TW202340246A - D3-binding molecules and uses thereof - Google Patents

Abstract

Provided D3-binding molecules, including anti-D3antibodies, and their uses.

Description

D3 binding molecules and their uses

Cross-references to related applications

This application claims priority from the international application PCT/CN2021/119011 submitted on September 17, 2021 and the Chinese application 202211115105.2 submitted on September 14, 2022, the entire contents of which are incorporated herein by reference. sequence list

This application is submitted electronically along with the Sequence Listing. The entire contents of the Sequence Listing are incorporated herein by reference.

The present application relates generally to Delta-like classical Notch ligand 3 (D3) binding molecules, including anti-D3 antibodies, and uses thereof.

Delta-like classical Notch ligand 3 (D3 or DLL3) is a type I transmembrane protein that belongs to the DSL family of Notch ligands. It usually only manifests on intracellular membranes, especially the Golgi apparatus. Other Notch family ligands include Delta-like classical Notch ligand 1 (D1), Delta-like classical Notch ligand 4 (D4), Jagged classical Notch ligand 1 (J1), and Jagged classical Notch ligand 2 (J2). Except for D3, other ligands can initiate 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 cell surface of lung tumors, including small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC). Although D3 is normally expressed only on intracellular membranes, D3 is a potential therapeutic tumor target in any tumor expressing D3, including SCLC and LCNEC.

Lung cancer is the most common cause of cancer death, with approximately 2 million people diagnosed with lung cancer worldwide each year; 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 the first-line treatment of SCLC, but the limited 2-month benefit highlighted the need to develop other therapies.

There is still a need to find new D3-targeting agents, including D3-binding molecules such as monoclonal antibodies, for various purposes, such as use as antibody conjugates (such as ADCs) or for the development of bispecific antibodies and CAR-T therapies .

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 can be applied broadly to the field of antibody therapeutics and diagnostics, and can be used in conjunction with other therapeutic agents such as antibodies that react with a variety of targets.

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

In the present disclosure, D3-binding molecules, such as monoclonal antibodies directed against D3, are developed that can be used to treat tumors expressing D3.

The present invention provides D3 binding molecules, nucleic acid molecules encoding them, expression vectors and host cells for expressing D3 binding molecules, and methods of using D3 binding molecules. The D3 binding molecules of the present disclosure provide effective agents for the treatment of various cancers, including lung cancer, by modulating human immune function.

In some embodiments, the present disclosure provides a D3 binding molecule comprising at least one immunoglobulin single variable domain (e.g., VHH domain). In some embodiments, the single variable domain comprises CDR1, CDR2, and CDR3, and wherein: The CDR1 includes the amino acid sequence shown in SEQ ID NO: 1, 4, 7 or 10; The CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, 5, 8 or 11; and The CDR3 includes the amino acid sequence shown in SEQ ID NO: 3, 6 or 9.

In some embodiments, a single variable domain as disclosed herein includes: (A) CDR1 as shown in SEQ ID NO:1; CDR2 as shown in SEQ ID NO:2; CDR3 as shown in SEQ ID NO:3; (B) CDR1 as shown in SEQ ID NO:4; CDR2 as shown in SEQ ID NO:5; CDR3 as shown in SEQ ID NO:6; (C) CDR1 as shown in SEQ ID NO:7; CDR2 as shown in SEQ ID NO:8; CDR3 as shown in SEQ ID NO:9; or (D) CDR1 as shown in SEQ ID NO:10; CDR2 as shown in SEQ ID NO:11; CDR3 as shown in SEQ ID NO:6.

In some embodiments, a single variable domain as disclosed herein includes: (A) CDR1 as shown in SEQ ID NO:27; CDR2 as shown in SEQ ID NO:28 or 56; and CDR3 as shown in SEQ ID NO:29; (B) CDR1 as shown in SEQ ID NO:38; CDR2 as shown in SEQ ID NO:39; and CDR3 as shown in SEQ ID NO:40; or (C) CDR1 as shown in SEQ ID NO:49; CDR2 as shown in SEQ ID NO:50; and CDR3 as shown in SEQ ID NO:51; The CDR number is based on the Contact numbering system.

In some embodiments, a single variable domain as disclosed herein includes: (A) The amino acid sequence shown in any one of SEQ ID NOs: 12-18 and 55; (B) Having at least 85%, at least 90%, or at least 95% identity with the amino acid sequence set forth in any one of SEQ ID NOs: 12-18 and 55, while retaining (e.g., substantially retaining, e.g., at least 50%, or (C) Compared with the amino acid sequence shown in any one of SEQ ID NOs: 12-18 and 55, one or a plurality (for example, 1, 2 or 3) of amino acids are added, deleted and/or substituted, while maintaining ( For example, an amino acid sequence that substantially maintains, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) specific binding affinity for D3.

In some embodiments, a D3 binding molecule as disclosed herein comprises one or more amino acid additions, deletions and /or replace. In some embodiments, the N-terminal FRW1 and/or the C-terminal FRW4 of the single variable domain is truncated, e.g., by no more than 5, 4, 3, 2, or 1 amino acid.

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

In some embodiments, a D3 binding molecule as disclosed herein further comprises one or more human IgG constant domains, such as one or more human IgGl, 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 the IgG1 constant domain is shown in SEQ ID NO: 19. In some embodiments, the D3 binding molecule comprises one or more variants of the human IgG1 constant domain, such as an IgG1 Fc with L234A/L235A (according to EU numbering) substitutions.

In some embodiments, a D3 binding molecule as disclosed herein has one or more of the following properties: (a) Binds human D3, cynomolgus monkey D3 and/or mouse D3 with an EC50 in the nM range, as measured by ELISA or FACS; (b) Demonstrate dose-dependent internalization efficacy in cells expressing human D3; and (c) Binds human D3 with a KD of no more than 0.1 nM, as measured by SPR.

In some embodiments, a D3 binding molecule as disclosed herein is a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the D3 binding molecule is a dimer.

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

In some embodiments, the present disclosure provides nucleic acid molecules comprising nucleic acid sequences encoding a D3 binding molecule as disclosed herein, e.g., a D3 binding molecule comprising a single variable domain (eg, VHH).

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

In some embodiments, the present disclosure provides host cells comprising expression vectors or nucleic acid molecules as disclosed herein.

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

In some embodiments, the present disclosure provides methods of making a D3 binding molecule, comprising expressing the D3 binding molecule in a host cell as disclosed herein, and isolating the D3 binding molecule from the host cell.

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

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

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

In some embodiments, the present disclosure provides D3-binding molecules as disclosed herein for use in diagnosing, treating, or preventing D3-positive cancers.

In some aspects, the present disclosure relates to kits or devices and related methods employing D3 binding molecules as disclosed herein, or pharmaceutical compositions as disclosed herein.

The foregoing is a summary and therefore contains necessary simplifications, generalizations and omitted details that are further described below in the detailed description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as a statement in determining the scope of the claimed subject matter.

While the invention may be embodied in many different forms, specific illustrative embodiments thereof are disclosed herein that demonstrate the principles of the invention. It should be emphasized that this invention is not limited to the specific embodiments illustrated. Additionally, any section headings used in this article are for organizational purposes only and are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used in connection with this invention shall have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, terms in the singular shall include the plural and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes a mixture of cells, etc. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "comprises" as well as other forms such as "includes" and "contains" is not limiting. In addition, the ranges provided in the specification and accompanying request include all values between the endpoint and the interruption point.

In general, the terms associated with cell and tissue culture, molecular biology, immunology, microbiology, genetics and proteins, and nucleic acid chemistry and hybridization and the techniques described herein are well-known and commonly used terms in the art. Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, 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 terminology and laboratory procedures and techniques associated with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well-known and commonly used terms in the art. definition

For a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

As used herein, 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, which contain only one chain, usually similar to a heavy chain), as well as fragments of any of the above, as long as they exhibit the required Antigen binding activity, this term includes, for example, antibodies comprising at least one VHH domain. Conventional antibodies contain heavy and light chains. Heavy chains can be divided into μ, δ, γ, α, and ε, which define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The heavy chain may comprise a heavy chain variable region ( _VH ) and a heavy chain constant region ( _CH ). The heavy chain constant region may comprise one or a plurality of constant regions, for example 3 constant regions ( _CH 1, _CH 2 and _CH 3). The light chain may comprise a light chain variable region ( _VL ) and a light chain constant region ( _CL ). The V _H and V _L regions can be further divided into hypervariable regions called complementarity determining regions (CDRs) separated by relatively conserved regions called framework regions (FRW). V _H and V _L can contain 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. Antibodies can be of different antibody isotypes, such as IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

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

A "functional Fc region" has the "effector function" 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; cell surface receptors (e.g., B cells Receptor (i.e. BCR) downregulation, etc. Such effector functions typically require combining an Fc region with a binding region or domain (e.g., an antibody variable region or domain, including a VHH domain) and can be assessed using the various assays disclosed.

A "native sequence Fc region" includes an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature and has not been artificially manipulated, modified and/or altered (e.g., isolated, purified, selected, including other sequences such as variable region sequences or in combination 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 variations thereof body.

"Variant Fc region" includes an amino acid sequence that differs from the native sequence Fc region by at least one amino acid modification (eg, 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, e.g., from about 1 to about 10 amino acid substitutions, compared to a native sequence Fc region or an Fc region of a parent polypeptide, e.g., preferably, a native sequence Fc region or a parent polypeptide. From about 1 to about 5 amino acid substitutions in the Fc region of the polypeptide. A variant Fc region may have at least about 80% homology to a native sequence Fc region and/or to an Fc region of a parent polypeptide, or at least about 90% homology thereto, e.g., at least about 95% homology thereto. Homology. Variant Fc regions described herein can have a loss of effector function (eg, silenced Fc).

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, cellular Endobody, single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibody, Fab fragment, F(ab') fragment, disulfide-linked Fv (sdFv), anti-idiotype (Anti-Id) antibodies, and epitope-binding fragments of any of the above.

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 to refer to the A single-chain antigen-binding domain that binds to an antigen or epitope independently of distinct variable domains. VHH domains (e.g., variable domains of heavy chain antibodies) represent the smallest known antigen-binding units produced by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov; 21(13):3490- 8. Epub 2007 Jun 15 (2007)). VHH domains may be human domains, but also include individual domains from other species, such as rodent, shark and camelid VHH domains. Camelidae VHHs are immunoglobulin single variable domain polypeptides derived from species including camels, llamas, alpacas, dromedaries, and guanacos that produce heavy chain antibodies that naturally lack light chains. Such VHH domains can 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.

The term "humanized antibody" is intended to refer to an antibody in which CDR sequences derived from the germline of another mammalian species, such as mouse, llama or alpaca, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequence.

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

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

As used herein, the term "high affinity" means that a D3 binding molecule, such as an antibody, has 1×10 ^-7 M or less, more preferably 5×10 ^-8 M or less, even more preferably 1× for a target antigen. KD of 10 ^-8 M or less, even more preferably 5 × 10 ^-9 M or less, and even more preferably 1 × 10 ^-9 M or less.

As used herein, the term " _EC50 ", also known as "half maximal effective concentration", refers to the concentration of a drug, antibody or agent that induces a response of 50% between baseline and maximum after a specified exposure time . In the context of this application, the unit of _EC50 is usually "nM".

As used herein, the term "epitope" refers to the portion of an antigen to which an immunoglobulin or antibody specifically binds. "Epitope" is also called "antigenic determinant". Epitopes or antigenic determinants typically consist of chemically active surface groups on molecules such as amino acids, carbohydrates, or sugar side chains, and often have a specific three-dimensional structure and specific charge characteristics. For example, an epitope typically contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique three-dimensional conformation, which may be "linear ” or “conformational” epitope. 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 (such as an antibody) exist linearly along the protein's primary amino acid sequence. In a conformational epitope, the site of interaction spans amino acid residues that are separated from each other in the protein. Antibodies can be screened depending on their competitiveness for binding to the same epitope as determined by routine 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. A high-throughput method for obtaining antibodies binding to the same epitope, based on their cross-competition, is described in international patent application WO 03/48731.

As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds a D3 protein is substantially free of specific binding to a protein other than D3 Antibodies to the antigen). However, isolated antibodies that specifically bind human D3 protein may be cross-reactive to other antigens, such as D3 proteins from other species. Furthermore, isolated antibodies may be substantially free of other cellular material and/or chemicals.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. A vector is called an expression vector when it permits the expression of a protein encoded by a polynucleotide inserted therein. The vector can be transformed, transduced or transfected into a host cell so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); phages such as lambda phage or M13 bacteriophage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyspace viruses (such as SV40). Vectors may contain a plurality of elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain an origin of replication.

As used herein, the term "host cell" refers to cells into which vectors can be introduced, including but not limited to prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast cells, Aspergillus, and insect cells such as Drosophila S2 cells and Sf9, and Animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

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 aligning and comparing the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably addressed by specific mathematical models or computer programs (i.e., "algorithms"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides are included in Computational Molecular Biology, (ed. Lesk, A. M.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (ed. Smith, D. W.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M. and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology , New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAMJ. Applied Math. 48:1073 those described.

