is not intended to identify the key features or essential 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

[0053] Figure 1 Exemplary binding results of antibodies to immobilized WT115-hPro1.ECD.his measured by ELISA are shown.

[0054] Figure 2a and 2b Exemplary binding results of antibodies to WT115-293F.hPro1.2E5 cells measured by FACS are shown.

[0055] Figure 3a and 3b Exemplary binding results of antibodies to WT115-Flpin293.cPro1.pool cells measured by FACS are shown.

[0056] Figure 4a and 4b Exemplary binding results of antibodies to immobilized WT115-MBP-mPro1.ECD.hFc measured by ELISA are shown.

[0057] Figure 5a and 5b Exemplary internalization results of antibodies by WT115-293F.hPro1.2E5 cells are shown.

[0058] Figure 6a-6d Exemplary epitope binning results of antibodies on immobilized WT115-hPro1.ECD.his measured by ELISA are shown. Figure 6a , WT1156-P3R2-1C2-uIgG1 bin; Figure 6b , WT1156-P3R2-1H6-uIgG1 bin; Figure 6c , WT1156-P8R2-1H1-uIgG1 bin; Figure 6d , WT115-BMK1 box.

[0059] Figure 7aExemplary ELISA binding results of antibodies to soluble WT115-hPro1.ECD.his or truncated proteins measured by ELISA are shown. Figure 7b ELISA binding of antibodies to immobilized WT115-hPro1.ECD.his or truncated proteins measured by ELISA is shown. Figure 7c Diagrams of truncated proteins are shown.

[0060] Figure 8 Exemplary cross-family binding results of antibodies to human D1 and human D4 measured by ELISA are shown.

[0061] Fig. 9 Exemplary serum stability test results of WT1156-P3R2-1C2-z109-uIgG1 are shown.

[0062] Fig.10 Shown are the alignments 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 the CDRs are indicated by Kabat, AbM, Chothia, Contact and IMGT numbering. DETAILED DESCRIPTION

[0063] Although the present disclosure can be implemented in many different forms, specific illustrative embodiments of the principles of the present disclosure are disclosed herein. It should be emphasized that the present disclosure is not limited to the specific embodiments shown. In addition, any section headings used herein are only for organizational purposes and should not be interpreted as limiting the subject matter described.

[0064] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include pluralities, and plural terms shall include the singular. More specifically, unless the context clearly dictates otherwise, the singular forms "a / an" and "the" as used in this specification and the appended claims include plural indicators. Thus, for example, reference to "a protein" includes multiple proteins; reference to "a cell" includes mixtures of cells, etc. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms, such as "comprises / comprised", is not restrictive. In addition, the ranges provided in the specification and the appended claims include both endpoints and all points between the endpoints.

[0065] In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. Unless otherwise indicated, 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 this specification. See, e.g., Abbas et al., Cellular and Molecular Immunology, 6th ed., WB Saunders 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 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.

[0066] definition

[0067] In order to better understand the present disclosure, definitions and explanations of relevant terms are provided below.

[0068] 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 any form of antibody that exhibits the desired biological or binding activity. It encompasses, but is not limited to, humanized antibodies, fully human antibodies, chimeric antibodies, and single domain antibodies (sdAbs, comprising only one chain, typically similar to the heavy chain), as well as fragments of any of the foregoing as long as they exhibit the desired antigen binding activity, including, for example, antibodies comprising at least one VHH domain. Conventional antibodies comprise one or more heavy chains and one or more light chains. Heavy chains can be classified as μ, δ, γ, α, and ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. A heavy chain may comprise a heavy chain variable region (V H ) and the heavy chain constant region (C H The heavy chain may contain one or more constant regions, for example three constant regions (C H 1. C H 2 and C H 3). The light chain may include a light chain variable region (V L ) and the light chain constant region (C L ). H and V L The V regions can be further divided into regions of hypervariability, termed complementarity determining regions (CDRs), separated by regions that are relatively conserved, termed framework regions (FRWs). H and V L It may comprise 3 CDRs (complementarity determining regions) and 4 FRs (framework regions) in the following order: from N-terminus to C-terminus: FRW1, CDR1, FRW2, CDR2, FRW3, CDR3, FRW4. The antibody may have different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0069] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may be different, the human IgG heavy chain Fc region is generally defined as the amino acid residue from position Cys226 (according to the EU numbering system) or from Pro230 (according to the EU numbering system) to the segment of its carboxyl terminal. The C-terminal lysine (according to the residue 447 of the EU numbering system) in the Fc region can be removed, for example, during antibody production or purification, or by removing by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody.

[0070] A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require that the Fc region be bound to a binding region or binding domain (e.g., an antibody variable region or domain, including a VHH domain) and can be assessed using the various assays disclosed.

[0071] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature, and has not been manipulated, modified, and / or altered by humans (e.g., isolated, purified, selected, including other sequences, 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.

[0072] A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region due to at least one amino acid modification (e.g., substitution, addition or deletion), preferably one or more amino acid substitutions. In some embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parent polypeptide, for example, about one to about ten amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide, and preferably about one to about five amino acid substitutions. The variant Fc region may have at least about 80% homology with the native sequence Fc region and / or with the Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith. The variant Fc region described herein may lose effector function (e.g., silencing Fc).

[0073] The antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0074] The terms "immunoglobulin single variable domain" or "single variable domain" or "VHH domain" or "VHH" or "heavy chain antibody variable domain only" are used interchangeably herein and refer to a single chain antigen binding domain that is capable of binding to an antigen or epitope independently of a different variable domain. A VHH domain (e.g., a variable domain of a heavy chain antibody) represents the smallest known antigen binding unit produced by an adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13):3490-8. Epub 2007 Jun 15 (2007)). VHH domains can 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 derived from species including camels, llamas, alpacas, dromedaries, and guanacos that produce heavy chain antibodies that are naturally free 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.

[0075] The term "humanized antibody" refers to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, llama or alpaca, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0076] As used herein, the term "Ka" means the association rate of a particular antibody-antigen interaction, while the term "Kd" as used herein means the dissociation rate of a particular antibody-antigen interaction. The Kd value of an antibody can be determined using methods well established in the art. The term "KD" as used herein means 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 system.

[0077] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as, for example, an antibody and an antigen.

[0078] As used herein, the term "high affinity" refers to a D3 binding molecule, such as one that has a 1×10 affinity for a target antigen. - 7 M or less, more preferably 5×10 -8 M or less, even more preferably 1x10 -8 M or less, even more preferably 5×10 -9 M or less and even more preferably 1×10-9 Antibodies with a KD of M or less.

[0079] As used herein, the term “EC 50 ” Also known as “half-maximal effective concentration”, it refers to the concentration of a drug, antibody or toxicant that induces a response intermediate between baseline and maximum after a specified exposure time. In the context of this disclosure, EC 50 The unit is "nM".

[0080] As used herein, the term "epitope" refers to a part of an antigen that specifically binds to an immunoglobulin or antibody. "Epitope" is also referred to as an "antigenic determinant". An epitope or antigenic determinant generally comprises a chemically active surface group of a molecule, such as an amino acid, a carbohydrate or a sugar side chain, and generally has a specific three-dimensional structure and a specific charge characteristic. For example, an epitope generally comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or discontinuous amino acids in a unique spatial conformation, which may be "linear" or "conformation". See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, ed. (1996). In a linear epitope, all interaction sites between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction site spans amino acid residues separated from each other in a protein. Antibodies can be screened according to the competitiveness of binding to the same epitope by conventional techniques known to those skilled in the art. For example, competition or cross-competition studies can be performed to obtain antibodies that compete or cross-compete with each other for binding to the antigen. International patent application WO 03 / 48731 describes a high throughput method for obtaining antibodies that bind to the same epitope based on cross-competition of antibodies.

[0081] As used herein, the term "isolated antibody" means an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to a D3 protein is substantially free of antibodies that specifically bind to antigens other than a D3 protein). However, an isolated antibody that specifically binds to a human D3 protein may have cross-reactivity with other antigens (such as D3 proteins from other species). In addition, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0082] As used herein, the term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. A vector can carry genetic material elements expressed in a host cell by transformation, transduction or transfection into a host cell. Vectors are well known to those skilled in the art, including but not limited to plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phages 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, papillomaviruses, papovaviruses (such as SV40). A vector may include multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector may include an origin of replication.

[0083] 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 Escherichia coli (E. coli) or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, and animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0084] 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, as determined by alignment and comparison of the sequences. "Identity percentage" means the percentage of identical residues between the amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest molecule compared. For these calculations, gaps in the alignment, if any, are preferably resolved by a specific mathematical model or computer program (e.g., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, 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.

[0085] As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It not only refers to the properties of antigens stimulating the activation, proliferation and differentiation of specific immune cells to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also refers to the specific immune response of antibodies or sensitized T lymphocytes that can be formed in the immune system of an organism after the organism is stimulated with an antigen. Immunogenicity is an important property of an antigen. Whether an antigen can successfully induce an immune response in a host depends on a variety of factors, including the characteristics of the antigen, the reactivity of the host and the means of immunity.

[0086] As used herein, the term "transfection" refers to the process of introducing nucleic acids 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 techniques are well known in the art and 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.

[0087] As used herein, the term "SPR" or "surface plasmon resonance" refers to and includes an optical phenomenon that allows real-time biospecific interactions to be analyzed by detecting changes in protein concentration within a biosensor matrix (e.g., using a BIAcore system) (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For additional descriptions, see Examples and U., et al. (1993) Ann. Biol. Clin. 51: 19-26; 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.

[0088] As used herein, the term "fluorescence activated cell sorting" or "FACS" 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 based on the specific light scattering and fluorescence properties of each cell, one cell at a time (FlowMetric. "Sorting Out FluorescenceActivated Cell Sorting". Retrieved on 2017-11-09). Instruments for performing FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include FACS Star Plus, FACScan and FACSort instruments from Becton Dickinson (FosterCity, Calif.), Epics C from Coulter Epics Division (Hialeah, Fla.), and MoFlo from MoFlo from Cytomation (Colorado Springs, Colo.).

[0089] The term "subject" includes any human or non-human animal, preferably a human.

[0090] As used herein, the term "D3-associated disorder" or "D3-related disorder" refers to any disorder caused, exacerbated or otherwise associated with an increase or decrease (generally an increase) in the expression or activity of D3 (eg, human D3).

[0091] As used herein, the term "cancer" refers to any tumor or any malignant cell growth or proliferation, primary or metastatic, including solid tumors and non-solid tumors such as leukemias.

[0092] As used herein in the context of treating a disorder, the terms "treatment / treating / treated" generally relate to treatment or therapy, whether human or animal, in which some desired therapeutic effect is achieved, e.g., inhibiting the progression of the disorder, and includes reducing the rate of progression, stopping the rate of progression, regression of the disorder, ameliorating the disorder, and curing the disorder. Also included are treatments that are preventive measures (e.g., prophylaxis, prevention). For cancer, "treatment" may refer to suppressing or slowing tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof. For tumors, "treatment" includes removing all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

[0093] As used herein, the term "therapeutically effective amount" relates to an amount of an active compound, or a material, composition or dosage form comprising an active compound, that is effective to produce some desired therapeutic effect commensurate with a reasonable benefit / risk ratio when administered according to the desired treatment regimen. For example, a "therapeutically effective amount" of a D3 binding molecule refers to an amount or concentration that is effective to treat a D3-related disease or disorder in a human.

[0094] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide is introduced.

[0095] As used herein, the term "pharmaceutically acceptable" means that the vehicle, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

[0096] As used herein, the term "pharmaceutically 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, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Gennaro AR, 19th edition Pennsylvania: Mack Publishing Company, 1995), and includes, but is 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; ionic strength enhancers include, but are not limited to, sodium chloride. The carrier, excipient or stabilizer is nontoxic to cells or mammals exposed thereto at the doses and concentrations used. Typically, 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 nonionic surfactants such as TWEEN®. TM , polyethylene glycol (PEG) and PLURONICS TM. The term "carrier" may also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, or vehicle administered with the therapeutic agent. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the composition (e.g., pharmaceutical composition) is administered intravenously, water is an exemplary carrier. Saline solutions and aqueous dextrose solutions and glycerol solutions may also be used as liquid carriers, especially for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. If necessary, the composition may also contain a trace amount of a wetting agent or emulsifier, or a pH buffer. The compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral compositions, including formulations, can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable carriers are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Compositions, including pharmaceutical compounds, can contain a prophylactically or therapeutically effective amount of a D3 binding agent (e.g., an anti-D3 antibody), e.g., in isolated or purified form, and a suitable amount of a carrier so as to provide a form for proper administration to a subject (e.g., a patient). The formulation should be appropriate for the mode of administration.

[0097] As used herein, the term "adjuvant" refers to a non-specific immunopotentiator that, when delivered to an organism together with an antigen or delivered to an organism in advance, can enhance the immune response to the antigen or change the type of immune response in the organism. There are a variety of 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), anaerobic coryne bacterium parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

[0098] D3 binding molecules

[0099] In some aspects, the present disclosure provides D3 binding molecules. In a general sense, a D3 binding molecule may include any molecule that specifically binds to D3. In some cases, a "D3 binding molecule" may include a "D3 antagonist" and an "anti-D3 antibody". A "D3 antagonist" refers to any chemical compound or biological molecule that blocks the activity of D3. 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 protein, and may include other components, such as nucleotides, hybrids, dextran, and combinations thereof. As exemplified herein, a D3 binding molecule may be an anti-D3 antibody or an anti-D3 fusion protein.

[0100] In some embodiments, the D3 binding molecules disclosed herein comprise at least one VHH that specifically binds to D3. In addition, the D3 binding molecule can be a single domain antibody and comprise one VHH. For example, a single domain antibody is capable of selectively binding to a specific antigen (e.g., D3). In some embodiments, the D3 binding molecule comprises a VHH fused to an immunoglobulin Fc region, such as an Fc region of an IgG (e.g., IgG4 or IgG1). In some embodiments, the Fc region is the Fc region of human IgG1. By fusing the VHH to the Fc region, effector functions may be recruited more effectively. In addition, the fusion of the VHH to the Fc region can help the D3 binding molecule form a dimer and can also help extend the in vivo half-life of the D3 binding molecule.

[0101] As is known in the art, VHH molecules derived from camelid antibodies are among the smallest complete antigen-binding domains known (approximately 15 kDa, or 10-fold smaller than conventional IgG) and are therefore well suited for delivery into dense tissues and into the confined spaces between macromolecules.

[0102] VHH as disclosed herein can be prepared by a person skilled in the art according to methods known in the art or any future methods. For example, VHH can be obtained using methods known in the art, such as by immunizing camels and obtaining hybridomas therefrom, or by cloning the VHH library of the present disclosure using molecular biology techniques known in the art and then selecting using phage display.

[0103] For example, VHH can be obtained by immunizing a llama or alpaca with the desired antigen and then isolating the mRNA encoding the heavy chain antibody. By reverse transcription and polymerase chain reaction, a gene library of single domain antibodies containing millions of clones is generated. Screening techniques such as phage display and ribosome display help to identify clones that bind to antigens. One technique is phage display, in which a (e.g., human) antibody library is synthesized on a phage, the library is screened with a target antigen or its antibody binding portion, and the phage that binds to the antibody is separated, from which immunoreactive fragments can be obtained. 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, catalog number 27-9400-01; and Stratagene SurfZAP TM Phage Display Kit, Catalog No. 240612). There are other methods and reagents that can be used to generate and screen antibody display libraries (see, e.g., Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991)).

[0104] Once effective clones are identified, their DNA sequences are optimized, for example through affinity maturation or humanization, which prevents the human organism from developing an immune response to the antibody.

[0105] Thus, a VHH may be obtained: (1) by isolating a 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 (as described below) or by expression of a nucleic acid encoding such a humanized VHH domain; (4) by "camelization" of a naturally occurring VH domain from any animal species, in particular a mammalian species, such as a human, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by as described by Ward et al. (see above) "Camelization" of the "domain antibody" or "Dab" described above, or by expression of a nucleic acid encoding such a camelized VH domain; (6) using synthetic or semi-synthetic techniques to prepare a protein, polypeptide or other amino acid sequence; (7) preparing a nucleic acid encoding a VHH using techniques for nucleic acid synthesis and then expressing the nucleic acid thus obtained; (8) subjecting the heavy chain antibody or VHH to affinity maturation, mutagenesis (e.g., random mutagenesis or site-directed mutagenesis) and / or any other technique in order to increase the affinity and / or specificity of the VHH; and / or (9) by any combination of the foregoing. Based on the disclosure herein, suitable methods and techniques for performing the foregoing will be clear to the skilled person, and include, for example, the methods and techniques described in more detail herein.

[0106] Single domain antibodies are usually produced by PCR cloning the variable domain library from blood, lymph nodes or spleen cDNA obtained from immune animals into phage display vectors. Antigen-specific single domain antibodies are usually selected by panning the library on fixed antigens, such as antigens coated on the plastic surface of test tubes, biotinylated antigens fixed on streptavidin beads, or membrane proteins expressed on cell surfaces. The affinity of sdAb can usually be improved by simulating this strategy in vitro, for example, by site-directed mutagenesis in CDR regions and under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH and low antigen concentration), the fixed antigen is further selected (Wesolowski et al., Singledomain antibodies:promising experimental and therapeutic tools ininfection and immunity.Med Microbiol Immunol (2009) 198: 157-174).

[0107] Methods for preparing VHHs that specifically bind to an antigen or epitope are described in references, 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), p. 2645, June 17, 2009 and WO 94 / 04678.

[0108] In some embodiments, the VHH may be truncated at the N-terminus or C-terminus such that it comprises only a portion of FRW1 and / or FRW4, or lacks one or both of those framework regions, as long as the VHH substantially maintains 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%).

[0109] The present disclosure also provides D3 binding molecules with masking moieties and / or cleavable moieties, wherein one or more D3 binding domains of the D3 binding molecule are masked (e.g., by a masking moiety) and / or are activatable (e.g., by a cleavable moiety). Techniques for masking D3 binding molecules (e.g., antibodies) are well known in the art, including 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) can be used to prepare conjugates, including immunoconjugates, antibody-drug conjugates (ADCs), masked ADCs, and activatable antibody-drug conjugates (AADCs), comprising any of the D3 binding molecules (e.g., antibodies) of the present disclosure, including those directly or indirectly linked to another agent such as a drug. For example, the D3-binding molecules of the present disclosure can be covalently bound to one or more agents, such as drugs, via synthetic linkers.

[0110] If desired, the D3 binding molecule is linked or conjugated (directly or indirectly) to a moiety with effector function, such as cytotoxic activity (e.g., a chemotherapeutic moiety or a radioisotope) or immune recruitment activity. The moiety linked or conjugated (directly or indirectly) includes a cytotoxic drug (e.g., a toxin, such as an 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 activation of the D3 binding molecule by cleavage of the cleavable moiety to expose one or more binding domains of the D3 binding molecule in the tumor microenvironment, in the form of a masked conjugate). 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 additionally, the D3 binding molecule is optionally linked or conjugated (directly or indirectly) to a moiety that facilitates separation from a mixture (e.g., a tag) or a moiety with reporter activity (e.g., a detection marker or reporter protein). It should be understood that the features of the D3 binding molecules described herein also extend to polypeptides comprising fragments of the D3 binding molecule.

[0111] In some embodiments, the D3 binding molecules described herein can be linked or conjugated (directly or indirectly) to a polypeptide, which can result in the production of an activatable antibody. In some embodiments, the D3 binding molecules are linked or conjugated (directly or indirectly) to an agent. In some embodiments, the agent is a drug, resulting in the production of an ADC or AADC when the antibody of the ADC comprises a masking moiety and a cleavable moiety.

[0112] 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. The conjugated or recombinantly linked antibodies, including masked or activatable conjugates, can be used, for example, to treat or prevent a disease, disorder, or condition, such as cancer or a tumor.

[0113] For example, diagnosis and detection can be accomplished by coupling the D3 binding molecule to a detectable substance, which detectable substance includes, for example: 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 material, including but not limited to umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin; a luminescent material, including but not limited to luminol; a bioluminescent material, including but not limited to luciferase, luciferin, or aequorin; a chemiluminescent material, including but not limited to acridine-based compounds or HALOTAG; a radioactive material, including but not limited to 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 a, 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; and non-radioactive paramagnetic metal ions.

[0114] Conjugates of antibodies and agents, including those in which the agent is a drug for preparing 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-vinyl sulfone) benzoate). The present disclosure further contemplates conjugates of antibodies and agents, including those in which the agent is a drug for preparing ADC or AADC, which can be prepared using any suitable method disclosed in the art (see, e.g., Bioconjugate Techniques (Hermanson ed., 2nd ed. 2008)).

[0115] Conventional conjugation strategies for antibodies and agents, including drugs for preparing ADCs or AADCs, have been based on random conjugation chemistry involving the ε-amino group of Lys residues or the thiol group of Cys residues, which produces heterogeneous conjugates. Recently developed technologies allow site-specific conjugation with antibodies, thereby producing a uniform load and avoiding antigen binding or pharmacokinetic changes in conjugate subpopulations. These include the engineering of "thiomabs", which include cysteine ​​substitutions at positions on heavy and light chains, provide reactive thiol groups, and do not disrupt immunoglobulin folding and assembly or change 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). In another approach, selenocysteine ​​is co-translationally inserted into the antibody sequence by recoding the stop codon UGA from stop to selenocysteine ​​insertion, allowing site-specific covalent conjugation at the nucleophilic selenol group of selenocysteine ​​in the presence of other natural amino acids (see, e.g., Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105: 12451-56; and Hofer et al., 2009, Biochemistry 48(50): 12047-57).

[0116] The D3 binding molecules described herein may be monospecific, bispecific, trispecific, or have greater multispecificity. Such agents may include antibodies. Multispecific antibodies, such as bispecific antibodies, are monoclonal antibodies that have binding specificity for at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., a bispecific antibody for D3 having a first binding domain for a first epitope of D3 and a second binding domain for a second epitope of D3). In some embodiments, multispecific (e.g., bispecific) antibodies may 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 mouse antibodies, chimeric antibodies, human antibodies, or humanized antibodies. In some embodiments, one of the binding specificities of the multispecific antibody is for D3, and the other is for any other target (e.g., antigen). In some embodiments, a multispecific (e.g., bispecific) antibody may comprise more than one target (e.g., antigen) binding domain, wherein 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 modulator (e.g., a negative checkpoint modulator). In some embodiments, a multispecific (e.g., bispecific) antibody molecule may bind to more than one (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 one or more of the following: 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, T cell immunoreceptor with Ig and ITIM domains (TIGIT / Vstm3 / WUCAM / VSIG9), V-domain Ig inhibitor 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.

[0117] Methods for preparing multispecific antibodies are known in the art, such as by co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For more detailed information on generating multispecific antibodies (e.g., bispecific antibodies), see, e.g., bispecific antibodies (Kontermann ed., 2011).

[0118] The present disclosure provides humanized antibodies that bind D3. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically obtained from an "import" variable domain. Humanized antibodies that bind to D3 can be generated using techniques known to those of skill 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. Pat. Nos. 6,180,370; 6,054,927; 5,869,619; 5,861,155; 5,712,120; and 4,816,567).

[0119] In the following sections, D3 binding molecules may be described as anti-D3 antibodies.

[0120] Functional characterization of anti-D3 antibodies

[0121] Antibodies of the present disclosure (including, for example, antibodies comprising at least one VHH domain) are characterized by specific functional characteristics or properties of the antibodies. In some embodiments, the antibodies have one or more of the following properties:

[0122] (a) binds to human D3, cynomolgus D3, and mouse D3 with EC50 in nM as measured by ELISA or FACS;

[0123] (b) demonstrated dose-dependent internalization efficacy in human cells engineered to express D3; and

[0124] (c) Binds to human D3 ECD with a KD of no more than 0.1 nM as measured by SPR.

[0125] 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 the D3 protein, for example by flow cytometry. For example, the antibodies can be tested by flow cytometry assays (e.g., FACS), wherein the antibodies react with cell lines expressing human D3, such as CHO cells and 293 cells transfected to express D3 on their cell surfaces. Additionally or alternatively, the binding of the antibodies can be tested in BIAcore binding assays, including binding kinetics (e.g., Kd values). Still other suitable binding assays include ELISA assays, for example using recombinant D3 protein. For example, the antibodies of the present disclosure are tested at 1x10 -7 M or smaller, 5x10 -8 M or smaller, 2x10 -8 M or smaller, 5x10 -9 M or smaller, 4x10 -9 M or smaller, 3x10 -9 M or smaller, 2x10 -9 M or smaller, 1x10 -9 M or smaller, 5x10 -10 M or smaller or 1x10 -10 M or smaller K D Binds to cell surface D3 (eg, human D3 ECD) protein.

[0126] In some embodiments, the antibodies of the present disclosure have an EC of no more than or 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. 50 Binding to cynomolgus monkey or mouse D3 as measured by FACS.

[0127] Anti-D3 antibody containing VHH CDR

[0128] In some embodiments, an anti-D3 antibody disclosed herein comprises at least one immunoglobulin single variable domain (e.g., VHH), wherein the VHH comprises CDR1, CDR2, and CDR3, and 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.

[0129] The extent of the framework regions and CDRs can be precisely defined using methods known in the art, such as by 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, 5th ed., 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 USA. 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. The correspondence or alignment between the numberings according to different definitions can be found, for example, at www.imgt.org / (see also Giudicelli V et al. IMGT, the 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 Igsuperfamily V-like domains. Dev Comp Immunol. (2003) 27:55–77).