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 property of antigens to stimulate 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 stimulate organisms with antigens. Later formed in the organism's immune system. Immunogenicity is an important property of an antigen. Whether an antigen can successfully induce an immune response in the host depends on several factors, including the nature of the antigen, the host's reactivity, and the means of immunization.

As used herein, the term "transfection" refers to the procedure of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are 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.

As used herein, the term "SPR" or "surface plasmon resonance" refers to and includes optical phenomena that allow analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, such as using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For detailed description, see Examples and Jönsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B ., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnson, B., et al. (1991) Anal. Biochem. 198:268-277.

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 heterogeneous mixtures of biological cells, one cell at a time, into two or more containers based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. “Sorting Out Fluorescence Activated Cell Sorting” . 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 the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, CA), the Epics C from Coulter Epics Division (Hialeah, FL), and the MoFlo from Cytomation (Colorado Springs, Colorado).

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

As used herein, the term "D3-related condition" or "D3-related disorder" refers to any condition caused by, worsened by, or otherwise associated with an increase or decrease (usually an increase) in the expression or activity of D3 (e.g., human D3). condition.

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

Treatment as a preventive measure (e.g., protection, prevention) is also included. For cancer, "treatment" may mean inhibiting or slowing tumor or malignant cell growth, proliferation or metastasis, or some combination thereof. With respect to tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination thereof.

As used herein, the term "therapeutically effective amount" refers to an amount of an active compound, or an amount of material, composition, or dosage form containing an active compound, that is effective to produce a result that is associated with reasonable benefits/risks when administered in accordance with the desired treatment regimen. ratio commensurate with certain desired therapeutic effects. For example, a "therapeutically effective amount" of a D3-binding molecule refers to an amount or concentration effective to treat a D3-related disease or condition in humans.

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

As used herein, the term "pharmaceutically acceptable" means that the vehicle, diluent, excipient, and/or salts thereof are chemically and/or physically compatible with the other ingredients of the formulation, and physiologically compatible with the recipient. Compatible.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active agent, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and include, but are not limited to, pH adjusters, surfactants, adjuvants and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride. The carrier, excipient or stabilizer is non-toxic to the cells or mammal to which it is exposed at the doses and concentrations employed. The carrier is usually an aqueous pH buffer solution. Examples of carriers include buffers such as phosphates, citrates, 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 dextrin; 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™, polyethylene glycol ( PEG) and PLURONICS™. The term "carrier" may also refer to a diluent, adjuvant (such as Freund's adjuvant (complete or incomplete)), excipient, or vehicle with which the therapeutic agent is administered. Such carriers may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When administering a composition (eg, a pharmaceutical composition) intravenously, an exemplary carrier is water. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g. pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicone, sodium stearate, glyceryl monostearate, talc, chlorine Sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, ethanol, etc. If desired, the compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, and the like. Oral compositions, including formulations, may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable carriers are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA. Compositions including pharmaceutical compounds may contain a prophylactically or therapeutically effective amount of a D3 binding agent (e.g., an anti-D3 antibody), e.g., in an isolated or purified form, and a suitable amount of carrier to provide administration to a subject (e.g., a patient) Appropriate form of administration. The formulation should be suitable for the mode of administration.

As used herein, the term "adjuvant" refers to a non-specific immunopotentiator that, when delivered with an antigen to an organism or in advance of delivery to an organism, can enhance the immune response to the antigen in the organism or alter the immune response. type. A variety of adjuvants exist, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvant (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. . Freund's adjuvant is currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials. D3 binding molecule

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, "D3-binding molecules" may include "D3 antagonists" and "anti-D3 antibodies." "D3 antagonist" refers to any chemical compound or biological molecule that blocks D3 activity. "Anti-D3 antibody" includes, but is not limited to, chimeric, humanized, human, or single domain antibodies. D3 binding molecules are not limited to polypeptides or proteins and may include other components such as nucleotides, hybrids, dextran, and combinations thereof. As exemplified herein, the D3 binding molecule can be an anti-D3 antibody or an anti-D3 fusion protein.

In some embodiments, the D3 binding molecules disclosed herein comprise at least one VHH that specifically binds D3. Further, the D3 binding molecule can be a single domain antibody and contain a VHH. For example, single domain antibodies are capable of selectively binding to a specific antigen (such as D3). In some embodiments, the D3 binding molecule comprises a VHH fused to the Fc region of an immunoglobulin, such as the Fc region of an IgG (eg, IgG4 or IgGl). In some embodiments, the Fc region is that of a human IgG1. By fusing VHH to the Fc region, recruitment of effector functions may be more efficient. In addition, the fusion of VHH and Fc region may help D3-binding molecules form dimers and may also help extend the half-life of D3-binding molecules in the body.

As is known in the art, the VHH molecule derived from camelid antibodies is one of the smallest complete antigen-binding domains known (approximately 15 kDa, or 10 times smaller than conventional IgG) and is therefore well suited for delivery to dense tissues and for Reach the limited space between macromolecules.

VHHs as disclosed herein may be manufactured by the skilled person according to methods known in the art or any future methods. For example, methods known in the art may be used, such as by immunizing camels and obtaining hybridomas therefrom or by cloning a library of VHHs of the present disclosure using molecular biology techniques known in the art and subsequently by using phage display. Choose to get VHH.

For example, VHH can be obtained by immunizing a llama or alpaca with the desired antigen and subsequently isolating the mRNA encoding the heavy chain antibody. By reverse transcription and polymerase chain reaction, a gene library containing millions of clones of single-domain antibodies was generated. Screening techniques such as phage display and ribosome display can help identify clones that bind to the antigen. One technique is phage display, in which (e.g., human) antibody libraries are synthesized on phage, the library is screened with the antigen of interest or its antibody-binding portion, and the phage that bind the antigen are isolated 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 ^™ Phage Display kit, catalog number 240612). There are other methods and reagents that can be used to generate and screen antibody display libraries (see, eg, Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991)).

When effective clones are identified, their DNA sequences are optimized, for example, through affinity maturation or humanization. Humanization prevents the body's immune response to antibodies.

Therefore, VHH can be obtained by: (1) isolating the VHH domain of a naturally occurring heavy chain antibody; (2) representing the nucleotide sequence encoding the naturally occurring VHH domain; (3) the naturally occurring VHH structure "Humanization" of a domain (as described below), or the expression of a nucleic acid encoding such a humanized VHH domain; (4) "Camelization" from any animal species (especially mammalian species such as from humans) A naturally occurring VH domain, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by "camelization" of a "domain antibody" or "Dab" as described by Ward et al. (supra), Or by the expression of nucleic acids encoding such camelized VH domains; (6) using synthetic or semi-synthetic techniques to prepare proteins, polypeptides or other amino acid sequences; (7) using nucleic acid synthesis techniques to prepare nucleic acids encoding VHH, 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 to increase the affinity and/or specificity of the VHH; and/ or (9) any combination of the above. Suitable methods and techniques for performing the foregoing will be apparent to the skilled person based on the disclosure herein, and including, for example, the methods and techniques described in greater detail herein.

Single domain antibodies are typically produced by PCR cloning variable domain libraries from blood, lymph node, or spleen cDNA obtained from immunized animals into phage display vectors. Antigen-specific single domain antibodies are typically selected by panning staged libraries on immobilized antigens, such as antigens coated on the plastic surface of test tubes or biotinylated antigens immobilized on streptavidin beads. or membrane proteins expressed on the cell surface. The affinity of sdAbs can often be improved by mimicking this strategy in vitro, for example, by site-directed mutagenesis of CDR regions and under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH). and low antigen concentrations) on immobilized antigens (Wesolowski et al., Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol (2009) 198: 157-174).

Methods for preparing VHHs that specifically bind to antigens or epitopes 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), pp.2645, 17 June, 2009 and WO 94/04678.

In some embodiments, the VHH can be truncated at the N- or C-terminus such that it contains only a portion of FRW1 and/or FRW4, or lacks one or both of those framework regions, as long as the VHH substantially retains antigen binding and specificity (e.g., substantially maintain, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

The present disclosure also provides D3 binding molecules having a masking moiety and/or a cleavable moiety, wherein one or more D3 binding domains of the D3 binding molecule are masked (e.g., by the masking moiety) and/or activatable (e.g., by from cuttable parts). Techniques for masking D3 binding molecules (e.g., antibodies) are well known in the art and include SAFE body masking technology (see, eg, US 2019/0241886) and Probody masking technology (see, eg, US 2015/0079088). Such techniques can be used to generate masked and/or turnable D3-binding molecules (eg, antibodies). Such masked and/or programmable D3-binding molecules (e.g., antibodies) can be used to prepare conjugates, including immunoconjugates, antibody-drug conjugates (ADCs), masked ADCs, and programmable antibody- Drug conjugates (AADC), which comprise any D3-binding molecule (eg, antibody) of the present disclosure linked directly or indirectly 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.

If desired, the D3 binding molecule is linked or conjugated (directly or indirectly) to a moiety having effector function, such as cytotoxic activity (eg, a chemotherapeutic moiety or radioisotope) or immune recruitment activity. Linking or conjugating moieties (direct or indirect) include drugs that are cytotoxic (e.g., toxins, such as auristatins) or non-cytotoxic drugs (e.g., signal transduction modulators, such as kinases) or that mask D3 binding molecules A masking portion of the binding domain(s), or a cleavable portion that allows activation of the D3 binding molecule by cleaving the cleavable portion, in the form of a masking conjugate, by binding the one or plurality of binding domains of the D3 binding molecule Demasking in the tumor microenvironment. 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 the mixture (e.g., a tag) or a moiety that has reporter activity (e.g., a detection label or reporter protein). It will be understood that the characteristics of D3 binding molecules described herein also extend to polypeptides comprising fragments of D3 binding molecules.

In some embodiments, a D3 binding molecule described herein can be linked or conjugated (directly or indirectly) to a polypeptide, which can result in the production of an antibody that can be initiated. In some embodiments, the D3 binding molecule is linked or conjugated (directly or indirectly) to an agent. In some embodiments, the agent is a drug that produces an ADC or an AADC when the antibody to the ADC includes a masking moiety and a cleavable moiety.

In some embodiments, a D3 binding molecule described herein is conjugated or recombinantly linked (directly or indirectly) to a therapeutic (eg, cytotoxic agent) or diagnostic or detectable agent. Conjugated or recombinantly linked antibodies, including masked or activatable conjugates, may be used, for example, to treat or prevent diseases, disorders or conditions, such as cancer or tumors.

For example, diagnosis and detection can be accomplished by coupling a D3 binding molecule to a detectable substance, including, for example, enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-semi- Lactosidase or acetylcholinesterase; prosthetic groups, including but not limited to streptavidin/biotin or avidin/biotin; fluorescent materials, including but not limited to umbelliferone, luciferin, Luciferin isothiocyanate, rhodamine, luciferin dichlorotriazinamide, dansyl chloride or phycoerythrin; luminescent materials, including but not limited to luminol; bioluminescent materials, including but not limited to fluorescein enzyme, luciferin or aequorin; chemiluminescent materials, including but not limited to acridinyl compounds or HALOTAG; radioactive substances, including but not limited to iodine (131I, 125I, 123I and 121I), carbon (14C), sulfur (35S), Tritium (3H), Indium (115In, 113In, 112In and 111In), Xun (99Tc), Thallium (201Ti), Gallium (68Ga and 67Ga), Palladium (103Pd), Molybdenum (99Mo), Xenon (133Xe ), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 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 nonradioactive paramagnetic metal ions.

A variety of bifunctional protein coupling agents can be used 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-vinylstyrene) benzoate) to prepare antibody and agent conjugates substances, including cases where the pharmaceutical agent is a drug for the preparation of an ADC or AADC. The present disclosure further contemplates that conjugates of antibodies and agents may be prepared using any suitable method disclosed in the art, including where the agent is a drug used to prepare an ADC or AADC (see, e.g., Bioconjugate Techniques (Hermanson ed. , 2d ed. 2008)).

Conventional conjugation strategies for antibodies and reagents, including cases where the reagent is a drug used to prepare an ADC or AADC, are based on stochastic conjugation chemistry involving the ε-amino group of a Lys residue or the thiol group of a Cys residue, which yields Heterogeneous conjugation. Recently developed technologies allow site-specific conjugation to antibodies, thereby enabling homogeneous loading and avoiding subpopulations of conjugates with altered antigen binding or pharmacokinetics. These include engineering "thiomabs" that include cysteine substitutions at heavy and light chain positions that provide reactive thiol groups and do not disrupt immunoglobulin folding and assembly or alter antigen binding (see e.g., Junutula et al., 2008, J. Immunol. Meth. 332:41-52; and Junutula et al., 2008, Nature Biotechnol. 26:925-32). In another approach, selenocysteine was co-translationally inserted into the antibody sequence by recoding the stop codon UGA from stop to selenocysteine insertion, thereby allowing for the addition of selenocysteine in the presence of other natural amino acids. and Hofer et al., 2009, Biochemistry 48(50):12047-57).