[0130] As will be appreciated by those skilled in the art, the precise numbering and position of CDRs may be different in different numbering systems. However, it will be appreciated that the disclosure of variable heavy sequences, variable light sequences and / or VHH sequences includes the disclosure of related (intrinsic) CDRs. Therefore, the disclosure of each variable region is the disclosure of CDRs (e.g., CDR1, CDR2, and CDR3). Two antibodies with identical VH, VL, or VHH CDRs mean that their CDRs are identical when measured by the same method (e.g., Kabat, AbM, Chothia, Contact, and IMGT numbering methods known in the art).

[0131] The variable region and CDR in the antibody sequence can be identified according to the general rules (e.g., Kabat, AbM, Chothia, Contact and IMGT numbering systems) developed in the art or by comparing the sequence with a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, ed., 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 below and can be accessed by: the "Abysis" website www.bioinf.org.uk / abs (maintained by AC Martin of 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). The sequences are preferably analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein DataBank (PDB) with structural data from the PDB. See the book chapter Protein Sequence and Structure Analysis of Antibody Variable Domains by Dr. Andrew CR Martin. In: Antibody Engineering Lab Manual (Editors: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13:978-3540413547, also available at the website bioinforg.uk / abs). The Abysis database website also includes general rules that have been developed for identifying CDRs that can be used according to the teachings herein. Fig.10 An alignment of exemplary immunoglobulin single variable domains is shown, and the boundaries of the CDRs are indicated by Kabat, AbM, Chothia, Contact, and IMGT numbering.

[0132] In some embodiments, the D3 binding molecules disclosed herein comprise at least one immunoglobulin single variable domain (e.g., VHH), wherein the VHH comprises FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4, and wherein CDR1 has the amino acid sequence shown in SEQ ID NO: 1, 4, 7 or 10, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, 5, 8 or 11, and CDR3 has the amino acid sequence shown in SEQ ID NO: 3, 6 or 9. In some embodiments, FRW1 and FRW4 at the N-terminus and C-terminus of the VHH contained in the D3 binding molecule may be truncated so that it only comprises part of FRW1 and / or FRW4, or the VHH lacks one or two of these framework regions, as long as the VHH substantially maintains antigen binding and specificity.

[0133] In some embodiments, provided herein are anti-D3 antibodies (such as anti-D3 single domain antibodies) comprising one, two, or all three CDRs of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, provided herein are anti-D3 antibodies (such as anti-D3 single domain antibodies) comprising one, two, or all three CDRs of the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, provided herein are anti-D3 antibodies (such as anti-D3 single domain antibodies) comprising one, two, or all three CDRs of the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, provided herein are anti-D3 antibodies (such as anti-D3 single domain antibodies) comprising one, two, or all three CDRs of the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, provided herein are anti-D3 antibodies (such as anti-D3 single domain antibodies) comprising one, two, or all three CDRs of the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, provided herein are anti-D3 antibodies (such as anti-D3 single domain antibodies) comprising 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, comprising 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, comprising 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 of 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, e.g., a human immunoglobulin framework or a human consensus framework.

[0134] In some embodiments, an anti-D3 antibody (such as a single domain antibody) comprises a CDR1 having the amino acid sequence of CDR1 as shown 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 as shown in SEQ ID NO: 12. In other embodiments, an anti-D3 antibody (such as a single domain antibody) comprises a CDR3 having the amino acid sequence of CDR3 as shown 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 sequence of CDR1 and CDR2 as shown 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 sequence of CDR1 and CDR3 as shown 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 sequence of CDR2 and CDR3 as shown in SEQ ID NO: 12. In some embodiments, an anti-D3 antibody (such as a single domain antibody) comprises CDR1, CDR2, and CDR3 having the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 12. CDR sequences can be identified according to a well-known numbering system. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to Kabat. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to AbM. In some embodiments, the anti-D3 single domain antibody is of 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, e.g., a human immunoglobulin framework or a human consensus framework.

[0135] In some embodiments, the single domain antibody has a CDR1 having the amino acid sequence of CDR1 as shown in SEQ ID NO: 13. In some embodiments, the single domain antibody has a CDR2 having the amino acid sequence of CDR2 as shown in SEQ ID NO: 13. In other embodiments, the single domain antibody has a CDR3 having the amino acid sequence of CDR3 as shown in SEQ ID NO: 13. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as shown in SEQ ID NO: 13. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as shown in SEQ ID NO: 13. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as shown in SEQ ID NO: 13. In some embodiments, the single domain antibody has CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 have the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 13. The CDR sequence can be determined according to a well-known numbering system. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to Kabat. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to AbM. In some embodiments, the anti-D3 single domain antibody is of 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, e.g., a human immunoglobulin framework or a human consensus framework.

[0136] In some embodiments, the single domain antibody has a CDR1 having the amino acid sequence of CDR1 as shown in SEQ ID NO: 14. In some embodiments, the single domain antibody has a CDR2 having the amino acid sequence of CDR2 as shown in SEQ ID NO: 14. In other embodiments, the single domain antibody has a CDR3 having the amino acid sequence of CDR3 as shown in SEQ ID NO: 14. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as shown in SEQ ID NO: 14. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as shown in SEQ ID NO: 14. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as shown in SEQ ID NO: 14. In some embodiments, the single domain antibody has CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 have the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 14. The CDR sequence can be determined according to a well-known numbering system. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to Kabat. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to AbM. In some embodiments, the anti-D3 single domain antibody is of 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, e.g., a human immunoglobulin framework or a human consensus framework.

[0137] In some embodiments, the single domain antibody has a CDR1 having the amino acid sequence of CDR1 as shown in SEQ ID NO: 15. In some embodiments, the single domain antibody has a CDR2 having the amino acid sequence of CDR2 as shown in SEQ ID NO: 15. In other embodiments, the single domain antibody has a CDR3 having the amino acid sequence of CDR3 as shown in SEQ ID NO: 15. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as shown in SEQ ID NO: 15. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as shown in SEQ ID NO: 15. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as shown in SEQ ID NO: 15. In some embodiments, the single domain antibody has CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 have the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 15. The CDR sequence can be determined according to a well-known numbering system. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to Kabat. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to AbM. In some embodiments, the anti-D3 single domain antibody is of 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, e.g., a human immunoglobulin framework or a human consensus framework.

[0138] In some embodiments, the single domain antibody has a CDR1 having the amino acid sequence of CDR1 as shown in SEQ ID NO: 16. In some embodiments, the single domain antibody has a CDR2 having the amino acid sequence of CDR2 as shown in SEQ ID NO: 16. In other embodiments, the single domain antibody has a CDR3 having the amino acid sequence of CDR3 as shown in SEQ ID NO: 16. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as shown in SEQ ID NO: 16. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as shown in SEQ ID NO: 16. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as shown in SEQ ID NO: 16. In some embodiments, the single domain antibody has CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 have the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 16. The CDR sequence can be determined according to a well-known numbering system. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to Kabat. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to AbM. In some embodiments, the anti-D3 single domain antibody is of 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, e.g., a human immunoglobulin framework or a human consensus framework.

[0139] In some embodiments, the single domain antibody has a CDR1 having the amino acid sequence of CDR1 as shown in SEQ ID NO: 17. In some embodiments, the single domain antibody has a CDR2 having the amino acid sequence of CDR2 as shown in SEQ ID NO: 17. In other embodiments, the single domain antibody has a CDR3 having the amino acid sequence of CDR3 as shown in SEQ ID NO: 17. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as shown in SEQ ID NO: 17. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as shown in SEQ ID NO: 17. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as shown in SEQ ID NO: 17. In some embodiments, the single domain antibody has CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 have the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 17. The CDR sequence can be determined according to a well-known numbering system. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to Kabat. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to AbM. In some embodiments, the anti-D3 single domain antibody is of 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, e.g., a human immunoglobulin framework or a human consensus framework.

[0140] In some embodiments, the single domain antibody has a CDR1 having the amino acid sequence of CDR1 as shown in SEQ ID NO: 18. In some embodiments, the single domain antibody has a CDR2 having the amino acid sequence of CDR2 as shown in SEQ ID NO: 18. In other embodiments, the single domain antibody has a CDR3 having the amino acid sequence of CDR3 as shown in SEQ ID NO: 18. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having the amino acid sequences of CDR1 and CDR2 as shown in SEQ ID NO: 18. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having the amino acid sequences of CDR1 and CDR3 as shown in SEQ ID NO: 18. In some embodiments, the single domain antibody has a CDR2 and a CDR3 having the amino acid sequences of CDR2 and CDR3 as shown in SEQ ID NO: 18. In some embodiments, the single domain antibody has CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 have the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 18. The CDR sequence can be determined according to a well-known numbering system. In some embodiments, the CDRs are numbered according to IMGT. In some embodiments, the CDRs are numbered according to Kabat. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, the CDRs are numbered according to Contact. In some embodiments, the CDRs are numbered according to AbM. In some embodiments, the anti-D3 single domain antibody is of 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, e.g., a human immunoglobulin framework or a human consensus framework.

[0141] In some embodiments, provided herein is a single domain antibody that binds to D3, the antibody comprising the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein (i) CDR1 comprises the amino acid sequence shown 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; (ii) CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, 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: 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) the CDR3 comprises the amino acid sequence shown 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 of 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, e.g., a human immunoglobulin framework or a human consensus framework.

[0142] 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, according to IMGT numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:20; according to IMGT numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:21; and according to IMGT numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:22. In some embodiments, according to Kabat numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:23; according to Kabat numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:2; and according to Kabat numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, according to Chothia numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:24; according to Chothia numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:25; and according to Chothia numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, according to Contact numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:27; according to Contact numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:28 or 56; and according to Contact numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:29. In some embodiments, according to AbM numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:1; according to AbM numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:30; and according to AbM numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the anti-D3 single domain antibody is of 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, e.g., a human immunoglobulin framework or a human consensus framework.

[0143] 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, according to IMGT numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:31; according to IMGT numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:32; and according to IMGT numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:33. In some embodiments, according to Kabat numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:34; according to Kabat numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:5; and according to Kabat numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, according to Chothia numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:35; according to Chothia numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:36; and according to Chothia numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:37. In some embodiments, according to Contact numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:38; according to Contact numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:39; and according to Contact numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:40. In some embodiments, according to AbM numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:4; according to AbM numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:41; and according to AbM numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the anti-D3 single domain antibody is of 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, e.g., a human immunoglobulin framework or a human consensus framework.

[0144] 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, according to IMGT numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:42; according to IMGT numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:43; and according to IMGT numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:44. In some embodiments, according to Kabat numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:45; according to Kabat numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:8; and according to Kabat numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, according to Chothia numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:46; according to Chothia numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:47; and according to Chothia numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:48. In some embodiments, according to Contact numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:49; according to Contact numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:50; and according to Contact numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:51. In some embodiments, according to AbM numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:7; according to AbM numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:52; and according to AbM numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the anti-D3 single domain antibody is of 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, e.g., a human immunoglobulin framework or a human consensus framework.

[0145] 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, according to IMGT numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 53; according to IMGT numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32; and according to IMGT numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, according to Kabat numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 34; according to Kabat numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 11; and according to Kabat numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, according to Chothia numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 54; according to Chothia numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 36; and according to Chothia numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, according to Contact numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:38; according to Contact numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:39; and according to Contact numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:40. In some embodiments, according to AbM numbering, CDR1 comprises the amino acid sequence set forth in SEQ ID NO:10; according to AbM numbering, CDR2 comprises the amino acid sequence set forth in SEQ ID NO:41; and according to AbM numbering, CDR3 comprises the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the anti-D3 single domain antibody is of 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, e.g., a human immunoglobulin framework or a human consensus framework.

[0146] 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 a sequence as shown in SEQ ID NO: 12. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising a sequence as shown in SEQ ID NO: 13. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising a sequence as shown 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.

[0147] In some embodiments, the single domain antibodies provided herein are humanized single domain antibodies.

[0148] The framework regions described herein are determined based on the boundaries of the CDR numbering system. In other words, if CDRs are determined by, for example, IMGT, Kabat, Chothia, Contact or AbM, the framework regions are the amino acid residues surrounding the CDRs in the variable region from the N-terminus to the C-terminus in the following format: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residue N-terminal to the CDR1 amino acid residues as defined, e.g., by the IMGT numbering system, the Kabat numbering system, the Chothia numbering system, the Contact numbering system, or the AbM numbering system, FR2 is defined as the amino acid residues between the CDR1 and CDR2 amino acid residues as defined, e.g., by the IMGT numbering system, the Kabat numbering system, the Chothia numbering system, the Contact numbering system, or the AbM numbering system, FR3 is defined as the amino acid residues between the CDR2 and CDR3 amino acid residues as defined, e.g., by the IMGT numbering system, the Kabat numbering system, the Chothia numbering system, the Contact numbering system, or the AbM numbering system, and FR4 is defined as the amino acid residue C-terminal to the CDR3 amino acid residues as defined, e.g., by the IMGT numbering system, the Kabat numbering system, the Chothia numbering system, the Contact numbering system, or the AbM numbering system.

[0149] In some embodiments, an isolated anti-D3 single domain antibody comprising a VHH domain having an amino acid sequence as shown in SEQ ID NO: 12 is provided. In some embodiments, a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 12 is provided. In some embodiments, an isolated anti-D3 single domain antibody comprising a VHH domain having an amino acid sequence as shown in SEQ ID NO: 13 is provided. In some embodiments, a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 13 is provided. In some embodiments, an isolated anti-D3 single domain antibody comprising a VHH domain having an amino acid sequence as shown in SEQ ID NO: 14 is provided. In some embodiments, a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 14 is provided. In some embodiments, an isolated anti-D3 single domain antibody comprising a VHH domain having an amino acid sequence as shown in SEQ ID NO: 15 is provided. In some embodiments, a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 15 is provided. In some embodiments, an isolated anti-D3 single domain antibody comprising a VHH domain having an amino acid sequence as shown in SEQ ID NO: 16 is provided. In some embodiments, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16 is provided. In some embodiments, an isolated anti-D3 single domain antibody comprising a VHH domain having the amino acid sequence set forth in SEQ ID NO: 17 is provided. In some embodiments, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 17 is provided. In some embodiments, an isolated anti-D3 single domain antibody comprising a VHH domain having the amino acid sequence set forth in SEQ ID NO: 18 is provided. In some embodiments, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 18 is provided. In some embodiments, an isolated anti-D3 single domain antibody comprising a VHH domain having the amino acid sequence set forth in SEQ ID NO: 55 is provided. In some embodiments, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 55 is provided.

[0150] Anti-D3 antibodies comprising VHH sequences

[0151] In some embodiments, the anti-D3 antibody comprises at least one immunoglobulin single variable domain (e.g., VHH), wherein the VHH comprises or consists of:

[0152] (A) the amino acid sequence shown in any one of SEQ ID NOs: 12-18 and 55;

[0153] (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

[0154] (C) an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids added, deleted and / or substituted compared to any one of SEQ ID NOs: 12-18 and 55.

[0155] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0156] Additionally or alternatively, protein (e.g., antibody) sequences of the present disclosure may also be used as "query sequences" to search public databases, e.g., to identify related sequences. Such searches may be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215:403-10. BLAST protein searches may be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the antibody molecules of the present disclosure. In order to obtain gapped alignments for comparison purposes, gapped BLAST may be used as described in Altschul et al. (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using BLAST and gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) may be used. See www.ncbi.nlm.nih.gov.

[0157] In some embodiments, the amino acid sequence of the VHH may be at least 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.

[0158] In some other embodiments, the anti-D3 antibodies may contain 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 abrogate 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.

[0159] As mentioned above, as used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the basic properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which the amino acid residue is replaced by another amino acid residue with a similar side chain, such as a residue physically or functionally similar to the corresponding amino acid residue (such as having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with 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 beta-branched side chains (such as threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the corresponding amino acid residue is preferably substituted by 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 herein by reference).

[0160] In some embodiments, the anti-D3 antibody comprises at least one VHH, and the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 12- 18. In some embodiments, the anti-D3 antibody comprises a VHH, and the VHH has the amino acid sequence set forth in any one of SEQ ID NOs: 12-18.

[0161] In some embodiments, the anti-D3 antibody is a chimeric antibody comprising a VHH fused to the Fc region of human IgG1 or IgG4, wherein the VHH comprises the amino acid sequence shown 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 the Fc region of human IgG1. 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 other embodiments, the anti-D3 antibody is a humanized antibody comprising a VHH and the Fc region of human IgG1. Such antibodies are exemplified herein as “WT1156-P3R2-1C2-z102-uIgG1”, “WT1156-P3R2-1C2-z109-uIgG1”, and “WT1156-P3R2-1H6-z100-uIgG1”.

[0162] In some embodiments, the addition, deletion and / or substitution of at least one amino acid in the VHH region is not in any of the CDR sequences, but in the framework (FRW) sequence. For example, an antibody or antigen-binding portion thereof as described above may comprise one or more substitutions of amino acids in the framework sequence, such as FRW1, FRW2, FRW3 and / or FRW4 of the VHH region.

[0163] 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 can specifically bind to D3.

[0164] As described above, the antibody or its antigen-binding portion thereof may contain modifications of one or more amino acids in the variable region of the heavy chain and / or light chain, including where the modifications are conservative substitutions. It will be appreciated in the art that certain conservative sequence modifications may be made that will not remove 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.

[0165] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a VHH domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 12-18 and 55 and an Fc region comprising the amino acid sequence set forth in SEQ ID NO: 19.

[0166] The antigen binding domain of the D3 binding molecule is not limited to the VHH format and may take a variety of other formats such as, but not limited to, Fab, Fab', F(ab') 2 , Fv fragments, single-chain antibody molecules (scFv). In some embodiments, the antigen binding domain is an Fv fragment, which has VH and VL regions in separate chains held together by tight non-covalent interactions.

[0167] Fc region containing the IgG constant domain

[0168] The anti-D3 antibodies and antigen-binding fragments provided herein also comprise an Fc region comprising one or more human IgG constant domains. The human IgG constant domain can 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 region is shown 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 functions.

[0169] In some embodiments, the Fc modification comprises a LALA mutation, such as a mutation of L234A and L235A according to the EU numbering in Kabat et al. The Kabat numbering system is generally used when referring to the residues in the variable domain (about residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to the residues in the constant region of the immunoglobulin heavy chain (e.g., the EU index reported in Kabat et al. above). "EU numbering in Kabat" or "EU index in Kabat" refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise specified herein, reference to the residue numbering in the antibody constant domain means the residue numbering by the EU numbering system.

[0170] Nucleic acid molecules encoding the antibodies of the present disclosure

[0171] In some aspects, the disclosure provides nucleic acid molecules comprising a nucleic acid sequence encoding a D3 binding molecule disclosed herein, e.g., encoding a single variable domain of a D3 binding molecule disclosed herein. The nucleic acids of the disclosure can be obtained using standard molecular biology techniques.

[0172] 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 nucleic acid encoding the VHH region can be converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat et al. (1991), supra) and DNA fragments covering 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, such as an IgG1 constant region.

[0173] Once the nucleic acids encoding the VHH segments are obtained, these nucleic acids can be further manipulated by standard recombinant DNA techniques, for example, converting the variable region genes into full-length antibody chain genes. In these manipulations, the nucleic acids encoding the VHHs are operably linked to another nucleic acid encoding another protein, such as an antibody constant region or a flexible linker. As used herein, the term "operably linked" means that two or more nucleic acids are linked so that the amino acid sequences encoded by the two or more nucleic acids remain in frame.

[0174] In some embodiments, the disclosure relates to nucleic acid molecules comprising a nucleic acid sequence encoding a single variable domain (eg, VHH) of a D3-binding molecule disclosed herein.

[0175] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of:

[0176] (A) a nucleic acid sequence encoding a VHH region as shown in any one of SEQ ID NOs: 12-18 and 55;

[0177] (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

[0178] (C) A nucleic acid sequence that hybridizes to the complementary strand of the nucleic acid sequence of (A) under high stringency conditions.

[0179] In some embodiments, provided herein are nucleic acid molecules 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 are nucleic acid molecules 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 are nucleic acid molecules 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 are nucleic acid molecules 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 are nucleic acid molecules 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 are nucleic acid molecules 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 are nucleic acid molecules 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 are nucleic acid molecules comprising a nucleic acid sequence encoding an anti-D3 single domain antibody comprising the amino acid sequence set forth in SEQ ID NO: 55.

[0180] In some embodiments, the percent identity results from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0181] Exemplary high stringency conditions include hybridization at 45°C in 5X SSPE and 45% formamide, and final washing at 65°C in 0.1X SSC. It will be appreciated in the art that conditions of equal stringency can be achieved by changing the 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 to 6.4.10. The change in hybridization conditions can be determined empirically or accurately calculated based on the length and percentage of the guanosine / cytosine (GC) base pairing of the probe. 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 to 9.51.

[0182] Host cells

[0183] The host cells disclosed in the present disclosure can be any cell suitable for expressing the antibodies of the present disclosure, such as yeast, bacteria, plants and mammalian cells. Mammalian host cells for expressing the antibodies of the present disclosure include Chinese hamster ovary (CHO cells) (including dhfr CHO cells, as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci USA 77: 4216-4220, used with a DHFR selectable marker, for example, as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159: 601-621), 293F cells, NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036 and EP 338,841.Also included are monkey kidney CV1 cell lines transformed by SV40 (COS-7, ATCC CRL-1651); human embryonic kidney cell lines (293 cells or 293 cells subcloned for growth in suspension culture; Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL-10); Chinese hamster ovary 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 cancer cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); buffalo rat liver cells (BRL 3A, ATCC CRL1442); human lung cells (W138, ATCC CCL-75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68); MRC5 cells; FS4 cells; mouse myeloma cells, such as NSO (e.g., RCB0213, 1992, Bio / Technology 10:169) and SP2 / 0 cells (e.g., SP2 / 0-Ag14 cells, ATCC CRL 1581); rat myeloma cells, such as YB2 / 0 cells (e.g., YB2 / 3HL.P2.G11.16Ag.20 cells, ATCC CRL 1662); PER.C6 cells; and human hepatoma cell 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, these may be altered so that they lack the ability to fucosylate proteins expressed therein. In some embodiments, the host cell herein is selected from CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NSO cells or lymphocytes.

[0184] Prokaryotes suitable for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, typhimurium, Serratia such as Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

[0185] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody encoding vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among the lower eukaryotic host microorganisms. However, many other genera, species and strains are commonly available and can be used herein, such as 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. drosophila (ATCC 36,906), K. thermotolerans and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesei (EP 244, 234); Neurospora crassa; Schwannella such as Schwannella occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolylcoceras, and Aspergillus hosts such as Aspergillus nidulans and Aspergillus niger.

[0186] When a recombinant expression vector encoding an antibody is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell or secreted into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0187] Pharmaceutical composition

[0188] In some aspects, the present disclosure provides pharmaceutical compositions comprising a D3 binding molecule disclosed herein (e.g., 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 pharmaceutical compositions comprising a nucleic acid encoding a D3 binding molecule disclosed herein (e.g., 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 pharmaceutical compositions comprising a cell expressing a D3 binding molecule disclosed herein (e.g., comprising a single variable domain (e.g., VHH) of a D3 binding molecule disclosed herein) and a pharmaceutically acceptable carrier.

[0189] Components of the composition

[0190] The pharmaceutical composition may optionally contain one or more additional components, including one or more pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present disclosure may also be administered in combination therapy with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, including where the anti-D3 antibody enhances the immune response. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquids, gels, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending agents / dispersants, chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other various components known in the art, or a combination of multiple components.

[0191] Suitable components of pharmaceutical compositions may include, for example, antioxidants, fillers, adhesives, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, coloring agents, emulsifiers or stabilizers, such as sugar and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butyl methyl anisole, butylated hydroxytoluene and / or propyl gallate. As disclosed in the present disclosure, compositions may include antibodies or Fabs of the present disclosure, and also include one or more antioxidants such as methionine, to prevent or reduce the reduction of binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, the present disclosure provides compositions comprising one or more antibodies or Fabs thereof and one or more antioxidants such as methionine. The present disclosure also provides methods in which antibodies or antigen-binding fragments thereof are mixed with one or more antioxidants, such as methionine, so that the antibodies or antigen-binding fragments thereof can be prevented from oxidation to extend their shelf life and / or increase their activity.

[0192] To further illustrate, pharmaceutically 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 at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN-80), sequestering / chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, 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 multiple dose containers, including phenol or cresol, mercurial, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, glucose, glycerol or ethanol. Suitable nontoxic auxiliary substances may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, dissolution enhancers or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.

[0193] Administration, formulation and dosing

[0194] The pharmaceutical compositions of the present disclosure can be administered to subjects in need by a variety of routes, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or otherwise by implantation or inhalation. The subject composition can be formulated into a preparation in the form of a solid, semisolid, liquid or gas; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. Appropriate formulations and routes of administration can be selected according to the intended application and treatment regimen.

[0195] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalants and controlled release forms thereof.

[0196] Formulations suitable 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 also contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohol, polyols, glycerol, vegetable oils, etc. Examples of suitable isotonic carriers for use in the formulation include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific dosing regimens, including dosage, time schedule, and repetition, will depend on the specific individual and the individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0197] The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of proliferative or tumorigenic cells, maintaining a reduction in such tumor cells, reducing the proliferation of tumor cells, or delaying the development of metastases. In some embodiments, the dose administered can be adjusted or attenuated to manage potential side effects and / or toxicity. Alternatively, a sustained continuous release formulation of the subject therapeutic composition may be appropriate.