D3 binding molecules described herein can be monospecific, bispecific, trispecific or have greater multispecificity. Such reagents may include antibodies. Multispecific antibodies, such as bispecific antibodies, are monoclonal antibodies with binding specificities for at least two different targets (e.g., antigens) or two different epitopes on the same target (e.g., for D3 The bispecific antibody has a first binding domain directed against a first epitope of D3 and a second binding domain directed against a second epitope of D3). In some embodiments, multispecific (eg, bispecific) antibodies can be constructed based on the sequences of the antibodies described herein. In some embodiments, the multispecific antibodies described herein are bispecific antibodies. In some embodiments, the bispecific antibody is a mouse antibody, a chimeric antibody, a human antibody, or a humanized antibody. In some embodiments, one of the binding specificities of the multispecific antibody is for D3 and the other is for any other target (eg, antigen). In some embodiments, multispecific (e.g., bispecific) antibodies can comprise more than one target (e.g., antigen) binding domain, where different binding domains are specific for different targets (e.g., bind D3 and a second binding domain that binds another target (eg, an antigen), such as an immune checkpoint modulator (eg, a negative checkpoint modulator). In some embodiments, a multispecific (eg, bispecific) antibody molecule can bind to more than one (eg, two or more) epitopes on the same target (eg, antigen). In some embodiments, one binding specificity is D3 and the other is for one or more of: 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 priming gene 3 (LAG-3; also known as CD223), galectin-3, B and T lymphocyte attenuation factor (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, herpesvirus entry mediator (HVEM), OX40, CD27, CD28, CD137, CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052 and CGEN-15092.

Methods for preparing multispecific antibodies are known in the art, for example, by co-expressing two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello , 1983, Nature 305: 537-40). For more details on the generation of multispecific antibodies (e.g. bispecific antibodies) see e.g. Bispecific Antibodies (Kontermann ed., 2011).

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 from a non-human source. These non-human amino acid residues are often referred to as "import" residues, and they are usually taken from the "import" variable domain. Humanized antibodies that bind D3 can be generated using techniques known to those skilled in the art (e.g., Zhang et al., Molecular Immunology, 42(12):1445-1451, 2005; Hwang et al., Methods, 36(1): 35-42, 2005; Dall'Acqua et al., Methods, 36(1): 43-60, 2005; Clark, Immunology Today, 21(8): 397-402, 2000, and U.S. Patent Nos. 6,180,370; 6,054,927; 5,869,619 ; 5,861,155; 5,712,120; and 4,816,567).

D3 binding molecules may be described below as anti-D3 antibodies. Anti -D3 antibodies with functional properties

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 antibody has one or more of the following properties: (a) Binds to human D3, cynomolgus monkey D3, and mouse D3 with an EC50 in the nM range, as measured by ELISA or FACS; (b) Demonstrate dose-dependent internalization efficacy in human cells engineered to express D3; and (c) Binds to human D3 ECD with a KD of no more than 0.1 nM, as measured by SPR.

The antibodies of the present disclosure bind cell surface D3 with high affinity. Binding of the antibodies of the invention to D3 can be assessed using one or more techniques established in the art, such as ELISA. The binding specificity of the antibodies of the invention can also be determined by monitoring the binding of the antibodies to cells expressing the D3 protein, for example, by flow cytometry. For example, antibodies can be tested by flow cytometry assays in which the antibodies react with cell lines expressing human D3 such as CHO cells and 293 cells that have been transfected to express D3 on their cell surface. Additionally or alternatively, the binding of the antibody, including binding kinetics (e.g., Kd value), can be tested in a BIAcore binding assay. Other suitable binding assays include ELISA assays, for example using recombinant D3 protein. For example, the antibodies of the present disclosure are 1×10 ^-7 M or lower, 5×10 ^-8 M or lower, 2×10 ^-8 M or lower, 5×10 ^-9 M or lower, 4×10 ^-9 M or less, 3 × 10 ^-9 M or less, 2 × 10 ^-9 M or less, 1 × 10 ^-9 M or less, 5 × 10 ^-10 M or less, or 1 × 10 ^- Binds cell surface _D3 (e.g., human D3 ECD) proteins with a K of ¹⁰ M or less.

In some embodiments, the antibodies of the disclosure are present at 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 EC ₅₀ binds cynomolgus monkey or mouse D3 as measured by FACS. Anti- D3 antibody containing VHH CDRs

In some embodiments, an anti-D3 antibody as disclosed herein comprises at least one immunoglobulin single variable domain (e.g., VHH), wherein VHH comprises CDR1, CDR2, and CDR3, and wherein CDR1 comprises SEQ ID NO: 1 , 4, 7 or 10, CDR2 includes the amino acid sequence listed as SEQ ID NO: 2, 5, 8 or 11, CDR3 includes the amino acid sequence listed as SEQ ID NO: 3, 6 or 9 Amino acid sequence. In some embodiments, CDR numbering is based on a combination of Kabat and AbM numbering.

The scope of framework regions and CDRs can be accurately identified by methods known in the art, such as by Kabat definition, Chothia definition, AbM definition, Contact definition, IMGT definition (all well known in the art) and any combination thereof. See, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition , US Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; Edelman et al, Proc Natl Acad Sci US A. 1969 May, 63(1):78-85; and Martin and Allen, “ Handbook of Therapeutic Antibodies ”, chapter 5, 2007. See also hgmp.mrc.ac.uk and bioinf.org.uk/abs. Correspondences or alignments between numbers 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 Ig superfamily V-like domains. Dev Comp Immunol. (2003) 27:55–77).

As those skilled in the art will appreciate, the exact numbering and location of CDRs may differ in different numbering systems. However, it is understood that disclosure of variable heavy chain sequences, variable light chain sequences and/or VHH sequences includes disclosure of the relevant (intrinsic) CDRs. Therefore, the disclosure of each variable region is the disclosure of a CDR (eg, CDR1, CDR2, and CDR3). Two antibodies having the same VH, VL or VHH CDR means that their CDRs are identical when determined by the same method (e.g., Kabat, AbM, Chothia, Contact, and IMGT numbering methods known in the art).

Variable regions and CDRs in antibody sequences can be determined according to general rules that have been developed in the art (as mentioned above, such as the Kabat, AbM, Chothia, Contact and IMGT numbering systems) or by comparing the sequence to a database of known variable regions. Compare to identify. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary libraries of antibody sequences are described and available from the "Abysis" website at www.bioinf.org.uk/abs (maintained by AC Martin, Department of Biochemistry & Molecular Biology University College London, London, England) and VBASE2 Website www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). It is better to use the Abysis database to analyze sequences, which integrates sequence data from Kabat, IMGT and Protein Data Bank (PDB) with structural data from PDB, see Protein Sequence and Structure Analysis in the book by Dr. Andrew CR Martin of Antibody Variable Domains . In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-VerTIM-3, Heidelberg, ISBN-13: 978-3540413547, also available at bioinforge.uk/abs obtained on). The Abysis repository website also includes general rules that have been developed for identifying CDRs that may be used in accordance with the teachings of this article. Figure 10 shows an exemplary alignment of immunoglobulin single variable domains, with CDR boundaries represented by Kabat, AbM, Chothia, Contact and IMGT numbers.

In some embodiments, the D3 binding molecules disclosed herein comprise at least one immunoglobulin single variable domain (e.g., VHH), wherein VHH comprises FRW1-CDR1-FRW2-CDR2-FRW3-CDR3-FRW4, and wherein CDR1 has as 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 the CDR3 has the amino acid sequence shown in SEQ ID NO: 3, 6 or 9 The amino acid sequence shown. In some embodiments, FRW1 and FRW4 at the N and C termini of a VHH contained in a D3 binding molecule can be truncated so that it contains only part of FRW1 and/or FRW4, or the VHH lacks one or both of these framework regions. , as long as the VHH substantially maintains antigen binding and specificity.

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

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

In some embodiments, an anti-D3 antibody (eg, a single domain antibody) comprises a CDR1 having the amino acid sequence of CDR1 as set forth in SEQ ID NO: 13. In some embodiments, an anti-D3 antibody (eg, a single domain antibody) comprises a CDR2 having the amino acid sequence of the CDR2 as set forth in SEQ ID NO: 13. In other embodiments, an anti-D3 antibody (eg, a single domain antibody) comprises a CDR3 having the amino acid sequence of the CDR3 as set forth in SEQ ID NO: 13. In some embodiments, an anti-D3 antibody (eg, a single domain antibody) comprises CDR1 and CDR2 having the amino acid sequence of CDR1 and CDR2 as set forth in SEQ ID NO: 13. In some embodiments, an anti-D3 antibody (eg, a single domain antibody) comprises CDR1 and CDR3 having the amino acid sequence of CDR1 and CDR3 as set forth in SEQ ID NO: 13. In some embodiments, an anti-D3 antibody (e.g., a single domain antibody) comprises CDR2 and CDR3 having the amino acid sequence of CDR2 and CDR3 as set forth in SEQ ID NO: 13. In some embodiments, an anti-D3 antibody (eg, a single domain antibody) comprises CDR1, CDR2, and CDR3 having the amino acid sequence of CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 13. CDR sequences can be determined according to well-known numbering systems. In some embodiments, CDRs are numbered according to IMGT. In some embodiments, CDRs are numbered according to Kabat. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, CDRs are numbered by Contact. In some embodiments, CDRs are numbered according to AbM. In some embodiments, the anti-D3 single domain antibody is a camelid antibody. In some embodiments, anti-D3 antibodies (eg, anti-D3 single domain antibodies) are humanized. In some embodiments, an anti-D3 antibody (eg, an anti-D3 single domain antibody) comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

In some embodiments, an anti-D3 antibody (eg, a single domain antibody) comprises a CDR1 having the amino acid sequence of CDR1 as set forth in SEQ ID NO: 14. In some embodiments, an anti-D3 antibody (eg, a single domain antibody) comprises a CDR2 having the amino acid sequence of the CDR2 as set forth in SEQ ID NO: 14. In other embodiments, an anti-D3 antibody (eg, a single domain antibody) comprises a CDR3 having the amino acid sequence of the CDR3 set forth in SEQ ID NO: 14. In some embodiments, an anti-D3 antibody (eg, a single domain antibody) comprises CDR1 and CDR2 having the amino acid sequence of CDR1 and CDR2 as set forth in SEQ ID NO: 14. In some embodiments, an anti-D3 antibody (eg, a single domain antibody) comprises CDR1 and CDR3 having the amino acid sequence of CDR1 and CDR3 as set forth in SEQ ID NO: 14. In some embodiments, an anti-D3 antibody (e.g., a single domain antibody) comprises CDR2 and CDR3 having the amino acid sequence of CDR2 and CDR3 as set forth in SEQ ID NO: 14. In some embodiments, an anti-D3 antibody (eg, a single domain antibody) comprises CDR1, CDR2, and CDR3 having the amino acid sequence of CDR1, CDR2, and CDR3 as set forth in SEQ ID NO: 14. CDR sequences can be determined according to well-known numbering systems. In some embodiments, CDRs are numbered according to IMGT. In some embodiments, CDRs are numbered according to Kabat. In other embodiments, the CDRs are numbered according to Chothia. In other embodiments, CDRs are numbered by Contact. In some embodiments, CDRs are numbered according to AbM. In some embodiments, the anti-D3 single domain antibody is a camelid antibody. In some embodiments, anti-D3 antibodies (eg, anti-D3 single domain antibodies) are humanized. In some embodiments, an anti-D3 antibody (eg, an anti-D3 single domain antibody) comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

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

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

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

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

In some embodiments, provided herein are D3-binding single domain antibodies comprising the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein (i) CDR1 comprises, for example, 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. The amino acid sequence; (ii) CDR2 includes 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: 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, The amino acid sequence shown in SEQ ID NO: 50 or SEQ ID NO: 52; and/or (iii) CDR3 includes 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 Amino acid sequence. In some embodiments, the anti-D3 single domain antibody is a camelid antibody. In some embodiments, anti-D3 single domain antibodies are humanized. In some embodiments, anti-D3 single domain antibodies comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

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

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

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

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

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

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

The frame areas described in this article are determined based on the boundaries of the CDR numbering system. In other words, if the CDR is determined by, for example, IMGT, Kabat, Chothia, Contact or AbM, the framework region is the amino acid residues surrounding the CDR in the variable region in the form, from N-terminus to C-terminus: FR1-CDR1-FR2 -CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residue N-terminal to the CDR1 amino acid residue as defined by, e.g., the IMGT numbering system, Kabat numbering system, Chothia numbering system, Contact numbering system, or AbM numbering system, and FR2 is defined as, e.g., the IMGT numbering system The amino acid residues between CDR1 and CDR2 amino acid residues defined by the IMGT numbering system, Kabat numbering system, Chothia numbering system, Contact numbering system or AbM numbering system, FR3 is defined as by, for example, the IMGT numbering system, Kabat numbering system, Chothia numbering system, The amino acid residue between CDR2 and CDR3 as defined by the Contact numbering system or the AbM numbering system, and FR4 is defined as an amino acid within the CDR3 as defined by, e.g., the IMGT numbering system, the Kabat numbering system, the Chothia numbering system, the Contact numbering system, or the AbM numbering system The amino acid residue at the C-terminal end of the residue.