[0198] It will be appreciated by those skilled in the art that appropriate dosage may vary from patient to patient. Determining the optimal dosage will generally involve balancing between the level of therapeutic benefit and any risk or harmful side effects. The selected dosage level will depend on various factors, including but not limited to the activity of the specific compound, the route of administration, the time of administration, the excretion rate of the compound, the duration of treatment, other drugs, compounds and / or materials used in combination, the severity of the illness, and the species, sex, age, body weight, illness, general health and previous medical history of the patient. The amount of the compound and the route of administration will ultimately be determined by a physician, veterinarian or clinician, but the dosage will generally be selected to reach a local concentration at the site of action, thereby achieving the desired effect without causing substantial harmful or toxic side effects.

[0199] In general, the D3 binding molecules of the present disclosure can be administered in various 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 the described range. 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.

[0200] In any case, the antibodies or antigen-binding portions thereof of the present disclosure are preferably administered to subjects in need as needed. The determination of the frequency of administration can be made by a person skilled in the art, such as an attending physician, based on considerations of the disorder being treated, the age of the subject being treated, the severity of the disorder being treated, and the overall health of the subject being treated.

[0201] In some embodiments, a course of treatment involving a D3 binding molecule of the present disclosure will include multiple doses of a selected drug product over a period of weeks or months. For example, a D3 binding molecule of the present disclosure may be administered once a day, every two days, every four days, every week, every ten days, every two weeks, every three weeks, every month, every six weeks, every two months, every ten weeks, or every three months. In this regard, it should be understood that the dosage may be varied or the interval adjusted based on patient response and clinical practice.

[0202] The dosage and regimen of the disclosed therapeutic composition can also be determined empirically in individuals who have received one or more administrations. For example, an increasing dose of a therapeutic composition produced as described herein can be given to an individual. In some embodiments, the dosage can be gradually increased or decreased or weakened based on the side effects or toxicity determined or observed empirically, respectively. In order to evaluate the efficacy of the selected composition, markers of a specific disease, disorder or illness can be tracked as previously described. For cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques; improvement assessed by direct tumor biopsy and microscopic examination of tumor samples; measurement of indirect tumor markers (e.g., PSA for prostate cancer) or tumorigenic antigens, relief of pain or paralysis; improvement of speech, vision, breathing or other disabilities associated with tumors; increased appetite; or improvement of quality of life or extension of survival measured by recognized tests. It is obvious to those skilled in the art that the dosage will vary according to the individual, the type of tumor disorder, the stage of the tumor disorder, whether the tumor disorder has begun to metastasize to other locations of the individual, and the past and concurrent treatments used.

[0203] Compatible formulations for parenteral administration (e.g., intravenous injection) may include a D3 binding molecule 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., antibody or antigen-binding portion thereof) will include 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., antibody or antigen binding portion thereof) will include 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.

[0204] Applications of the present disclosure

[0205] The antibodies, antibody compositions and methods disclosed herein have a variety of in vitro and in vivo utilities and uses, including, for example, detecting D3 or enhancing immune responses. For example, these molecules can be administered to cells in culture in vitro or ex vivo, or, for example, administered to human subjects in vivo, to enhance immunity in various situations. The immune response can be modulated, for example, enhanced, stimulated or upregulated.

[0206] For example, the subject includes a human patient in need of an enhanced immune response. The methods are particularly suitable for treating a human patient suffering from a condition that can be treated by enhancing an immune response (e.g., a T cell-mediated immune response). In some embodiments, the methods are particularly suitable for treating cancer in vivo. To achieve antigen-specific enhancement of immunity, the anti-D3 antibody can be administered with the target antigen, or the antigen may already be present in the subject to be treated (e.g., a tumor-bearing or virus-bearing subject). When an antibody to D3 is administered with another agent, the two can be administered sequentially or simultaneously.

[0207] The present disclosure also provides a method for detecting the presence of human D3 antigen in a sample or measuring the amount of human D3 antigen, comprising contacting a sample and a control sample, for example, with a human monoclonal antibody or an antigen-binding portion thereof that specifically binds to human D3, under conditions that allow formation of a complex between the antibody or a portion thereof and human D3. The formation of the complex is then detected, wherein a difference in complex formation between the sample and the control sample indicates the presence of human D3 antigen in the sample. In addition, the anti-D3 antibodies of the present disclosure can be used to purify human D3 by immunoaffinity purification.

[0208] Treatment of conditions including cancer

[0209] In some aspects, the disclosure provides methods of treating a condition 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 an antigen binding portion thereof, disclosed herein. In some aspects, the disclosure provides an anti-D3 antibody, or an antigen binding portion thereof, disclosed herein for use in treating a disease or condition. In some aspects, provided herein is a use of an anti-D3 antibody, or an antigen binding portion thereof, disclosed herein for the manufacture of a medicament for treating a disease or condition. The condition or disease may be cancer.

[0210] A variety of cancers involving D3, whether malignant or benign and whether primary or secondary, can be treated or prevented using the methods provided by the present disclosure. Cancers can include, but are not limited to, lung cancer (including various subtypes, such as small cell and non-small cell lung cancer), adrenal cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, stomach 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. Exemplary cancers include, for example, small cell lung cancer, large cell neuroendocrine cancer, glioblastoma, Ewing's sarcoma, and cancers with a neuroendocrine phenotype.

[0211] The anti-D3 antibodies disclosed herein can be used to treat lung cancer, such as bronchial carcinoma, non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma, e.g., lung adenocarcinoma. 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).

[0212] Cancers to be treated by the anti-D3 antibodies disclosed herein may also be large cell neuroendocrine carcinoma (LCNEC), medullary thyroid carcinoma, glioblastoma, neuroendocrine prostate cancer (NEPC), high-grade gastroenteropancreatic cancer (GEP), and malignant melanoma. The anti-D3 antibodies disclosed herein may be used to treat neuroendocrine tumors (NETs and pNETs) arising in the kidney, genitourinary tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid carcinoma), and lung (small cell lung cancer and large cell neuroendocrine carcinoma).

[0213] As noted above, anti-D3 antibodies are particularly effective for treating lung cancer, including the following subtypes: small cell lung cancer and non-small cell lung cancer (eg, squamous cell non-small cell lung cancer or squamous cell small cell lung cancer) and large cell neuroendocrine carcinoma.

[0214] Stimulation of immune response

[0215] In some aspects, the disclosure also provides methods of enhancing (e.g., stimulating) an immune response in a subject, comprising administering to the subject a D3 binding molecule of the disclosure, such as an anti-D3 antibody or an antigen binding portion thereof, such that the immune response in the subject is enhanced. In some aspects, the disclosure provides an anti-D3 antibody or an antigen binding portion thereof disclosed herein for use in enhancing (e.g., stimulating) an immune response in a subject. In some aspects, provided herein is a use of an anti-D3 antibody or an antigen binding portion thereof disclosed herein for the manufacture of a medicament for enhancing (e.g., stimulating) an immune response in a subject. For example, in some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0216] The term "enhancing an immune response" or grammatical variations thereof means any response that stimulates, elicits, increases, improves or enhances the immune system of a mammal. The immune response can 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 can be a primary or secondary immune response. Examples of enhancing an immune response include increased CD4 + Helper T cell activity and the production of cytolytic T cells. The enhancement of the immune response can be evaluated using a variety of in vitro or in vivo measurements known to those skilled in the art, including but not limited to cytotoxic T lymphocyte assays, the release of cytokines (e.g., IL-2 production or IFN-γ production), tumor regression, survival of tumor-bearing animals, antibody production, immune cell proliferation, cell surface marker expression, and cytotoxicity. For example, when compared to the immune response of an untreated mammal or a mammal not treated with the methods disclosed herein, the methods disclosed herein can be used to enhance the immune response of a mammal.

[0217] The D3 binding molecules can be used alone as a monotherapy, or can be used in combination with chemotherapy, radiotherapy, targeted therapy or cellular immunotherapy, etc.

[0218] Combination with chemotherapy

[0219] D3 binding molecules (eg, anti-D3 antibodies) can be used in combination with chemotherapy, including, for example, anti-cancer agents, cytotoxic agents, or chemotherapeutic agents.

[0220] The term "anti-cancer agent" or "anti-proliferative agent" means any agent useful for treating a cell proliferative disorder such as cancer, and includes, but is 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, hormone therapy, radiotherapy, and anti-metastatic and immunotherapeutic agents. It should be understood that in some embodiments as described above, such anti-cancer agents may comprise a conjugate and may be combined with the disclosed anti-D3 antibodies prior to administration. For example, in some embodiments, the selected anti-cancer agent will be linked to an unpaired cysteine ​​of an engineered antibody to provide an engineered conjugate (e.g., an antibody-drug conjugate) as described herein. Therefore, such engineered conjugates are explicitly encompassed within the scope of the present disclosure. In some embodiments, the disclosed anti-cancer agents will be administered in combination with an anti-D3 conjugate comprising a different therapeutic agent as described above.

[0221] As used herein, the term "cytotoxic agent" means a substance that is toxic to cells and reduces or inhibits cell function and / or causes cell destruction. 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 toxins or enzyme active toxins from bacteria (e.g., diphtheria toxin, Pseudomonas endotoxins and exotoxins, Staphylococcus enterotoxin A), fungi (e.g., α-sarcin, limitans), plants (e.g., abrin, ricin, modeccin, viscumin, pokeweed anti-viral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin, Aleurites fordii protein, dianthin protein, pokeweed proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor inhibitor, gelonin, mitegellin, restrictocin, phenomycin, neomycin and tricothecenes) or animals (e.g., cytotoxic RNases, such as extracellular pancreatic RNase; DNase I, including fragments and / or variants thereof).

[0222] For the purposes of this disclosure, "chemotherapeutic agents" include compounds (e.g., cytotoxic agents or cytostatic agents) that non-specifically reduce or inhibit the growth, proliferation and / or survival of cancer cells. Such chemicals are generally directed to the intracellular processes necessary for cell growth or division, and are therefore particularly effective for cancer cells that usually grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. In general, chemotherapeutic agents may include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become cancerous or produce tumorigenic progeny (e.g., TICs). Such agents are often administered in combination, and are generally the most effective, for example in regimens such as CHOP or FOLFIRI.

[0223] 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 in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethyleneimine and methylmelamine, acetogenins, camptothecins, bryostatins, callystatins, CC-1065, cryptophycins, dolastatins, duocarmycins, eleutherobin, pancratistatins, sarcodictyins, spongistatins, nitrogen mustards, antibiotics, enediyne antibiotics, danemycins, ynemicin), bisphosphonates, esperamicin, chromophores of chromoprotein enediyne antibiotics, aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin in), ubenimex, zinostatin, zorubicin; antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folate analogs, purine analogs, androgens, antiadreners, folate supplements such as folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate acetate), epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoid, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, Polysaccharide complex (JHS Natural Products, Eugene, OR), razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxoid, chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11), topoisomerase inhibitor RFS2000; difluoromethylornithine; retinoid; capecitabine; combretastatin; leucovorin; oxaliplatin; PKC-α, Raf, H-Ras, EGFR and VEGF-A inhibitors that reduce cell proliferation, and pharmaceutically acceptable salts, acids or derivatives of any of the above. This definition also includes antihormonal agents that modulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase, which regulates the production of estrogen in the adrenal glands, and antiandrogens; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, rIL-2; Topoisomerase 1 inhibitors; rmRH; vinorelbine and esperamicin and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

[0224] Combination with radiation therapy

[0225] The present disclosure also provides for the combination of D3 binding molecules and radiation therapy (e.g., any mechanism for inducing DNA damage locally within tumor cells, such as gamma-radiation, X-rays, UV-radiation, microwaves, electron emission, etc.). Combination therapies using directed delivery of radioisotopes to tumor cells are also contemplated, and the disclosed D3 binding molecules can be used in combination with targeted anticancer agents or other targeting means. Typically, radiation therapy is administered in pulses over a period of about 1 to about 2 weeks. Radiation therapy can be administered to a subject with head and neck cancer for about 6 to 7 weeks. Optionally, radiation therapy can be administered as a single dose or as multiple consecutive doses.

[0226] diagnosis

[0227] The present disclosure provides in vitro and in vivo methods for detecting, diagnosing or monitoring proliferative disorders and methods for screening cells from a patient to identify tumor cells, including tumorigenic cells. Such methods include identifying individuals with cancer for treatment or monitoring the progression of cancer, the methods comprising contacting the patient or a sample obtained from the patient (in vivo or in vitro) with an anti-D3 antibody described herein, and detecting the presence or absence or level of association of the antibody with a bound or free target molecule in the sample. In some embodiments, the anti-D3 antibody will comprise a detectable label or reporter molecule as described herein.

[0228] In some embodiments, association of an anti-D3 antibody with specific cells in a sample can indicate that the sample is likely to contain tumorigenic cells, thereby indicating that an individual with cancer can be effectively treated with an anti-D3 antibody described herein.