In some embodiments, an isolated anti-D3 single domain antibody is provided comprising a VHH domain having the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 12 is provided. In some embodiments, isolated anti-D3 single domain antibodies are provided that comprise a VHH domain having the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13 is provided. In some embodiments, an isolated anti-D3 single domain antibody is provided comprising a VHH domain having the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 14 are provided. In some embodiments, isolated anti-D3 single domain antibodies are provided that comprise a VHH domain having the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 15 are provided. In some embodiments, isolated anti-D3 single domain antibodies are provided that comprise a VHH domain having the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 16 are provided. In some embodiments, isolated anti-D3 single domain antibodies are provided that comprise a VHH domain having the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 17 are provided. In some embodiments, isolated anti-D3 single domain antibodies are provided that comprise a VHH domain having the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 18 are provided. In some embodiments, an isolated anti-D3 single domain antibody is provided comprising a VHH domain having the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 55 are provided. Anti -D3 antibodies containing VHH sequences

In some embodiments, an anti-D3 antibody comprises at least one immunoglobulin single variable domain (e.g., VHH), wherein the VHH comprises or consists of: (A) The amino acid sequence shown in any one of SEQ ID NOs: 12-18 and 55; (B) An amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs: 12-18 and 55; or (C) The addition of one or a plurality (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) of amino acids compared to any one of SEQ ID NOs: 12-18 and 55 , deleted and/or substituted amino acid sequences.

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 weighted residue table, with a gap length penalty of 12 and a gap penalty of 4. Additionally, 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 GCG suite of software (available from http In the GAP program in (obtained from http://www.gcg.com), Blossum 62 matrix or PAM250 matrix is used, the gap weight is 16, 14, 12, 10, 8, 6 or 4, and the length weight is 1, 2, 3, 4, 5 or 6.

Additionally or alternatively, protein (eg, antibody) sequences of the invention may further be used as "query sequences" to perform searches against public repositories, eg, to identify relevant sequences. This search can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215:403-10. A BLAST protein search can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and gapped BLAST programs, you can use the default parameters for each program (for example, XBLAST and NBLAST). See www.ncbi.nlm.nih.gov.

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

In some further embodiments, anti-D3 antibodies may comprise conservative substitutions or modifications of amino acids in the variable and/or constant regions. It is understood in the art that certain conservative sequence modifications can be made that do not eliminate antigen binding. See, for example, 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.

The term "conservative substitution" as used herein refers to amino acid substitutions that do not adversely affect or alter the essential 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 an amino acid residue is replaced by another amino acid residue with a similar side chain, such as a physically or functionally similar residue (e.g., having similar size, shape, charge, chemical properties, including formation of covalent bonds or hydrogen bonding ability, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar sides Chains of amino acids (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 (e.g., threonine, valine, isoleucine ) and amino acids with aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Therefore, the corresponding amino acid residue is preferably replaced by another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids 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 is incorporated herein by reference).

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 having the amino acid sequence set forth in any one of SEQ ID NOs: 12-18.

In some embodiments, the anti-D3 antibody is a chimeric antibody comprising a VHH fused to the Fc region of human IgGl or IgG4, wherein the VHH comprises the amino acids set forth in any of SEQ ID NOs: 12-18 and 55 sequence. In some embodiments, the anti-D3 antibody is a chimeric antibody comprising VHH and the Fc region of human IgGl. Examples of such antibodies herein are "WT1156-P3R2-1C2-uIgG1", "WT1156-P3R2-1H6-uIgG1", "WT1156-P8R2-1H1-uIgG1" and "WT1156-P3R2-1C9-uIgG1". In some additional embodiments, the anti-D3 antibody is a humanized antibody comprising VHH and the Fc region of human IgG1. Examples of such antibodies herein are "WT1156-P3R2-1C2-z102-uIgG1", "WT1156-P3R2-1C2-z109-uIgG1" and "WT1156-P3R2-1H6-z100-uIgG1".

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

In some embodiments, an antibody or antigen-binding portion thereof as provided herein includes any suitable framework region (FRW) sequence so long as the antigen-binding domain is capable of specifically binding D3. As noted above, an antibody, or an antigen-binding portion thereof, may contain one or more amino acid modifications in the variable region of the heavy and/or light chain, including where the modification is a conservative substitution. It is understood in the art that certain conservative sequence modifications can be made that do not eliminate antigen binding. See, for example, 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.

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 NO: 12-18 and 55, and comprising the amino acid sequence set forth in SEQ ID NO: 19 sequence of the Fc region.

The antigen-binding domain of the D3 binding molecule is not limited to the VHH form, and can be in a variety of other forms, such as but not limited to Fab, Fab', F(ab')2, Fv fragments, and single-chain antibody molecules (scFv). In some embodiments, the antigen-binding domain is an Fv fragment having the VH and VL regions in separate chains, held together by tight non-covalent interactions. Fc region containing IgG constant domain

The anti-D3 antibodies and antigen-binding fragments thereof provided herein also comprise an Fc region comprising one or more human IgG constant domains. The human IgG constant domain may be a human IgG1, IgG2, IgG3 or IgG4 constant domain, preferably a human IgG1 constant domain. An example of the amino acid sequence of the Fc region comprising the human IgG1 constant region is listed 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 (e.g., Leu234Ala/Leu235Ala or LALA) ADCC) or other effector functions.

In some embodiments, the Fc modifications comprise LALA mutations, such as the mutations L234A and L235A according to EU numbering in Kabat et al. The Kabat numbering system is generally used to refer to residues in variable domains (approximately residues 1–107 for the light chain and residues 1–113 for the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" is typically used (eg, the EU index reported in Kabat et al., supra). "EU numbering in Kabat" or "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbering in an antibody constant domain refer to the residue numbering according to the EU numbering system. Nucleic acid molecules encoding antibodies of the present disclosure

In some aspects, the present disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding a D3 binding molecule as disclosed herein, eg, encoding a single variable domain of a D3 binding molecule as disclosed herein. The nucleic acids of the present disclosure can be obtained using standard molecular biology techniques.

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

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

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

In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of: (A) A nucleic acid sequence encoding a VHH region as shown in any one of SEQ ID NOs: 12-18 and 55; (B) has at least 80% (such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, A nucleic acid sequence with a sequence identity of 96%, 97%, 98% or 99%); and (C) A nucleic acid sequence that hybridizes to the complementary strand of the nucleic acid sequence of (A) under highly stringent conditions.

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.

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

Exemplary highly stringent conditions include hybridization in 5X SSPE and 45% formamide at 45°C, with final washes in 0.1X SSC at 65°C. It will be understood in the art that the temperature and buffer or salt concentration can be adjusted as described in Ausubel et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pages 6.0.3 to 6.4.10 changes to achieve equivalent stringent conditions. Changes in hybridization conditions can be determined empirically or calculated precisely based on the length of the probe and the percentage of guanosine/cytosine (GC) base pairing. Hybridization conditions can be calculated as described in Sambrook et al. (Eds.), Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pages 9.47 to 9.51. host cell

The host cells disclosed in the present disclosure can be any cell suitable for expressing the antibodies of the present disclosure, such as yeast, bacterial, plant and mammalian cells. Mammalian host cells for expressing the antibodies of the present disclosure include Chinese hamster ovary (CHO cells) (including dhfr CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, and DHFR selection markers are used together, for example as described by R. J. Kaufman and P. A. 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 WO87/04462, WO89/01036 and EP338,841. Also included are monkey kidney CV1 cell lines transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cells, subcultured 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 CRL 1442); Human lung cells (W138, ATCC CCL 75); Human liver Cells (Hep G2, HB 8065); Mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383:44-68); MRC 5 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 useful 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 the proteins expressed therein. In some embodiments, the host cells herein are selected from CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NSO cells or lymphocytes.

Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Escherichia, such as Escherichia coli, Enterobacter, Erwinia , Klebsiella, Proteus, Salmonella such as Salmonella typhimurium, Serratia such as Serratia marcescans, Shigella Shigella, Bacillus such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

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

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 expression of the antibody in the host cell or secretion of the antibody into the medium in which the host cell is cultured. Antibodies can be recovered from the culture medium using standard protein purification methods. pharmaceutical composition

In some aspects, the disclosure provides a pharmaceutical composition comprising a D3 binding molecule as disclosed herein, e.g., a single variable domain (eg, VHH) comprising a D3 binding molecule as disclosed herein and a pharmaceutically acceptable carrier. In some aspects, the present disclosure provides a pharmaceutical composition comprising a nucleic acid encoding a D3 binding molecule as disclosed herein, and a pharmaceutically acceptable carrier, the D3 binding molecule comprising, for example, a monoconjugate of a D3 binding molecule as disclosed herein. Variable domain (e.g. VHH). In some aspects, the present disclosure provides pharmaceutical compositions comprising cells expressing a D3 binding molecule as disclosed herein, and a pharmaceutically acceptable carrier, the D3 binding molecule comprising, for example, a monoconjugate of a D3 binding molecule as disclosed herein. Variable domain (e.g. VHH). Components of the composition

Pharmaceutical compositions may optionally contain one or more additional components, including one or more pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the present invention may also be administered in combination with, for example, another immunostimulatory agent, an anticancer agent, an antiviral agent, or a vaccine, including where anti-D3 antibodies enhance the immune response. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspension/dispersion agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, combinations of various components known in the art or more.

Suitable components of pharmaceutical compositions may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers, such as Sugar and cyclodextrin. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylbenzene Methyl ether, butylated hydroxytoluene and/or propyl gallate. As disclosed herein, compositions may include an antibody or antigen-binding fragment of the disclosure, and also include one or more antioxidants, such as methionine, to prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and prolonging Shelf life. Accordingly, in some embodiments, the present invention provides compositions comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants, such as methionine. The present invention further provides methods in which the antibody or antigen-binding fragment thereof is mixed with one or more antioxidants such as methionine, thereby preventing oxidation of the antibody or antigen-binding fragment thereof to extend its shelf life and/or increase activity.

To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactate Grignard injection, non-aqueous vehicle such as fixed oil of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, antimicrobial agent in bacteriostatic or fungistatic concentration, isotonic agent such as sodium chloride or dextrose, buffering agent 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, hydroxypropylmethylcellulose or polyvinylpyrrolidone , emulsifiers such as polysorbate 80 (TWEEN-80), barrier agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethylene glycol, polyethylene glycol, propylene glycol, hydrogen Sodium oxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multi-dose containers, and the compositions can contain phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, methylparaben and parahydroxybenzene. Propyl formate, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, or auxiliary substances such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins. Reagents. Administration, formulation and dosage

The pharmaceutical composition of the present invention can be administered to a subject in need via various routes, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, cardiac. Intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or by implantation or inhalation. The composition of the present invention can be formulated into preparations in the form of solid, semi-solid, liquid or gas; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. The appropriate formulation and route of administration can be selected based on the intended application and treatment regimen.

Suitable preparations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups or inhalations and controlled release dosage forms thereof.

Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free formulations in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other particulates) , sterile liquids (such as solutions, suspensions). Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickening agents and agents that render the preparation compatible with the blood (or other related substances) of the intended recipient. body fluids) isotonic solutes. Examples of excipients include, for example, water, alcohols, polyols, glycerin, vegetable oils, and the like. Examples of isotonic carriers suitable for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific dosing regimens (including dose, timing, 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.).

The frequency of administration can be determined and adjusted during the treatment program and is based on reducing the number of proliferating or tumorigenic cells, maintaining this reduction of tumor cells, reducing the proliferation of tumor cells, or delaying the development of metastases. In some embodiments, the dose administered can be adjusted or reduced to control potential side effects and/or toxicity. Alternatively, sustained continuous release formulations of the therapeutic compositions of the present invention may be suitable.

Those skilled in the art will understand that appropriate dosages may vary from patient to patient. Determining the optimal dosage often involves balancing the level of therapeutic benefit against any risks or harmful side effects. The dosage level selected will depend on a variety of factors, including, but not limited to, the activity of the particular compound, route of administration, time of administration, rate of compound clearance, duration of treatment, other concomitant drugs, compounds and/or materials, and the severity of the condition. , as well as the patient's type, gender, age, weight, condition, general health and past medical history. The amount of compound and route of administration are ultimately determined by the physician, veterinarian, or clinician, but dosages are generally selected to achieve local concentrations at the site of action that achieve the desired effect without causing substantial harmful or adverse side effects.

In general, the D3 binding molecules of the invention can be administered in various ranges. These include from about 5 μg/kg body weight to about 100 mg/kg body weight per dose; from about 50 μg/kg body weight to about 5 mg/kg body weight per dose; from about 100 μg/kg body weight to about 10 mg/kg body weight per dose; and any within this range. value. 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.

In any event, the D3 binding molecules of the invention are preferably administered to a subject in need thereof on an as-needed basis. Frequency of administration can be determined by those skilled in the art, such as by the attending physician, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, and the like.