[0229] Samples can be analyzed by a variety of assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblotting assays, Western blot analysis, and flow cytometry assays. Compatible in vivo therapeutic or diagnostic assays may include art-recognized imaging or monitoring techniques, such as magnetic resonance imaging, computed tomography (e.g., CAT scans), positron emission tomography (e.g., PET scans), radiography, ultrasound, etc., as will be known to those skilled in the art.

[0230] Drug kits and test kits

[0231] Also provided are pharmaceutical packs and kits comprising one or more containers containing one or more doses of the D3 binding molecule. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, a D3 binding molecule, with or without one or more additional agents. In some embodiments, such a unit dose is provided in a disposable prefilled syringe for injection. In some embodiments, the composition contained in the unit dose may comprise saline, sucrose, or the like; a buffer, such as phosphate, or the like; and / or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder, which may be reconstituted after the addition of an appropriate liquid (e.g., sterile water or saline solution). In some embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. Any label on or associated with the container indicates that the enclosed composition is used to treat a selected neoplastic disease condition.

[0232] The present disclosure also provides kits for producing single-dose or multi-dose administration units of a D3 binding molecule and optionally one or more anticancer agents. The kit comprises 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 may be made of a variety of materials such as glass or plastic, and contains a pharmaceutically effective amount of a disclosed D3 binding molecule in conjugated or unconjugated form. In some embodiments, the container comprises a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). Such kits will generally contain a pharmaceutically acceptable formulation of a D3 binding molecule in conjugated or unconjugated form in a suitable container, and optionally, one or more anticancer agents in the same or different container. The kit may also contain other pharmaceutically acceptable formulations for diagnosis or combination therapy. For example, in addition to the D3-binding molecules of the present disclosure, such kits may contain any one or more of a range of anti-cancer agents, such as chemotherapeutic or radiotherapeutic drugs; anti-angiogenic agents; anti-metastatic agents; targeted anti-cancer agents; cytotoxic agents; and / or other anti-cancer agents.

[0233] For example, the kits may have a single container containing the D3 binding molecule, with or without additional components, or they may have different containers for each desired agent. When providing a combination therapeutic agent for conjugation, a single solution may be premixed in a molar equivalent combination or with one component in excess of another. Alternatively, the conjugate and any optional anticancer agent of the kit may be stored separately in different containers prior to administration to a patient. The kits may also include a second / third container member for holding a sterile, pharmaceutically acceptable buffer or other diluent, such as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution, and dextrose solution.

[0234] When the components of the kit are provided in the form of one or more liquid solutions, the liquid solution is preferably an aqueous solution, such as a sterile aqueous solution or a saline solution. However, the components of the kit may be provided as dry powders. When the reagents and / or components are provided as dry powders, such powders may be reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in another container.

[0235] As briefly noted above, the kits may also contain means for administering the D3 binding molecules and any optional components to a patient, such as one or more needles, IV bags or syringes or even eye droppers, pipettes or other similar devices from which the formulation can be injected or introduced into an animal or applied to an affected area of ​​the body. The kits of the present disclosure also typically include means for containing vials and the like, as well as other components for close confinement for commercial sale, such as injection or blow molded plastic containers to house and retain the required vials and other equipment.

[0236] Sequence Listing Overview

[0237] Attached to this application is a sequence listing containing a number of amino acid sequences. Tables AF below provide an overview of the included sequences. Exemplary antibodies are collectively referred to in this disclosure as "WBPT1156 antibodies".

[0238] Table A. Amino acid sequences of CDR regions

[0239]

[0240] Table B. Amino acid sequences of the VHH region and the Fc region of the human IgG1 heavy chain

[0241]

[0242] Table C. Amino acid sequences of antibody clones WT1156-P3R2-1C2, WT1156-P3R2-1C2-z102, WT1156-P3R2-1C2-z109 and WT1156-P3R2-1C2-z109'

[0243]

[0244] Table D. Amino acid sequence of antibody clone WT1156-P3R2-1C9

[0245]

[0246] Table E. Amino acid sequences of antibody clones WT1156-P3R2-1H6, WT1156-P3R2-1H6-z100

[0247]

[0248] Table F. Amino acid sequence of antibody clone WT1156-P8R2-1H1

[0249]

[0250] Example

[0251] The present disclosure thus generally described will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present disclosure.The examples are not intended to represent that the following experiments are all or the only experiments performed.

[0252] Example 1

[0253] Preparation of antigens, reference antibodies and cell lines

[0254] 1.1 Antigen generation

[0255] DNA sequences encoding the extracellular domain (ECD) sequences of cynomolgus monkey D3 (Uniprot No. A0A2K5WSR4) and mouse D3 (Uniprot No. O88516) were synthesized at Sangon Biotech (Shanghai, China) and then subcloned into modified pcDNA3.3 expression vectors with an MBP tag at the N-terminus and an AVI-His tag or a human Fc tag at the C-terminus. Human D3 (Uniprot No. Q9NYJ7) was purchased from AcroBiosystems (Catalog No. DL3-H52H4).

[0256] The DNA sequence encoding the truncated isoform of human D3 (disclosed in WO 2017 / 021349) was synthesized at Sangon Biotech (Shanghai, China) and then subcloned into a modified pcDNA3.3 expression vector with an MBP tag and an AVI-His tag at the C-terminus.

[0257] Expi293 cells (Invitrogen-A14527) were transfected with the purified expression vector. The cells were cultured for 5 days, and the supernatant was collected and used for protein purification using a Ni-NTA column (GE Healthcare, catalog number 175248) or a protein A column (GE Health, catalog number 175438). The obtained mouse D3, cynomolgus monkey D3, and truncated human D3 were analyzed by SDS-PAGE and SEC and then stored at -80°C.

[0258] Human D3 (ACRO DL3-H52H4) was named WT115-hPro1.ECD.His. The obtained mouse D3 was named 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. Truncated isoforms of human D3 were named 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), WT115-hPro1. V4.ECD.MBP.AVI.His (EGF3-6 domain + membrane proximal end), WT115-hPro1.V5.ECD.MBP.AVI.His (EGF4-6 domain + membrane proximal end), WT115-hPro1.V6.ECD.MBP.AVI.His (EGF5-6 domain + membrane proximal end), WT115-hPro1.V7.ECD.MBP.AVI.His (EGF6 domain + membrane proximal end). The diagram of the truncated protein is as follows Figure 7c shown.

[0259] 1.2 Construction of BMK antibody expression vector

[0260] 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 then subcloned into a modified pcDNA3.3 expression vector encoding the Fc region of human IgG1.

[0261] Plasmids containing VH and VL genes were co-transfected into Expi293 cells. The cells were cultured for 5 days, and the supernatant was collected and used for protein purification using a protein A column (GE Health, 175438). The obtained antibodies were analyzed by SDS-PAGE and SEC and then stored at -80°C.

[0262] 1.3 Establishment of stable cell lines / cell pools

[0263] 293F cells were transfected with an expression vector containing a gene encoding full-length human D3 (UniProt, Q9NYJ7-1) using Lipofectamine 2000. Flpin293 cells were transfected with an expression vector containing a gene encoding full-length cynomolgus monkey D3. The cells were cultured in a medium containing an appropriate selection marker. A cell line with high expression of human D3 (WT115-293F.hPro1.2E5) was selected after limiting dilution, and a cell pool with high expression of cynomolgus monkey D3 (WT115.Flpin293.cPro1.pool) was selected with appropriate selection antibiotics.

[0264] 1.4 Antibody biotinylation

[0265] For NHS-PEO4-biotinylation, 1-10 mg / mL antibody (IgG) was incubated with 20-fold molar excess of NHS-PEO4-Biotin reagent at 25°C in a metal bath for 75 minutes or on ice for two hours. Excess biotin was then removed using a desalting spin column, and the purified protein sample was collected from the effluent solution. The level of biotin incorporation in the protein was determined by the HABA assay: the biotinylated sample was diluted 10 times with a HABA / avidin solution and the absorbance of the mixed solution was measured at A500. The number of moles of biotin per mole of protein was calculated based on the A500 value.

[0266] Example 2

[0267] Generation of the VHH-containing WBPT1156 antibody

[0268] 2.1 Generation of anti-D3 VHH

[0269] Anti-D3 VHHs were generated by immunization of camelids and phage display technology. Briefly, alpacas (Alpaca / Vicugna pacos) were subcutaneously immunized with hFc-tagged human D3 ECD protein (ACRO, DL3-H5255). After immunization, peripheral blood was collected for the construction of phage libraries displaying VHH fragments. After bio-panning with the corresponding target ECD protein, positive VHH clones binding to D3 were selected.

[0270] 2.2 VHH sequencing

[0271] The E. coli supernatant was sent to Biosune (Shanghai, China) for nucleotide sequencing of VHH genes by target-specific binding ELISA and FACS-selected positive E. coli clones. The sequencing results were analyzed using the CLC main workbench (Qiagen, Hilden, Germany). The sequences of the 4 unique positive VHH clones were WT1156-P3R2-1C2, WT1156-P3R2-1C9, WT1156-P3R2-1H6, and WT1156-P8R2-1H1 shown in Table A and Table B.

[0272] 2.3 Generation of human Fc fusion antibodies containing VHH

[0273] Four unique positive VHH clones were converted into VHH-Fc (hIgG1) fusion antibodies. In brief, VHH genes were PCR amplified from the pET-bac vector using VHH-specific cloning primers containing appropriate restriction sites and then cloned into the modified human hIgG1 expression pcDNA3.3 vector by fusion to produce the corresponding clones of VHH-Fc (hIgG1) chimeric antibodies. 293F or Expi293 cells were transiently transfected with the vector for antibody expression. Cell culture supernatants containing antibodies were harvested 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 obtained antibodies were analyzed by SDS-PAGE and HPLC-SEC and then stored at -80°C.

[0274] 2.4 Humanization

[0275] VHH humanization is accomplished by the "best fit" method. In short, the amino acid sequence of the VHH framework region is attacked against the human germline V gene database, and the humanized VHH sequence is generated by replacing the most hit human CDR sequence with the VHH CDR sequence using the Kabat CDR definition. Then, the key residues in the framework that play an important role in antibody affinity or developability are mutated back to parent residues alone or in combination. The variant codons are optimized for mammalian expression and then synthesized by GENEWIZ (SuZhou, China). The designed VHH variants and parent VHH proteins are cloned into human IgG1 expression vectors to produce human IgG1 constructs. The antibodies are produced in HEK293 cells and purified using protein A chromatography. The variants with the desired affinity are finally selected as humanized leader sequences.

[0276] 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.

[0277] Example 3

[0278] Characterization of D3-binding antibodies

[0279] 3.1 Human D3 binding by ELISA

[0280] The plates were pre-coated with 1 μg / mL, 100 μL per well of WT115-hPro1.ECD.His overnight at 4°C. Antigen was diluted from stock solution into coating buffer (0.02 M Na 2 CO 3 and 0.18 M NaHCO 3 , pH 9.2). The next day, the plates were washed once with 1×PBST (PBS containing 0.05% tween-20) and blocked by adding 200 μL of 1×PBS / 2% BSA. The antibodies were serially diluted in blocking buffer (serial dilution 5-fold from 20 nM to 0.00128 nM). After blocking for 1 hour, the plates were washed 3 times with 1×PBST, and then the antibodies were added to the plates and incubated for 1 hour at ambient temperature. The binding of the antibodies to immobilized human D3 was detected by HRP-labeled secondary antibodies (Bethyl, A80-304P), which were diluted at a concentration of 1:10000 in 1×PBS / 2% BSA. After incubation, the plates were washed 6 times with 1×PBST. Color was developed by dispensing 100 μL of TMB substrate, and the reaction was terminated by adding 100 μL of 2M HCl. The absorbance at 450 nm and 540 nm was read using a microplate spectrophotometer. Anti-human D3 antibodies WT115-BMK1 and WT115-BMK2 were used as positive controls. Human IgG1 isotype antibody was used as a negative control. All samples were tested in duplicate.

[0281] like Figure 1As shown in Table 1, WT1156-P3R2-1C2-uIgG1, WT1156-P3R2-1C9-uIgG1, WT1156-P3R2-1H6-uIgG1, and WT1156-P8R2-1H1-uIgG1 can strongly bind to immobilized human D3, and the binding is comparable to WT115-BMK1 and WT115-BMK2. The EC50 of the WT1156 antibody is in the range of 0.013nM to 0.026nM. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.0094nM and 0.011nM, respectively.

[0282] Table 1. Overview of characterization of the WBPT1156 antibody.

[0283]

[0284] na: EC50 value not fitted.

[0285] 3.2 Human D3 binding by FACS

[0286] WT115-293F.hPro1.2E5 cells (1×10 5 Cells / well) were incubated with different concentrations of antibodies (serial dilution 5-fold from 200nM to 0.0128nM) at 4°C for 1 hour. After washing with 1×PBS / 1% BSA, the secondary antibody, R-PE-labeled goat anti-human IgG (1:150, Jackson ImmunoResearch, 109-115-098) was added and incubated with the cells in the dark at 4°C for 1 hour. Anti-human D3 antibodies WT115-BMK1 and WT115-BMK2 were used as positive controls. Human IgG1 isotype antibody was used as a negative control. The cells were washed and resuspended in 4% paraformaldehyde. The MFI of the cells was measured by flow cytometry and analyzed by FlowJo.