In some embodiments, a therapeutic regimen involving a D3-binding molecule of the invention will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. For example, the D3 binding molecules of the invention can be used every 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 Apply every three months. In this regard, it is understood that dosages may be changed or intervals may be adjusted based on patient response and clinical practice.

Doses and regimens of the disclosed therapeutic compositions in subjects administered one or more administrations can also be determined empirically. For example, an individual may be administered incremental doses of a therapeutic composition produced as described herein. In some embodiments, dosage may be gradually increased or decreased or mitigated based on empirically determined or observed side effects or toxicity, respectively. To assess the efficacy of a selected composition, markers of a particular disease, disorder or condition can be tracked as described above. 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; improvements in assessment by direct tumor biopsies and microscopic examination of tumor samples; indirect measurement of tumor size Markers (such as PSA for prostate cancer) or tumorigenic antigens; reduction in pain or numbness; improvement in speech, vision, breathing, or other disability related to the tumor; increase in appetite; or as measured by tests administered Improvement of quality of life or extension of survival period. One skilled in the art will appreciate that dosage will vary depending on the individual, the type of tumor condition, the stage of the tumor condition, whether the tumor condition has begun to metastasize to other locations in the individual, and previous and current treatments.

Compatible formulations for parenteral administration (eg, intravenous injection) may contain 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 D3 binding molecules (e.g., antibodies or antigen-binding portions thereof) will include 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, 100 μg/ml, 200 μg/ml, 300 μg/ml, 400μg/ml, 500μg/ml, 600μg/ml, 700μg/ml, 800μg/ml, 900μg/ml or 1mg/ml. In some embodiments, the concentration of D3 binding molecules (e.g., antibodies or antigen-binding portions thereof) will include 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 8 mg/ml, 10 mg/ml, 12mg/ml, 14mg/ml, 16mg/ml, 18mg/ml, 20mg/ml, 25mg/ml, 30mg/ml, 35mg/ml, 40mg/ml, 45mg/ml, 50mg/ml, 60mg/ml, 70mg/ ml, 80mg/ml, 90mg/ml or 100mg/ml. Applications of the Disclosure

The antibodies, antibody compositions and methods of the invention have many in vitro and in vivo applications, including, for example, the detection of D3 or the enhancement of immune responses. For example, these molecules can be administered to cultured cells in vitro or ex vivo, or, for example, in vivo to human subjects to enhance immunity in various situations. The immune response can be modulated, such as enhanced, stimulated or up-regulated.

For example, subjects include human patients in need of enhanced immune response. The method is particularly suitable for treating human patients with conditions that are treatable by enhancing immune responses, such as T cell-mediated immune responses. In some embodiments, the method is particularly suitable for treating cancer in vivo. To achieve antigen-specific enhancement of immunity, anti-D3 antibodies may be administered together with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., tumor- or virus-bearing subjects). When an anti-D3 antibody is administered with another agent, the two may be administered in any order or simultaneously.

The invention further provides a method for detecting the presence of human D3 antigen or measuring the amount of human D3 antigen in a sample, comprising contacting the sample and a control sample with specific Contact with, for example, a human monoclonal antibody or an antigen-binding portion thereof that binds human D3. Complex formation is then detected, where differential complex formation between samples compared to control samples indicates the presence of human D3 antigen in the sample. In addition, the anti-D3 antibodies of the invention can be used to purify human D3 by immunoaffinity purification. Treat conditions including cancer

In some aspects, the present invention 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, as disclosed herein. In some aspects, the present disclosure provides anti-D3 antibodies, or antigen-binding portions thereof, as disclosed herein for use in treating a disease or disorder. In some aspects, provided herein is the use of an anti-D3 antibody, or antigen-binding portion thereof, disclosed herein, in the manufacture of a medicament for treating a disease or disorder. The condition or disease may be cancer.

A variety of cancers involving D3, whether malignant or benign, primary or secondary, can be treated or prevented using the methods provided by the present disclosure. Cancers may 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, carcinoid, sarcoma, glioblastoma and various head and neck tumors. Exemplary cancers include, for example, small cell lung cancer, large cell neuroendocrine carcinoma, glioblastoma, Ewing's sarcoma, and cancers with a neuroendocrine phenotype.

Anti-D3 antibodies as 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, such as lung adenocarcinoma. Lung cancer may be refractory, relapsed, or responsive to platinum-based drugs (e.g., carboplatin, cisplatin, oxaliplatin, topotecan) and/or taxanes (e.g., docetaxel, paclitaxel, latin Rotaxel or Cabazitaxel) are resistant.

Cancers treated with 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. Anti-D3 antibodies as disclosed herein may be used to treat tumors in the kidney, urogenital tract (bladder, prostate, ovary, cervix, and endometrium), gastrointestinal tract (colon, stomach), thyroid (medullary thyroid carcinoma), and lung ( Neuroendocrine tumors (NETs and pNETs) originating in small cell lung cancer and large cell neuroendocrine carcinoma).

As mentioned above, anti-D3 antibodies are particularly effective in treating lung cancer and large cell neuroendocrine cancer, which include the following subtypes: small cell lung cancer and non-small cell lung cancer (such as squamous cell non-small cell lung cancer or squamous cell small cell lung cancer) . stimulation of immune response

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

The term "enhanced immune response" or its grammatical variations means any response that stimulates, induces, increases, improves or enhances the mammalian immune system. The immune response may be a cellular response (eg, cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (eg, antibody-mediated response), and may be a primary or secondary immune response. Examples of enhanced immune responses include increased CD4 ⁺ helper T cell activity and the production of cytolytic T cells. Enhancement of the immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, cytokine release (e.g., IL-2 production or IFN-γ production), Tumor regression, survival of tumor-bearing animals, antibody production, immune cell proliferation, expression of cell surface markers and cytotoxicity. For example, the methods disclosed herein enhance the immune response of a mammal compared to the immune response of an untreated mammal or a mammal not treated using the methods disclosed herein.

D3-binding molecules can be used alone as monotherapy or in combination with chemotherapy, radiation therapy, targeted therapy, or cellular immunotherapy. Used in combination with chemotherapy

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

The term "anticancer agent" or "antiproliferative agent" means any agent useful in the treatment of cell proliferative disorders, such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, tumor reducing agents, chemotherapy agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapies, anti-metastatic agents and immunotherapeutic agents. It will be appreciated that in some embodiments, as described above, such anti-cancer agents can comprise conjugates and can be combined with the disclosed anti-D3 antibodies prior to administration. For example, in some embodiments, a selected anti-cancer agent is linked to an unpaired cysteine of an engineered antibody to provide an engineered conjugate as described herein. Accordingly, such engineered conjugates are expressly considered within the scope of the present invention. In some embodiments, the disclosed anti-cancer agents will be administered in combination with anti-D3 conjugates comprising different therapeutic agents as described above.

As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells and reduces or inhibits cell function and/or causes cells to be destroyed. 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, bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxins, staphylococcal enterotoxin A), fungi (e.g., alpha-sarcinin, aspergillin), plants (Abrin, ricin, caprytoxin, mistletoe, pokeweed antiviral protein, saporin, gelonin, momoridin, trichosanthin, hordetoxin, Aleurites fordii protein, dianthus Phytolacca mericana proteins, Phytolacca mericana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitors, Jatropha curcas toxins, crotonin, phytolacca inhibitors, gelonin, mitegellin, aspergillin, phenomycin, new small molecule toxins or enzymatically active toxins of animals (e.g., cytotoxic RNases such as extracellular pancreatic RNase; DNase I, including fragments and/or variants thereof) or animals (e.g., cytotoxic RNases such as extracellular pancreatic RNase; DNase I, including fragments and/or variants thereof).

For purposes of the present invention, "chemotherapeutic agents" include chemical compounds (eg, cytotoxic agents or cytostatics) that non-specifically reduce or inhibit the growth, proliferation and/or survival of cancer cells. These chemical agents often target intracellular programs required for cell growth or division, and are therefore particularly effective against cancer cells, which often 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 (eg, TICs). These agents are often used in combination and are often most effective in regimens such as CHOP or FOLFIRI.

Examples of anti-cancer agents (either as components of site-specific conjugates or in the unconjugated state) that can be used in combination with the D3 binding molecules (e.g., anti-D3 antibodies) of the invention include, but are not limited to, alkylating agents, alkyl Sulfonates, aziridines, ethyleneimine and methylmelamine, acetogenins, camptothecin, bryostatin, callystatin, CC-1065, cryptophycins ), dolastatin, becanomycin, eleuterobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustard, antibiotics, enediynes Antibiotics, dynemicin, bisphosphonates, espomycin, chromoprotein enediyne antibiotic chromophore, aclacinomysins, actinomycin, antrimycin, azoserine, bleomycin, Actinomycin C, carabicin, carcinogen, chromomycin, dactinomycin, daunorubicin, ditobicin, 6-diazo-5-oxo- L-Norleucine, ^ADRIAMYCIN® Doxorubicin, Epirubicin, Isobicin, Idarubicin, Masiciromycin, Mitomycin, Mycophenolic Acid, Nocardiomycin, Olive mold antibiotics, pelomycin, potfiromycin, puromycin, quelamycin, rhodobicin, streptomycin, streptozotocin, tuberculin, eben Mesi, zistinib, zorubicin; antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogues , purine analogues, androgens, anti-adrenaline, folic acid supplements such as frolinic acid, acetoglucuronide, aldehyde phosphatide glycosides, aminoacetate, enuracil, amsacridine, bestrabucil, bisantrene, idatroxate, defofamine, colcemid, acriquinone, elfornithine, etrine, eboxilon, etoglu , gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoids, mitoguanine hydrazone, mitoxantrone, mopidanmol, nitraerine, pentostat Methylmethamine, pirarubicin, loxantrone, podophylline acid, 2-ethylhydrazine, procarbazine, PSK® Polysaccharide Complex (JHS Natural Products, Eugene, OR), Razoxane; Rhizopus trichothecenes (especially T-2 toxin, vera verracurin A (verracurin A, bacillisporin A and anguidine); urethane; vindesine; dacarbazine; manromustine; dibromomannitol; dibromodulconol; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes; chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine, NAVELBINE® vinorelbine ; Nostrolin; Teniposide; Edatroxate; Daunorubicin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11); Topoisomerase inhibitor Agent RFS 2000; difluoromethylornithine; retinoids; capecitabine; combretastatin; leucovorin; oxaliplatin; PKC-α, Raf, H-Ras, EGFR and VEGF -Inhibitors of A (which reduce cell proliferation), and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that modulate or inhibit hormonal effects on tumors, such as antiestrogens and selective estrogen receptor modulators, which inhibit the aromatase enzyme that regulates estrogen production in the adrenal gland. enzyme inhibitors, and anti-androgens; and troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes such as inhibitors of VEGF expression and HER2 expression Inhibitors; vaccines, PROLEUKIN ^® rIL-2; LURTOTECAN ^® topoisomerase 1 inhibitors; ABARELIX ^® rmRH; vinorelbine and espotromycin, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing things. Used in combination with radiation therapy

The present invention also provides for combinations of D3 binding molecules with radiation therapy (i.e., any mechanism used to induce DNA damage locally within tumor cells, such as gamma-irradiation, X-rays, UV-irradiation, microwaves, electron emission, etc.). Combination therapies using targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed D3 binding moieties may be used in conjunction with targeted anti-cancer agents or other targeting means. Typically, radiation therapy is administered in pulses over a period of about 1 week to about 2 weeks. Radiation therapy can be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy may be administered as a single dose or as multiple sequential doses. Diagnosis

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

In some embodiments, binding of an anti-D3 antibody to specific cells in a sample may indicate that the sample may contain tumorigenic cells, thereby indicating that an individual with cancer may be effectively treated with the anti-D3 antibodies described herein.

Samples can be analyzed by a variety of assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblot assays, Western blot analysis, and flow cytometry assays . Compatible in vivo theranostics or diagnostic assays may include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scan), positron tomography (e.g., PET scan) known to those skilled in the art ), radiography, ultrasound, etc. Drug packaging and kits

Pharmaceutical packages and kits containing one or more containers containing one or more doses of a D3 binding molecule are also provided. In some embodiments, unit dosages are provided, wherein the unit dosage 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 unit doses are supplied in single-use, prefilled injection syringes. In some embodiments, the composition contained in the unit dose may contain saline, sucrose, or the like; a buffering agent, such as phosphate, etc.; and/or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition can be provided as a lyophilized powder, which can be reconstituted after the addition of a suitable liquid (eg, sterile water or saline solution). In certain embodiments, the compositions comprise 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 packaged composition is for use in treating the selected neoplastic disease condition.

The invention also provides kits for generating single or multiple dose administration units of D3 binding molecules and optionally one or more anti-cancer agents. The kit includes a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic, and contain a pharmaceutically effective amount of the disclosed D3 binding molecule in conjugated or unconjugated form. In some embodiments, the container includes a sterile access port (eg, the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). Such kits typically contain a pharmaceutically acceptable formulation of the D3 binding molecule in conjugated or unconjugated form in a suitable container, and optionally one or more anti-cancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable preparations for diagnosis or combination therapy. For example, in addition to the D3-binding molecule of the invention, such a kit may contain any one or more anti-cancer agents, such as chemotherapeutic agents or radiotherapy agents; anti-angiogenic agents; anti-metastasis agents; targeted anti-cancer agents; Cytotoxic agents; and/or other anticancer agents.