[0287] like Figure 2a As shown in 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, and the binding is comparable to that of WT115-BMK2. The EC50 of the WT1156 antibody is in the range of 0.15nM to 0.52nM. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.019nM and 0.54nM, respectively.

[0288] like Figure 2bAs shown in, WT1156-P3R2-1C2-z102-uIgG1 and WT1156-P3R2-1C2-z109-uIgG1 can strongly bind to human D3 expressing cells, with EC50 of 0.088nM and 0.14nM, respectively. The EC50 of WT115-BMK1 and WT115-BMK2 were 0.03nM and 0.52nM, respectively. The data of WT1156-P3R2-1C2-z109-uIgG and two BMK antibodies are also summarized in Table 2.

[0289] Table 2. Overview of characterization of WT1156-P3R2-1C2-z109-uIgG1.

[0290]

[0291] 3.3 Cynomolgus D3 binding by FACS

[0292] WT115-Flpin293.cPro1.pool cells (1×10 5 Cells / well) were incubated with different concentrations of antibodies (serial dilution 4-fold from 10nM to 0.00061nM) at 4°C for 1 hour. After washing with 1×PBS / 1%BSA, the secondary antibody, Alexa Fluor 647-labeled goat anti-human IgG (1:150, Jackson ImmunoResearch, 109-605-098) was added and incubated with the cells in the dark at 4°C for 1 hour. Anti-human D3 antibodies WT115-BMK1 and WT115-BMK2 were used as positive controls. Human IgG1 isotype antibody was used as a negative control. The cells were washed with 1×PBS / 1%BSA and resuspended in 4% paraformaldehyde, and incubated with the cells at 4°C in the dark for 0.5 hours. The buffer was then replaced with 1×PBS / 1%BSA and the cells were filtered. The MFI of the cells was measured by flow cytometry and analyzed by FlowJo.

[0293] like Figure 3a As shown in Table 1, WT1156-P3R2-1C2-uIgG1, WT1156-P3R2-1C9-uIgG1, WT1156-P3R2-1H6-uIgG1, and WT1156-P8R2-1H1-uIgG1 can bind to cynomolgus monkey D3 expressing cells, and the binding is comparable to WT115-BMK1 and WT115-BMK2. The EC50 of the WT1156 antibody is in the range of 0.12nM to 0.44nM. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.019nM and 0.20nM, respectively.

[0294] like Figure 3bAs shown in, WT1156-P3R2-1C2-z102-uIgG1, WT1156-P3R2-1C2-z109-uIgG1 and WT1156-P3R2-1H6-z100-uIgG1 can strongly bind to cynomolgus monkey D3 expressing cells, with EC50 of 0.083nM, 0.096nM and 0.42nM, respectively. The EC50 of WT115-BMK1 and WT115-BMK2 were 0.019nM and 0.20nM, respectively. The data of WT1156-P3R2-1C2-z109-uIgG and two BMKs are also summarized in Table 2.

[0295] 3.4 Mouse D3 binding by ELISA

[0296] The plate was pre-coated with 1 μg / mL, 100 μL of WT115-MBP-mPro1.ECD.hFc per well at 4°C overnight. The antigen was diluted into coating buffer from the stock solution. The next day, the plate was washed once with 1×PBST and blocked by adding 200 μL of 1×PBS / 2%BSA. The antibody was serially diluted in blocking buffer (from 20nM to 0.000256nM, serially diluted 5 times). After blocking for 1 hour, the plate was washed 3 times with 1×PBST, and then the antibody was added to the plate and incubated at ambient temperature for 1 hour. The anti-human D3 antibodies WT115-BMK1-Biotin and WT115-BMK2-Biotin were used as positive controls. The WT114-BMK1-Biotin antibody was used as a negative control. The binding of the antibody to immobilized mouse D3 was detected by HRP-labeled secondary antibody (Invitrogen, SNN1004), which was diluted at a concentration of 1:30000 in 1×PBS / 2%BSA. After incubation, the plate was washed 6 times with 1×PBST. The reaction was terminated by dispensing 100 μL of TMB substrate and then adding 100 μL of 2M HCl. The absorbance at 450 nm and 540 nm was read using a microplate spectrophotometer. All samples were tested in duplicate.

[0297] like Figure 4a As shown in , WT1156-P3R2-1C2-uIgG1 can strongly bind to mouse D3, and the binding is comparable to that of 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.

[0298] like Figure 4bAs shown in , WT1156-P3R2-1C2-z102-uIgG1 and WT1156-P3R2-1C2-z109-uIgG1 can strongly bind to mouse D3 protein, with EC50 of 0.0067nM and 0.0075nM, respectively. The EC50 of WT115-BMK1 and WT115-BMK2 were 0.0039nM and 0.014nM, respectively. The data of WT1156-P3R2-1C2-z109-uIgG and two BMKs are also summarized in Table 2.

[0299] 3.5 Internalization

[0300] WT115-293F.hPro1.2E5 cells (4×10 4 Cells / well) were plated in 96-well plates, and the medium was removed from the plates after centrifugation. The prepared 1× final maximum concentration of primary antibody (serial dilution 5-fold from 40nM to 0.00256nM, or serial dilution 5-fold from 200nM to 0.0128nM) 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 = 1:1 molecule) dilution was 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 a negative control. After incubation, the cells were stained with reagents (nuclei - Hoechst 33342, 1000 ng / ml; cytoplasm - Calcein AM, diluted 1:2000 in DPBS) and the plates were incubated for 15 min at 37° C. Finally, the cells were photographed with Operatta CLS and the antibody internalization was analyzed by the “number of spots per cell” parameter.

[0301] like Figure 5a As shown in Table 1, WT1156-P3R2-1C2-uIgG1, WT1156-P3R2-1C9-uIgG1, WT1156-P3R2-1H6-uIgG1, and WT1156-P8R2-1H1-uIgG1 showed dose-dependent internalization potency in human D3-expressing cells, which was comparable to WT115-BMK1 and WT115-BMK2. The EC50 of the WT1156 antibody ranged from 0.46 nM to 0.95 nM. The EC50 of WT115-BMK1 and WT115-BMK2 were 0.19 nM and 0.58 nM, respectively.

[0302] like Figure 5b As shown in Table 2, WT1156-P3R2-1C2-z109-uIgG1 showed a dose-dependent internalization potency 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.

[0303] 3.6 Kinetic binding affinity of anti-D3 antibodies

[0304] The binding affinity of anti-D3 antibodies to human D3 ECD protein was detected by SPR assay using Biacore T200. Each antibody tested was captured on a CM5 sensor chip (GE) immobilized with anti-human IgG Fc antibody. Different concentrations of WT115-hPro1.ECD.His were injected onto the sensor chip at a flow rate of 30 μl / min, with an association period of 180 s and then a dissociation period of 3600 s. After each binding cycle, the chip was regenerated with 10 mM glycine (pH 1.5).

[0305] As shown in Table 3, the experimental data for human D3 were fitted by the steady-state affinity model. The experimental data for WT-115-BMK1 against human D3 were fitted by the heterogeneous ligand model. The other experimental data were fitted by the 1:1 model using Langmiur analysis. The sensorgrams of the blank surface and the buffer channel were subtracted from the test sensorgrams. The molar concentration of the analyte was calculated using a molecular weight of 34 KDa. The affinities of the tested antibodies for human D3 are shown in Table 3.

[0306] Table 3. Binding kinetics of WBPT1156 antibody.

[0307]

[0308] 3.7 Epitope binning by competition ELISA

[0309] The plate was pre-coated with 1 μg / mL, 100 μL of WT115-hPro1.ECD.His in each well at 4 ° C overnight. The antigen was diluted from the stock solution into the coating buffer (0.02M Na2CO3 and 0.18M NaHCO3, pH9.2). The next day, the plate was washed once with 1×PBST and blocked with 200 μL of 1×PBS / 2%BSA. The VHH antibody was serially diluted in the blocking buffer (serial dilution 5 times from 10nM to 0.00013nM) and pre-mixed with a constant concentration of full IgG antibody (0.02nM). After blocking for 1 hour, the plate was washed 3 times with 1×PBST, and then the VHH antibody / full IgG antibody mixture was added to the plate and incubated at ambient temperature for 1 hour. Binding of full IgG antibody to immobilized human D3 was detected by HRP-labeled secondary antibody (Bethyl, A80-304P), which was diluted at a concentration of 1:10000 in 1×PBS / 2%BSA. After incubation, the plate was washed 6 times with 1×PBST. Color was developed by dispensing 100 μL of TMB substrate, and the reaction was terminated by adding 100 μL of 2M HCl. The absorbance at 450 nm and 540 nm was read using a microplate spectrophotometer. All samples were tested in duplicate.

[0310] like Figure 6a As shown in , WT1156-P3R2-1C2.uIgG1 does not compete with the other three WBPT1156 antibodies for binding to human D3-ECD protein by ELISA test. Figure 6b As shown in , 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 It was shown that WT1156-P8R2-1H1-uIgG1 could compete with WT1156-P3R2-1C9-uIgG1. Figure 6d As shown in , the four antibodies to WBPT1156 did not compete with WT115-BMK1.

[0311] 3.8 Binding to truncated D3 protein by ELISA

[0312] The binding epitope of WT1156 antibody to truncated D3 protein, such as 1.1 and Figure 7c ELISA tests are performed by pre-coating plates with antibodies or antigens. The results are as follows Figure 7a and Figure 7b as shown in .

[0313] For the ELISA of pre-coated antibodies, the ELISA plates were pre-coated with 2 μg / mL, 100 μL of antibodies per well at 4 ° C overnight. The antibodies were diluted from the stock solution into the coating buffer (0.02M Na2CO3 and 0.18M NaHCO3, pH 9.2). The next day, the plates were washed once with 1 × PBST and blocked with 200 μL of 1 × PBS / 2% BSA. The 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 the blocking buffer were then added at a constant concentration. After blocking for 1 hour, the plates were washed 3 times with 1 × PBST, and then the antigens were added to the plates and incubated for 1 hour at ambient temperature. The binding of antigen to the immobilized WBPT1156 antibody was detected by HRP-labeled secondary antibody (GenScript, A00612), which was diluted at a concentration of 1:2000 in 1×PBS / 2% BSA. After incubation, the plate was washed 6 times with 1×PBST. Color was developed by dispensing 100 μL of TMB substrate. The color reaction was terminated by 2M HCl, and the absorbance at 450 nm and 540 nm was read using a microplate spectrophotometer. All samples were tested in duplicate. The results are shown in Figure 7a as shown in .

[0314] For ELISA with pre-coated antigen, the ELISA plate was pre-coated with 100 μL of WT115-hPro1.ECD.His (2 μg / mL) or truncated D3 protein (2 μg / mL or 5 μg / mL) per well at 4°C overnight. The antigen was diluted from the stock solution into coating buffer (0.02M Na2CO3 and 0.18M NaHCO3, pH 9.2). The next day, the plate was washed once with 1×PBST and blocked with 200 μL of 1×PBS / 2%BSA. A constant concentration of antibody (2 μg / mL) was diluted in blocking buffer. After blocking for 1 hour, the plate was washed 3 times with 1×PBST, and then the antibody was added to the plate and incubated at ambient temperature for 1 hour. The binding of the antibody to immobilized human D3 was detected by an HRP-labeled secondary antibody (Bethyl, A80-304P), which was diluted at a concentration of 1:10000 in 1×PBS / 2%BSA. After incubation, the plates were washed 6 times with 1× PBST. Color was developed by dispensing 100 μL of TMB substrate, and the reaction was terminated by adding 100 μL of 2 M HCl. The absorbance at 450 nm and 540 nm was read using a microplate spectrophotometer. All samples were tested in duplicate. The results are shown in Table 1. Figure 7b as shown in .

[0315] like Figure 7a and 7bAs shown in, 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 at the N-terminus. For WT1156-P8R2-1H1-uIgG1, when tested by ELISA with pre-coated antibodies, it showed binding to WT115-hPro1.ECD.His, but not to the truncated D3 isoform ( Figure 7a ); When using pre-coated antigen for ELISA test, such as Figure 7b As shown in , WT1156-P8R2-1H1-uIgG1 showed binding to WT115-hPro1.V1.ECD.MBP.AVI.His and WT115-hPro1.V2.ECD.MBP.AVI.His, but not to other isoforms, indicating that its binding epitope may be located at the N-terminal-DSL-EGF-1. The ELISA binding results showed that WT115-BMK1 bound to the DSL domain and WT115-BMK2 bound to the EGF-3 domain, which were consistent with the results shown in US2019 / 0046656 and WO 2017 / 021349, respectively.