For example, 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. Where combined therapeutics are provided for conjugation, single solutions may be premixed in molar equivalent combinations or with more of one component than another. Alternatively, the conjugates of the kit and any optional anti-cancer agent can be stored separately in separate containers prior to administration to a patient. The kit may also contain a second/second buffer for containing sterile, pharmaceutically acceptable buffers or other diluents such as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution, and dextrose solution. Third container device.

When the components of the kit are provided in 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 a reagent or component is provided as a dry powder, the powder can be reconstituted by adding an appropriate solvent. It is envisaged that the solvent may also be provided in another container.

As briefly mentioned above, the kit may also contain means for administering the D3 binding molecule and any optional components to the patient, such as one or more needles, IV bags or syringes, or even eye drops, pipettes or other similar devices , by which the formulation can be injected or introduced into an animal or applied to a diseased area of the body. Kits of the present invention also typically include means for holding vials or the like and other tightly closed components for commercial sale, such as injection or blow molded plastic containers, in which the desired vials and other means are placed and retained. Sequence Listing Overview

This application is accompanied by a sequence listing containing a number of amino acid sequences. Table AF below provides an overview of the included sequences. The exemplified antibodies are collectively referred to in this disclosure as "WBPT1156 antibodies." Table A. Amino acid sequences of CDR regions VHH CDR1 CDR2 CDR3 WT1156-P3R2-1C2 WT1156-P3R2-1C2-z102 WT1156-P3R2-1C2-z109 WT1156-P3R2-1C2-z109' SEQ ID NO: 1GLTFSTATVG SEQ ID NO: 2 AIPAYYSTYYASSVKG SEQ ID NO: 3DDTPPSRSPFYKH WT1156-P3R2-1C9 SEQ ID NO: 4 GRTTSRYSMV SEQ ID NO: 5GNSADHDGRSAYADSVKG SEQ ID NO: 6DTNPPYGPPWSTPSEYEY WT1156-P3R2-1H6 WT1156-P3R2-1H6-z100 SEQ ID NO: 7GRTFRSYAMG SEQ ID NO: 8 AISWIGGGTYYADSVKG SEQ ID NO: 9 SSLLRHGHMFEESDY WT1156-P8R2-1H1 SEQ ID NO: 10GRTASRYSMV SEQ ID NO: 11GNSADHDGRSAYTDSVKG SEQ ID NO: 6DTNPPYGPPWSTPSEYEY Table B. Amino acid sequences of VHH region and human IgG1 heavy chain Fc region Amino acid sequence of VHH region Amino acid sequence of human IgG1 Fc region WT1156-P3R2-1C2 SEQ ID No: 12 EVQLVESGGGLVQTGDSLRLSCAASGLTFSTATVGWFRQAPGKERDLIAAIPAYYSTYYASSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAADDTPSPSRSPFYKHRGQGTQVTVSS SEQ ID No: 19 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK WT1156-P3R2-1C9 SEQ ID No: 13 QVQLVESGGGLVQAGGSLRLSCAASGRTTSRYSMVWFRQAPGQEREFVGGNSAHDGRSAYADSVKGRTFSRDNAKNTGYLQMSSLRPDDTAVYYCAADTNPPYGPPWSTPSEYEYWGHGTQVTVSS WT1156-P3R2-1H6 SEQ ID No: 14 QVQLVESGGGLVQAGGSLRLSCAASGRTFRSYAMGWFRQAPGKEREFVAAISWIGGGTYYADSVKGRFTISGDNAKNTLYLQMNSLKPEDTAVYYCAASSLLRHGHMFEESDYWGQGTQVTVSS WT1156-P8R2-1H1 SEQ ID No: 15 EVDLVESGGGLVQPGGSLRLSCAASGRTASRYSMVWFRQAPGQEREFVGGNSAHDGRSAYTDSVKGRFTFSRDNAKNTGYLQMNSLRPDDTAVYYCAADTNPPYGPPWSTPSEYEYWGHGTQVTVSS WT1156-P3R2-1C2-z102 SEQ ID No: 16 EVQLVESGGGLVQPGGSLRLSCAASGLTFSTATVGWFRQAPGKGRELIAAIPAYYSTYYASSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAADDTPSPSRSPFYKHRGQGTMVTVSS WT1156-P3R2-1C2-z109 SEQ ID No: 17 EVQLVESGGGLVQPGGSLRLSCAASGLTFSTATVGWFRQAPGKGRELVAAIPAYYSTYYASSVKGRFTISRDNAKNSLYLQMNSLRPEDTAVYYCAADDTPSPSRSPFYKHRGQGTMVTVSS WT1156-P3R2-1H6-z100 SEQ ID No: 18 QVQLVESGGGVVQPGGSLRLSCAASGRTFRSYAMGWFRQAPGKEREFVAAISWIGGGTYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCAASSLLRHGHMFEESDYWGQGTMVTVSS Table C. Amino acid sequences of antibody strains WT1156-P3R2-1C2, WT1156-P3R2-1C2-z102, WT1156-P3R2-1C2-z109 and WT1156-P3R2-1C2-z109' Example IMGT Kabat Chothia Contact ikB VHH CDR Seq. VHH CDR1 GLTFSTATVG (SEQ ID NO: 1) GLTFSTAT (SEQ ID NO: 20) TATVG (SEQ ID NO: 23) GLTFSTA (SEQ ID NO: 24) STATVG (SEQ ID NO: 27) GLTFSTATVG (SEQ ID NO: 1) VHH CDR2 AIPAYYSTYYASSVKG (SEQ ID NO: 2) IPAYYST (SEQ ID NO: 21) AIPAYYSTYYASSVKG (SEQ ID NO: 2) AYY (SEQ ID NO: 25) LIAAIPAYYSTY (SEQ ID NO: 28) or LVAAIPAYYSTY (SEQ ID NO: 56) AIPAYYSTY (SEQ ID NO: 30) VHH CDR3 DDTPPSRSPFYKH (SEQ ID NO: 3) AADDTPSRSPFYKH (SEQ ID NO: 22) DDTPPSRSPFYKH (SEQ ID NO: 3) DTPSPSRSPFYK (SEQ ID NO: 26) AADDTPSPSRSPFYK (SEQ ID NO: 29) DDTPPSRSPFYKH (SEQ ID NO: 3) VHH sequence WT1156-P3R2-1C2: EVQLVESGGGLVQTGDSLRLSCAASGLTFSTATVGWFRQAPGKERDLIAAIPAYYSTYYASSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAADDTPSPSRSPFYKHRGQGTQVTVSS (SEQ ID No: 12) VHH sequence WT1156-P3R2-1C2-z102 EVQLVESGGGLVQPGGSLRLSCAASGLTFSTATVGWFRQAPGKGRELIAAIPAYYSTYYASSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCAADDTPSPSRSPFYKHRGQGTMVTVSS (SEQ ID No: 16) VHH sequence WT1156-P3R2-1C2-z109: EVQLVESGGGLVQPGGSLRLSCAASGLTFSTATVGWFRQAPGKGRELVAAIPAYYSTYYASSVKGRFTISRDNAKNSLYLQMNSLRPEDTAVYYCAADDTPSPSRSPFYKHRGQGTMVTVSS (SEQ ID No: 17) VHH sequence WT1156-P3R2-1C2-z109': EVQLVESGGGLVQPGGSLRLSCAASGLTFSTATVGWFRQAPGKGRELIAAIPAYYSTYYASSVKGRFTISRDNAKNSLYLQMNSLRPEDTAVYYCAADDTPSPSRSPFYKHRGQGTMVTVSS (SEQ ID No: 55) Table D. Amino acid sequence of antibody clone WT1156-P3R2-1C9 Example IMGT Kabat Chothia Contact ikB VHH CDR Seq. VHH CDR1 GRTTSRYSMV (SEQ ID NO: 4) GRTTSRYS (SEQ ID NO: 31) RYSMV (SEQ ID NO: 34) GRTTSRY (SEQ ID NO: 35) SRYSMV (SEQ ID NO: 38) GRTTSRYSMV (SEQ ID NO: 4) VHH CDR2 GNSAHDGRSAYADSVKG (SEQ ID NO: 5) NSAHDGRS (SEQ ID NO: 32) GNSAHDGRSAYADSVKG (SEQ ID NO: 5) AHDG (SEQ ID NO: 36) FVGGNSAHDGRSA (SEQ ID NO: 39) GNSAHDGRSA (SEQ ID NO: 41) VHH CDR3 DTNPPYGPPWSTPSEYEY (SEQ ID NO: 6) AADTNPPYGPPWSTPSEYEY (SEQ ID NO: 33) DTNPPYGPPWSTPSEYEY (SEQ ID NO: 6) TNPPYGPPWSTPSEYE (SEQ ID NO: 37) AADTNPPYGPPWSTPSEYE (SEQ ID NO: 40) DTNPPYGPPWSTPSEYEY (SEQ ID NO: 6) VHH sequence WT1156-P3R2-1C9: QVQLVESGGGLVQAGGSLRLSCAASGRTTSRYSMVWFRQAPGQEREFVGGNSAHDGRSAYADSVKGRFTFSRDNAKNTGYLQMSSLRPDDTAVYYCAADTNPPYGPPWSTPSEYEYWGHGTQVTVSS (SEQ ID No: 13) Table E. Amino acid sequences of antibody strains WT1156-P3R2-1H6, WT1156-P3R2-1H6-z100 Example IMGT Kabat Chothia Contact ikB VHH CDR Seq. VHH CDR1 GRTFRSYAMG (SEQ ID NO: 7) GRTFRSYA (SEQ ID NO: 42) SYAMG (SEQ ID NO: 45) GRTFRSY (SEQ ID NO: 46) RSYAMG (SEQ ID NO: 49) GRTFRSYAMG (SEQ ID NO: 7) VHH CDR2 AISWIGGGTYYADSVKG (SEQ ID NO: 8) ISWIGGGT (SEQ ID NO: 43) AISWIGGGTYYADSVKG (SEQ ID NO: 8) WIGG (SEQ ID NO: 47) FVAAISWIGGGTY (SEQ ID NO: 50) AISWIGGGTY (SEQ ID NO: 52) VHH CDR3 SSLLRHGHMFEESDY (SEQ ID NO: 9) AASSLLRHGHMFEESDY (SEQ ID NO: 44) SSLLRHGHMFEESDY (SEQ ID NO: 9) SLLRHGHMFEESD (SEQ ID NO: 48) AASSLLRHGHMFEESD (SEQ ID NO: 51) SSLLRHGHMFEESDY (SEQ ID NO: 9) VHH sequence WT1156-P3R2-1H6: QVQLVESGGGLVQAGGSLRLSCAASGRTFRSYAMGWFRQAPGKEREFVAAISWIGGGTYYADSVKGRFTISGDNAKNTLYLQMNSLKPEDTAVYYCAASSLLRHGHMFEESDYWGQGTQVTVSS (SEQ ID No: 14) VHH sequence WT1156-P3R2-1H6-z100: QVQLVESGGGVVQPGGSLRLSCAASGRTFRSYAMGWFRQAPGKEREFVAAISWIGGGTYYADSVKGRFTISGDNSKNTLYLQMNSLRAEDTAVYYCAASSLLRHGHMFEESDYWGQGTMVTVSS (SEQ ID No: 18) Table F. Amino acid sequence of antibody clone WT1156-P8R2-1H1 Example IMGT Kabat Chothia Contact ikB VHH CDR Seq. VHH CDR1 GRTASRYSMV (SEQ ID NO: 10) GRTASRYS (SEQ ID NO: 53) RYSMV (SEQ ID NO: 34) GRTASRY (SEQ ID NO: 54) SRYSMV (SEQ ID NO: 38) GRTASRYSMV (SEQ ID NO: 10) VHH CDR2 GNSAHDGRSAYTDSVKG (SEQ ID NO: 11) NSAHDGRS (SEQ ID NO: 32) GNSAHDGRSAYTDSVKG (SEQ ID NO: 11) AHDG (SEQ ID NO: 36) FVGGNSAHDGRSA (SEQ ID NO: 39) GNSAHDGRSA (SEQ ID NO: 41) VHH CDR3 DTNPPYGPPWSTPSEYEY (SEQ ID NO: 6) AADTNPPYGPPWSTPSEYEY (SEQ ID NO: 33) DTNPPYGPPWSTPSEYEY (SEQ ID NO: 6) TNPPYGPPWSTPSEYE (SEQ ID NO: 37) AADTNPPYGPPWSTPSEYE (SEQ ID NO: 40) DTNPPYGPPWSTPSEYEY (SEQ ID NO: 6) VHH sequence WT1156-P8R2-1H1: EVDLVESGGGLVQPGGSLRLSCAASGRTASRYSMVWFRQAPGQEREFVGGNSAHDGRSAYTDSVKGRFTFSRDNAKNTGYLQMNSLRPDDTAVYYCAADTNPPYGPPWSTPSEYEYWGHGTQVTVSS (SEQ ID No: 15) Example

The invention generally described herein will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. These examples are not intended to represent that the following experiments are all or the only experiments performed. Example 1 Preparation of antigens, reference antibodies and cell lines 1.1 Generation of antigens

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 In the expression vector, there is an MBP tag at the N terminus and an AVI-His tag or human Fc tag at the C terminus. Human D3 (Uniprot No. Q9NYJ7) was purchased from AcroBiosystems (Cat. DL3-H52H4).