[0316] 3.9 Cross-family binding of anti-human D3 antibodies

[0317] The plate was pre-coated with 1 μg / mL, 100 μL of WT115-hPro1.ECD.His, WT115-hPro2.ECD.His (human D1, SinoBiological, catalog number: 11635-H08H) or WT115-hPro3.ECD.His (human D4, SinoBiological, catalog number: 10171-H08H) in each well at 4°C overnight. Antigen was diluted from stock solution into coating buffer (0.02M Na2CO3 and 0.18M NaHCO3, pH 9.2). The next day, the plate was washed once with 1×PBST (PBS containing 0.05% tween-20) and blocked by adding 200 μL of 1×PBS / 2%BSA in each well. During blocking, BMK and WT1156 antibodies were diluted to 10 nM in blocking buffer, and WT115-cAb (WT115-cAb1 is an anti-D1 antibody purchased from SinoBiological, catalog number: 11635-MM07; WT115-cAb2 is an anti-D4 antibody purchased from SinoBiological, catalog number: 10171-MM15) was diluted 1000 times. After blocking for 1 hour, the plate was washed 3 times with 1×PBST, and then the diluted antibodies were added to the plate and incubated at ambient temperature for 1 hour. The binding of antibodies to immobilized human D3 was detected by goat anti-human IgG-Fc fragment cross-adsorption antibody HRP (Bethyl, A80-304P) and mouse IgG-Fc fragment cross-adsorption antibody HRP (Bethyl, A90-231P), which were diluted 1:10000 in 1×PBS / 2% BSA. After incubation, the plate was washed 6 times with 1×PBST. The reaction was developed by dispensing 100 μL of TMB substrate, and then stopped by adding 100 μL of 2M HCl. The absorbance at 450 nm and 540 nm was read using a microplate spectrophotometer. WT115-cAb1 and cAb2 were used as anti-human D1 and D4 positive controls, respectively. Human IgG1 isotype antibody was used as a negative control. All samples were tested in duplicate.

[0318] like Figure 8 As shown in , the WBPT1156 antibody does not bind to human D1 or human D4.

[0319] 3.10 Human serum stability

[0320] Human serum was freshly isolated from healthy donors by centrifugation. The samples were diluted in serum and the serum volume accounted for more than 90% of the total volume. Five aliquots of the samples were incubated at 37°C. The samples were then collected on day 0, day 1, day 4, day 7 and day 14 and quickly frozen until analyzed together.

[0321] The stability of the samples was tested by binding to human D3 using ELISA. In brief, the plates were pre-coated with 1 μg / mL WT115-hPro1.ECD.His at 100 μL / well overnight at 4°C. The next day, the plates were washed once with 1×PBST (PBS containing 0.05% tween-20) and blocked by adding 200 μL of 1×PBS / 2%BSA per well. During blocking, test antibodies at different concentrations (serial dilution 4-fold from 3nM to 0.00018nM) were added to the plates. The plates were incubated at ambient temperature for 1 hour. The binding of antibodies to immobilized human D3 was detected by goat anti-human IgG-Fc fragment cross-adsorption antibody HRP (Bethyl, A80-304P) and mouse IgG-Fc fragment cross-adsorption antibody HRP (Bethyl, A90-231P), which were diluted 1:5000 in 1×PBS / 2%BSA. After incubation, the plates were washed 6 times with 1× PBST. The color was developed by dispensing 100 μL of TMB substrate, and then the reaction was stopped by adding 100 μL of 2M HCl. The absorbance at 450 nm and 540 nm was read using a microplate spectrophotometer. Human IgG1 isotype antibody was used as a negative control. All samples were tested in duplicate.

[0322] like Fig. 9 As shown in , the binding profile of WT1156-P3R2-1C2-z109-uIgG1 to human D3 protein did not change after incubation in human serum at 37°C for up to 14 days.

[0323] Implementation

[0324] 1. A D3 binding molecule comprising an immunoglobulin single variable domain, wherein the single variable domain comprises CDR1, CDR2 and CDR3 of a VHH as shown in SEQ ID NO: 12, 13, 14, 15, 16, 17, 55 or 18.

[0325] 2. The D3 binding molecule of embodiment 1, wherein CDR1, CDR2 and CDR3 are according to Kabat, Chothia, AbM, Contact, IMGT or any combination thereof.

[0326] 3. The D3 binding molecule of embodiment 1, wherein (i) the CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, 4, 7 or 10; (ii) the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, 5, 8 or 11; and (iii) CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, 6 or 9.

[0327] 4. The D3 binding molecule of embodiment 1, wherein the D3 binding molecule comprises:

[0328] (A) CDR1 shown in SEQ ID NO: 1; CDR2 shown in SEQ ID NO: 2; and CDR3 shown in SEQ ID NO: 3;

[0329] (B) CDR1 shown in SEQ ID NO:4; CDR2 shown in SEQ ID NO:5; and CDR3 shown in SEQ ID NO:6;

[0330] (C) CDR1 shown in SEQ ID NO:7; CDR2 shown in SEQ ID NO:8; and CDR3 shown in SEQ ID NO:9; or

[0331] (D) CDR1 shown in SEQ ID NO:10; CDR2 shown in SEQ ID NO:11; and CDR3 shown in SEQ ID NO:6.

[0332] 5. The D3 binding molecule of any one of embodiments 1 to 4, wherein the single variable domain comprises:

[0333] (A) the amino acid sequence shown in any one of SEQ ID NOs: 12-18 and 55;

[0334] (B) an amino acid sequence that is at least 85%, 90% or 95% identical to the amino acid sequence shown in any one of SEQ ID NOs: 12-18 and 55, but still retains specific binding affinity for D3; or

[0335] (C) an amino acid sequence in which one or more (e.g., 1, 2 or 3) amino acids are added, deleted and / or substituted compared to the amino acid sequence shown in any one of SEQ ID NOs: 12-18 and 55.

[0336] 6. The D3 binding molecule of any one of embodiments 1 to 5, comprising one or more amino acid substitutions, additions and / or deletions in the framework region of the single variable domain, e.g., FRW1, FRW2, FRW3 and / or FRW4.

[0337] 7. The D3 binding molecule of any one of embodiments 1 to 6, wherein the single variable domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 12-18 and 55.

[0338] 8. The D3 binding molecule of any one of embodiments 1 to 7, wherein the D3 binding molecule further comprises a human IgG constant domain.

[0339] 9. The D3 binding molecule of embodiment 8, 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.

[0340] 10. The D3 binding molecule of any one of embodiments 1 to 9, which has one or more of the following properties:

[0341] (a) binds to human D3, cynomolgus D3 and / or mouse D3 with an EC50 in nM as measured by ELISA or FACS;

[0342] (b) showed dose-dependent internalization potency in human D3-expressing cells; and

[0343] (c) Binds to human D3 with a KD of no more than 0.1 nM, as measured by SPR.

[0344] 11. The D3 binding molecule of any one of embodiments 1 to 10, wherein the D3 binding molecule is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0345] 12. The D3 binding molecule of any one of embodiments 1 to 11, comprising a single variable domain as shown in any one of SEQ ID NOs: 12-18 and 55 and an IgG constant domain as shown in SEQ ID NO: 19.

[0346] 13. The D3 binding molecule of any one of embodiments 1 to 12, which is a dimer.

[0347] 14. A fusion protein comprising a D3 binding molecule as defined in any one of embodiments 1 to 13 fused to a heterologous peptide such as an antigen binding domain targeting a different antigen.

[0348] 15. A nucleic acid molecule comprising a nucleic acid sequence encoding the single variable domain of the D3 binding molecule as defined in any one of embodiments 1 to 13.

[0349] 16. A vector comprising the nucleic acid molecule described in embodiment 15.

[0350] 17. A host cell comprising the vector described in embodiment 16.

[0351] 18. A pharmaceutical composition comprising at least one D3 binding molecule as defined in any one of embodiments 1 to 13 and a pharmaceutically acceptable carrier.

[0352] 19. A method for producing a D3 binding molecule as defined in any one of embodiments 1 to 13, comprising the following steps:

[0353] - expressing the D3 binding molecule in a host cell as described in embodiment 15; and

[0354] - isolating said D3 binding molecule from said host cell.

[0355] 20. A method of modulating a D3-related immune response in a subject, comprising administering to the subject a D3 binding molecule as defined in any one of embodiments 1 to 13 or a pharmaceutical composition as described in embodiment 18, such that the immune response in the subject is modulated.

[0356] 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 embodiments 1 to 13 or a pharmaceutical composition as described in embodiment 18, wherein the cancer is D3 positive or overexpressing.

[0357] 22. The method of embodiment 21, wherein the cancer is selected from lung cancer and neuroendocrine cancer.

[0358] 23. The method of embodiment 22, wherein the cancer is SCLC or LCNEC.

[0359] 24. Use of a D3 binding molecule as defined in any one of embodiments 1 to 13 in the manufacture of a medicament for diagnosing, preventing or treating a D3-positive cancer.

[0360] 25. A D3 binding molecule as defined in any one of embodiments 1 to 13 for use in treating or preventing a D3-positive cancer.

[0361] 26. A kit for treating or diagnosing cancer, comprising a container comprising a D3 binding molecule as defined in any one of embodiments 1 to 13.

[0362] It will also be appreciated by those skilled in the art that the present disclosure may be implemented in other specific forms without departing from the spirit or central attributes of the present disclosure. Since the foregoing description of the present disclosure discloses only exemplary embodiments thereof, it will be appreciated that other variations are also contemplated within the scope of the present disclosure. Therefore, the present disclosure is not limited to the specific embodiments described in detail herein. On the contrary, the scope and content of the present disclosure should be indicated with reference to the appended claims.

Claims

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

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

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

3.

5. 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).

6. The D3 binding molecule of claim 1, further comprising FRW1, FRW2, FRW3 and / or FRW4 as shown in the VHH comprising the amino acid sequence of SEQ ID NO: 17, 55, 12 or 16.

7. The D3 binding molecule of claim 1, wherein the single variable domain comprises: (A) the amino acid sequence of SEQ ID NO: 17, 55, 12 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, 12 or 16, but still retains specific binding affinity for D3; or (C) an amino acid sequence in which one or more amino acids are added, deleted and / or substituted compared to the amino acid sequence of SEQ ID NO: 17, 55, 12 or 16.

8. The D3 binding molecule of claim 1, wherein the single variable domain comprises the amino acid sequence of SEQ ID NO:

17.

9. The D3 binding molecule of claim 1, wherein the single variable domain comprises the amino acid sequence of SEQ ID NO:

55.

10. The D3 binding molecule of claim 1, wherein the single variable domain comprises the amino acid sequence of SEQ ID NO:

12.

11. The D3 binding molecule of claim 1, wherein the single variable domain comprises the amino acid sequence of SEQ ID NO:

16.

12. The D3 binding molecule of claim 1, wherein the D3 binding molecule further comprises a human IgG constant domain.

13. The D3 binding molecule of claim 12, 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.

14. The D3 binding molecule of claim 1, having one or more of the following properties: (a) binds to human D3, cynomolgus D3 and / or mouse D3 with an EC50 in nM as measured by ELISA or FACS; (b) showed dose-dependent internalization potency in human D3-expressing cells; and (c) Binds to human D3 with a KD of no more than 0.1 nM as measured by SPR.

15. The D3 binding molecule of claim 1, wherein the D3 binding molecule is a chimeric antibody, a humanized antibody or a fully human antibody.

16. The D3 binding molecule of claim 1, comprising a single variable domain comprising the amino acid sequence of SEQ ID NO: 17 and an IgG constant domain comprising the amino acid sequence of SEQ ID NO:

19.

17. The D3 binding molecule of claim 1, comprising a single variable domain comprising the amino acid sequence of SEQ ID NO: 55 and an IgG constant domain comprising the amino acid sequence of SEQ ID NO:

19.

18. The D3 binding molecule of claim 1, comprising a single variable domain comprising the amino acid sequence of SEQ ID NO: 12 and an IgG constant domain comprising the amino acid sequence of SEQ ID NO:

19.

19. The D3 binding molecule of claim 1, comprising a single variable domain comprising the amino acid sequence of SEQ ID NO: 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 targeting a different antigen.

22. A nucleic acid molecule comprising a nucleic acid sequence encoding the 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 according to 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 the steps of: - expressing the D3 binding molecule in a host cell according to claim 24; as well as - isolating said D3 binding molecule from said host cell.

27. Use of a D3 binding molecule as defined in any one of claims 1 to 20 in the preparation of a medicament for modulating a D3-related immune response in a subject, such that an immune response in the subject is modulated.

28. Use of a D3 binding molecule as defined in any one of claims 1 to 20 in the preparation of a medicament for treating or preventing cancer in a subject, wherein the cancer is D3 positive or overexpresses D3.

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

30. The use 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 a 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 comprising a D3 binding molecule as defined in any one of claims 1 to 20.

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