The DNA sequence encoding the human D3 truncated isoform (as 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 at the C terminus and AVI-His tag.

Expi293 cells (Invitrogen-A14527) were transfected with the purified expression vector. The cells were cultured for 5 days, and the supernatant was collected for protein purification using a Ni-NTA column (GE Healthcare, Cat. 175248) or a protein A column (GE Healthcare, Cat. 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.

Human D3 (ACRO DL3-H52H4) is called WT115-hPro1.ECD.His. The obtained mouse D3 was called WT115-MBP-mPro1.ECD.hFc. Human D3 protein is characterized by containing a Delta/Serrate/LAG-2 (DSL) domain, six epidermal growth factor (EGF)-like repeats (EGF domain), and a transmembrane domain. The truncated isoforms of human D3 are 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 end), WT115-hPro1.V3.ECD.MBP.AVI.His (EGF2-6 domain + membrane proximal end), 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). A map of the truncated protein is shown in Figure 7c. 1.2 Construction of BMK antibody expression vector

Two anti-D3 antibodies were used as baseline antibodies, referred to herein as WT115-BMK1 and WT115-BMK2. Variable regions encoding WT115-BMK1 (SEQ ID NO: 212 and SEQ ID NO: 213 in US 2019/0046656) and WT115-BMK2 (SEQ ID NO: 37 and SEQ ID NO: 38 in WO 2017/021349) The DNA sequence was synthesized at Sangon Biotech (Shanghai, China) and then subcloned into a modified pcDNA3.3 expression vector encoding the human IgG1 Fc region.

Plasmids containing VH and VL genes were co-transfected into Expi293 cells. The cells were cultured for 5 days, and the supernatant was collected for protein purification using a protein A column (GE Healthcare, 175438). Obtained antibodies were analyzed by SDS-PAGE and SEC and then stored at -80ºC. 1.3 Establishment of stable cell lines / cell pools

293F cells were transfected with an expression vector containing the gene encoding full-length human D3 (UniProt, Q9NYJ7-1) using Lipofectamine 2000. Flpin293 cells were transfected with an expression vector containing the gene encoding full-length cynomolgus D3. Cells are cultured in culture medium containing the correct selection markers. After limiting dilution, select the human D3 high-performance stable cell line (WT115-293F.hPro1.2E5), and use appropriate selection antibiotics to obtain the cynomolgus monkey D3 high-performance stable cell pool (WT115.Flpin293.cPro1.pool). 1.4 Antibody biotinylation

For NHS-PEO4-biotinylation, incubate 1-10 mg/mL antibody (IgG) with a 20-fold molar excess of NHS-PEO4-biotin reagent in a metal bath at 25°C for 75 min or on ice for 2 hours. Excess biotin is then removed using a desalting spin column and the purified protein sample is collected from the flow-through solution. Determination of biotin incorporation levels in proteins by HABA assay: Dilute biotinylated samples 10-fold with HABA/avidin solution and measure the absorbance of the mixed solution at A500. Moles of biotin per mole of protein were calculated based on the A500 value. Example 2 Production of WBPT1156 Antibody Containing VHH 2.1 Production of Anti- D3 VHH

Anti-D3 VHH is generated by camelid immunization and phage display technology. Briefly, alpacas (Vicugna pacos) were immunized subcutaneously with hFc-tagged human D3 ECD protein (ACRO, DL3-H5255). After immunization, peripheral blood was collected to construct a phage library displaying VHH fragments. After biopanning with the corresponding target ECD proteins, positive VHH clones that bind to D3 are selected. 2.2 VHH sequencing

Positive E. coli colonies screened by target-specific binding ELISA and FACS using E. coli supernatants were sent to Biosune (Shanghai, China) for nucleotide sequencing of the VHH gene. Sequencing results were analyzed using CLC Main Workbench (Qiagen, Hilden, Germany). The sequences of the four unique positive VHH clones are WT1156-P3R2-1C2, WT1156-P3R2-1C9, WT1156-P3R2-1H6 and WT1156-P8R2-1H1, as shown in Tables A and B. 2.3 Generation of human Fc fusion antibodies containing VHH

Four unique positive VHH clones were converted into VHH-Fc (hlgG1) fusion antibodies. Briefly, the VHH gene was PCR amplified from the pET-bac vector using VHH-specific clone primers containing appropriate restriction sites, followed by fusion of the clone into a modified human hIgG1 expressing pcDNA3.3 vector to generate Corresponding VHH-Fc (hlgG1) chimeric antibody clone. 293F or Expi293 cells were transiently transfected with vectors for antibody expression. Antibody-containing cell culture supernatants were harvested and purified using protein A chromatography. The antibodies produced were named “WT1156-P3R2-1C2-uIgG1”, “WT1156-P3R2-1C9-uIgG1”, “WT1156-P3R2-1H6-uIgG1” and “WT1156-P8R2-1H1-uIgG1” respectively. Obtained antibodies were analyzed by SDS-PAGE and HPLC-SEC and then stored at -80ºC. 2.4 Humanization

VHH humanization is accomplished using a “best fit” approach. Briefly, the amino acid sequences of the VHH framework regions were blasted against a human germline V gene database, and humanized VHH sequences were generated by replacing the highest-hit human CDR sequences with VHH CDR sequences using Kabat CDR definitions. Key residues in the framework that play an important role in antibody affinity or developability are then backmutated individually or in combination to the parent residues. These variants were codon-optimized for mammalian performance and then synthesized by GENEWIZ (Suzhou, China). The designed VHH variants and parental VHH proteins were cloned into human IgG1 expression vectors to generate human IgG1 constructs. Antibodies were produced in HEK293 cells and purified using protein A chromatography. The variant with the desired affinity is ultimately selected as the humanized leader.

The sequences of three unique humanized D3 antibodies, namely WT1156-P3R2-1C2-z102-uIgG1, WT1156-P3R2-1C2-z109-uIgG1, and WT1156-P3R2-1H6-z100-uIgG1, are also shown in Tables A and B. Example 3 Characterization of D3 - binding antibodies 3.1 Human D3 binding by ELISA

Precoat the plate with 100 μL per well of WT115-hPro1.ECD.His at 1 μg/mL overnight at 4 ºC. Antigens were diluted from stock solutions in coating buffer (0.02 M _NaCO and 0.18 M _NaHCO , pH 9.2 ₎ . The next day, wash the plate once with 1× PBST (PBS containing 0.05% tween-20), and add 200 μL of 1× PBS/2% BSA to block. Antibodies were serially diluted in blocking buffer (5-fold serial dilution from 20 nM to 0.00128 nM). After blocking for 1 hour, wash the plate 3 times with 1× PBST, then add the antibody to the plate and incubate for 1 hour at ambient temperature. Antibody binding to immobilized human D3 was detected by HRP-conjugated secondary antibody (Bethyl, A80-304P) diluted 1:10000 in 1x PBS/2% BSA. After incubation, wash the plate 6 times with 1× PBST. Color was developed by adding 100 μL of TMB substrate, and the reaction was stopped by adding 100 μL of 2M HCl. Read absorbance at 450 nm and 540 nm using a microplate spectrophotometer. Anti-human D3 antibodies WT115-BMK1 and WT115-BMK2 were used as positive controls. Human IgG1 isotype antibodies were used as negative controls. All samples were tested in duplicate.

As shown in Figure 1 and Table 1, WT1156-P3R2-1C2-uIgG1, WT1156-P3R2-1C9-uIgG1, WT1156-P3R2-1H6-uIgG1, and WT1156-P8R2-1H1-uIgG1 are able to strongly bind to immobilized human D3, Comparable to WT115-BMK1 and WT115-BMK2. The EC50 range of WT1156 antibody is 0.013 nM to 0.026 nM. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.0094 nM and 0.011 nM respectively. Table 1. Summary of characterization of WBPT1156 antibodies Antibody name Human D3 binding ELISA (EC50, nM) Human D3 binds FACS (EC50, nM) Cynomolgus D3 binds FACS (EC50, nM) Internalization (EC50, nM binding domain WT1156-P3R2-1C2-uIgG1 0.013 0.15 0.14 0.65 EGF1-EGF2 WT1156-P3R2-1H6-uIgG1 0.014 0.52 0.25 0.95 N-Term WT1156-P8R2-1H1-uIgG1 0.021 0.45 0.21 0.47 N-DSL-EGF1 WT1156-P3R2-1C9-uIgG1 0.026 0.25 0.07 0.46 N-Term WT115-BMK1 0.0094 0.019 na 0.19 DSL WT115-BMK2 0.011 0.54 na 0.58 EGF3 na: EC50 values are not fitted. 3.2 Human D3 binding determined by FACS

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

As shown in Figure 2a and Table 1, WT1156-P3R2-1C2-uIgG1, WT1156-P3R2-1C9-uIgG1, WT1156-P3R2-1H6-uIgG1, and WT1156-P8R2-1H1-uIgG1 were able to bind to cells expressing human D3, and WT115-BMK2 is comparable. The EC50 range of WT1156 antibody is 0.15 nM to 0.52 nM. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.019 nM and 0.54 nM respectively.

As shown in Figure 2b, WT1156-P3R2-1C2-z102-uIgG1 and WT1156-P3R2-1C2-z109-uIgG1 can strongly bind to cells expressing human D3 with EC50 of 0.088 nM and 0.14 nM, respectively. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.03 nM and 0.52 nM, respectively. The data of WT1156-P3R2-1C2-z109-uIgG and two BMK antibodies are also summarized in Table 2. Table 2. Summary of characterization of WT1156-P3R2-1C2-z109-uIgG1 Antibody name Human D3 binding FACS (EC50, nM) Cynomolgus D3 binding FACS (EC50, nM) Mouse D3 binding ELISA (EC50, nM) Internalization (EC50, nM) WT1156-P3R2-1C2-z109-uIgG1 0.14 0.096 0.0075 12.2 WT115-BMK1 0.03 0.019 0.0039 3.47 WT115-BMK2 0.52 0.20 0.014 14.4 3.3 Cynomolgus D3 binding determined by FACS

Incubate WT115-Flpin293.cPro1.pool cells (1 × ¹⁰ cells/well) with different concentrations of antibodies (4-fold serial dilutions from 10 nM to 0.00061 nM) for 1 hour at 4 ^o C. 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 at 4 ^° C in the dark for 1 hours. Anti-human D3 antibodies WT115-BMK1 and WT115-BMK2 were used as positive controls. Human IgG1 isotype antibodies were used as negative controls. Wash cells with 1×PBS/1% BSA and resuspend in 4% paraformaldehyde and incubate with cells for 0.5 hours at 4 ^° C in the dark. Then replace the buffer with 1×PBS/1% BSA and filter the cells. MFI of cells was measured by flow cytometry and analyzed by FlowJo.

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

As shown in Figure 3b, WT1156-P3R2-1C2-z102-uIgG1, WT1156-P3R2-1C2-z109-uIgG1 and WT1156-P3R2-1H6-z100-uIgG1 can strongly bind to cells expressing cynomolgus monkey D3, with EC50 of respectively 0.083 nM, 0.096 nM and 0.42 nM. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.019 nM and 0.20 nM respectively. The data of WT1156-P3R2-1C2-z109-uIgG and two BMKs are also summarized in Table 2. 3.4 Mouse D3 binding determined by ELISA

Precoat the plate with 100 μL per well of WT115-MBP-mPro1.ECD.hFc at 1 μg/mL overnight at 4 ºC. Antigens were diluted in coating buffer from stock solutions. The next day, wash the plate once with 1×PBST and add 200 μL of 1×PBS/2% BSA to block. Antibodies were serially diluted in blocking buffer (5-fold serial dilutions from 20 nM to 0.000256 nM). After blocking for 1 hour, wash the plate 3 times with 1× PBST, then add the antibody to the plate and incubate for 1 hour at ambient temperature. 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. Antibody binding to immobilized mouse D3 was detected by HRP-conjugated secondary antibody (Invitrogen, SNN1004) diluted 1:30000 in 1x PBS/2% BSA. After incubation, wash the plate 6 times with 1× PBST. Color was developed by adding 100 μL of TMB substrate, and the reaction was stopped by adding 100 μL of 2M HCl. Read absorbance at 450 nm and 540 nm using a microplate spectrophotometer. All samples were tested in duplicate.

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

As shown in Figure 4b, WT1156-P3R2-1C2-z102-uIgG1 and WT1156-P3R2-1C2-z109-uIgG1 can strongly bind to mouse D3 protein with EC50 of 0.0067 nM and 0.0075 nM, respectively. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.0039 nM and 0.014 nM, respectively. The data of WT1156-P3R2-1C2-z109-uIgG and two BMKs are also summarized in Table 2. 3.5 Internalization

WT115-293F.hPro1.2E5 cells (4 × 10 ⁴ cells/well) were plated in a 96-well plate, and the culture medium was removed from the plate after centrifugation. Prepare 1x final maximum concentration of primary antibody (serial 5-fold dilution from 40 nM to 0.00256 nM, or 5-fold serial dilution from 200 nM to 0.0128 nM) and pHrodo (amine reactive, Thermo Fisher, P36011) labeled secondary antibody. Anti-(Affinipure F(ab')2 fragment goat anti-human IgG, Jackson ImmunoResearch, 109-006-098, ratio = 1:1 molecule) dilution was added to the plate containing cell culture medium and incubated at 37 ºC for 5 hours. Anti-human D3 antibodies WT115-BMK1 and WT115-BMK2 were used as positive controls. Human IgG1 isotype antibodies were used as negative controls. After incubation, cells were stained with reagents (Nuclear - Hoechst33342, 1000 ng/ml; Cytoplasmic - Calcein AM, diluted 1:2000 in DPBS) and the plate was incubated at 37°C for 15 minutes. Finally, cells were photographed using Operatta CLS and antibody endocytosis was analyzed using the "Spots per cell" parameter.

As shown in Figure 5a and Table 1, WT1156-P3R2-1C2-uIgG1, WT1156-P3R2-1C9-uIgG1, WT1156-P3R2-1H6-uIgG1 and WT1156-P8R2-1H1-uIgG1 showed dose in cells expressing human D3 Dependent internalization potency, comparable to WT115-BMK1 and WT115-BMK2. The EC50 range of WT1156 antibody is 0.46 nM to 0.95 nM. The EC50 of WT115-BMK1 and WT115-BMK2 are 0.19 nM and 0.58 nM, respectively.

As shown in Figure 5b and Table 2, WT1156-P3R2-1C2-z109-uIgG1 showed dose-dependent internalization potency in cells expressing human D3 with an EC50 of 12.2 nM. The EC50 of WT115-BMK1 and WT115-BMK2 are 3.47 nM and 14.4 nM respectively. 3.6 Kinetic binding affinity of anti- D3 antibodies

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 seconds, followed by a dissociation period of 3600 seconds. After each binding cycle, the chip was regenerated with 10 mM glycine (pH 1.5).

As shown in Table 3, the experimental data of human D3 were fitted by the steady-state affinity model. The experimental data of WT-115-BMK1 on human D3 were fitted using a heterogeneous ligand model. Other experimental data were fitted using a 1:1 model using Langmiur analysis. The sensorgrams for the blank surface and the buffer channel were subtracted from the test sensorgrams. Use a molecular weight of 34 KDa to calculate the molar concentration of the analyte. The affinities of the tested antibodies for human D3 are shown in Table 3. Table 3. Binding kinetics of WBPT1156 antibody Antibody name k _a (1/Ms) k _d (1/s) KD(M) WT1156-P3R2-1C2-uIgG1 1.92E+05 1.28E-05 6.68E-11 WT1156-P3R2-1H6-uIgG1 1.08E+05 <1.00E-05 <9.26E-11 WT1156-P8R2-1H1-uIgG1 2.00E+05 5.14E-05 2.57E-10 WT1156-P3R2-1C9-uIgG1 1.80E+05 2.38E-04 1.32E-09 WT1156-P3R2-1C2-z102-uIgG1 1.83E+05 1.78E-05 9.77E-11 WT1156-P3R2-1C2-z109-uIgG1 1.85E+05 3.06E-05 1.65E-10 WT1156-P3R2-1H6-z100-uIgG1 8.02E+04 1.74E-05 2.17E-10 WT115-BMK1 (heterogeneous ligand) 2.10E+06 2.65E-03 1.26E-09 1.73E+06 1.50E-04 8.67E-11 WT115-BMK2 4.04E+05 3.98E-05 9.87E-11 3.7 Epitope binning determined by competitive ELISA

Pre-coat the plate with 1 μg/mL, 100 μL per well of WT115-hPro1.ECD.His at 4 ^° C overnight. Antigens were diluted from stock solutions in coating buffer (0.02 M Na2CO3 and 0.18 M NaHCO3, pH9.2). The next day, wash the plate once with 1×PBST and block with 200 μL 1×PBS/2% BSA. VHH antibodies were serially diluted in blocking buffer (5-fold serial dilutions from 10 nM to 0.00013 nM) and premixed with a constant concentration of full IgG antibody (0.02 nM). After blocking for 1 hour, wash the plate 3 times with 1× PBST, then add the VHH antibody/whole IgG antibody mixture to the plate and incubate for 1 hour at ambient temperature. The 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, wash the plate 6 times with 1× PBST. Color was developed by adding 100 μL of TMB substrate, and the reaction was stopped by adding 100 μL of 2M HCl. Read the absorbance at 450 nm and 540 nm using a microplate spectrophotometer. All samples were tested in duplicate.

As shown in Figure 6a, by ELISA test, WT1156-P3R2-1C2.uIgG1 did not compete with the other three WBPT1156 antibodies for binding to human D3-ECD protein. As shown in Figure 6b, WT1156-P3R2-1H6-uIgG1 can compete with WT1156-P8R2-1H1-uIgG1 and WT1156-P3R2-1C9-uIgG1 for binding to human D3-ECD protein. Figure 6c shows that WT1156-P8R2-1H1-uIgG1 can compete with WT1156-P3R2-1C9-uIgG1. As shown in Figure 6d, the 4 antibodies of WBPT1156 did not compete with WT115-BMK1. 3.8 Binding determined by ELISA using truncated D3 protein

The binding epitope of the WT1156 antibody was tested by ELISA using truncated D3 protein as described in 1.1 and Figure 7c. ELISA tests are performed by precoating the plates with antibodies or antigens. The results are shown in Figure 7a and Figure 7b respectively.

For pre-coated antibody ELISA, pre-coat the ELISA plate with 2 μg/mL, 100 μL antibody per well, overnight at 4 ºC. Antibodies were diluted from stock solutions in coating buffer (0.02 M Na2CO3 and 0.18 M NaHCO3, pH9.2). The next day, wash the plate once with 1× PBST and block with 200 μL 1× PBS/2% BSA. A constant concentration of full-length DLL ECD protein (WT115-hPro1.ECD.His) (3 μg/mL) or truncated D3 protein (3 μg/mL or 6 μg/mL) diluted in blocking buffer was then added. After blocking for 1 hour, wash the plate three times with 1× PBST, then add the antigen to the plate and incubate for 1 hour at ambient temperature. The binding of the antigen to the immobilized WBPT1156 antibody was detected by HRP-labeled secondary antibody (GenScript, A00612) diluted at a concentration of 1:2000 in 1×PBS/2% BSA. After incubation, wash the plate 6 times with 1× PBST. Develop color by dispensing 100 μL of TMB substrate. The color reaction was stopped with 2M HCl and the absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer. All samples were tested in duplicate. The results are shown in Figure 7a.

For ELISA with precoated antigen, precoat the ELISA plate with 100 μL per well of WT115-hPro1.ECD.His (2 μg/mL) or truncated D3 protein (2 μg/mL or 5 μg/mL), 4 ºC overnight. Antigens were diluted from stock solutions in coating buffer (0.02 M Na2CO3 and 0.18 M NaHCO3, pH9.2). The next day, wash the plate once with 1×PBST and block with 200 μL 1×PBS/2% BSA. Dilute a constant concentration of antibody (2 μg/mL) in blocking buffer. After blocking for 1 hour, wash the plate 3 times with 1× PBST, then add the antibody to the plate and incubate for 1 hour at ambient temperature. Binding of the antibody to immobilized human D3 was detected by HRP-conjugated secondary antibody (Bethyl, A80-304P) diluted 1:10000 in 1x PBS/2% BSA. After incubation, wash the plate 6 times with 1× PBST. Color was developed by adding 100 μL of TMB substrate, and the reaction was stopped by adding 100 μL of 2M HCl. Read the absorbance at 450 nm and 540 nm using a microplate spectrophotometer. All samples were tested in duplicate. The results are shown in Figure 7b.

As shown in Figure 7a and Figure 7b, WT1156-P3R2-1C2-uIgG1 binds to WT115-hPro1.V1.ECD.MBP.AVI.His, WT115-hPro1.V2.ECD.MBP.AVI.His, and partially binds to WT115- hPro1.V3.ECD.MBP.AVI.His, but did not bind other isotypes, indicating that its binding epitope is located in EGF1-2. WT1156-P3R2-1C9-uIgG1 and WT1156-P3R2-1H6-uIgG1 bound to WT115-hPro1.ECD.His but not to the truncated D3 isotype, 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 isotype (Fig. 7a); When tested by ELISA with pre-coated antigen, as shown in Figure 7b, WT1156-P8R2-1H1-uIgG1 showed the same relationship with WT115-hPro1.V1.ECD.MBP.AVI.His and WT115-hPro1.V2.ECD.MBP. .AVI.His binds, but not to other isoforms, indicating that its binding epitope may be located at N-term-DSL-EGF-1. The ELISA binding results show that WT115-BMK1 binds to the DSL domain and WT115-BMK2 binds to the EGF-3 domain, which are consistent with the results shown in US 2019/0046656 and WO 2017/021349 respectively. 3.9 Cross-family binding of anti-human D3 antibodies

Use 1 μg/mL, 100 μL per well of WT115-hPro1.ECD.His, WT115-hPro2.ECD.His (human D1, SinoBiological, Cat: 11635-H08H) or WT115-hPro3.ECD.His (human D4, SinoBiological, Cat: 10171-H08H) pre-coated plates at 4 ^o C overnight. Antigens were diluted from stock solutions in coating buffer (0.02 M Na2CO3 and 0.18 M NaHCO3, pH9.2). The next day, wash the plate once with 1× PBST (PBS containing 0.05% tween-20), and block by adding 200 μL of 1× PBS/2% BSA per well. During blocking, BMK and WT1156 antibodies were diluted to 10 nM in blocking buffer, and WT115-cAb (WT115-cAb1 is anti-D1 antibody, purchased from SinoBiological, Cat: 11635-MM07; WT115-cAb2 is anti-D4 antibody, Purchased from SinoBiological, Cat: 10171-MM15) and diluted 1000 times. After blocking for 1 hour, wash the plate 3 times with 1× PBST, then add the diluted antibody to the plate and incubate for 1 hour at ambient temperature. Antibody binding to immobilized human D3 was detected by goat anti-human IgG-Fc fragment cross-adsorbed antibody HRP (Bethyl, A80-304P) and mouse anti-human IgG-Fc fragment cross-adsorbed antibody HRP (Bethyl, A90-231P). , Antibody HRP was diluted 1:10000 in 1×PBS/2%BSA. After incubation, wash the plate 6 times with 1× PBST. Color was developed by adding 100 μL of TMB substrate, and the reaction was stopped by adding 100 μL of 2M HCl. Read the absorbance at 450 nm and 540 nm 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.

As shown in Figure 8, the WBPT1156 antibody did not bind human D1 or human D4. 3.10 human serum stability

Human serum was freshly isolated from healthy donors by centrifugation. Samples were diluted in serum, with the serum volume accounting for more than 90% of the total volume. Five aliquots of the sample were incubated at 37 ºC. Samples were then collected at day 0, day 1, day 4, day 7, and day 14 and snap frozen until analyzed together.

The stability of the samples was tested by binding to human D3 using ELISA. Briefly, pre-coat plates with 100 μL/well of 1 μg/mL WT115-hPro1.ECD.His overnight at 4 ºC. The next day, wash once with 1× PBST (PBS containing 0.05% tween-20), and add 200 μL 1× PBS/2% BSA to each well to block. During the blocking period, different concentrations of test antibodies were added to the plate (serial 4-fold dilutions from 3 nM to 0.00018 nM). Incubate the plate at ambient temperature for 1 hour. Antibody binding to immobilized human D3 was detected by goat anti-human IgG-Fc fragment cross-adsorbed antibody HRP (Bethyl, A80-304P) and mouse anti-human IgG-Fc fragment cross-adsorbed antibody HRP (Bethyl, A90-231P). , Antibody HRP was diluted 1:5000 in 1×PBS/2%BSA. After incubation, wash the plate 6 times with 1× PBST. Color was developed by adding 100 μL of TMB substrate, and the reaction was stopped by adding 100 μL of 2M HCl. Read the absorbance at 450 nm and 540 nm using a microplate spectrophotometer. Human IgG1 isotype antibodies were used as negative controls. All samples were tested in duplicate.

As shown in Figure 9, the binding profile of WT1156-P3R2-1C2-z109-uIgG1 to human D3 protein did not change after incubation in human serum for up to 14 days at 37 ºC.

Those skilled in the art will further recognize that the present invention may be embodied in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the invention discloses only exemplary embodiments thereof, it is to be understood that other variations are considered to be within the scope of the invention. Therefore, this invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims as an indication of the scope and content of the invention.

without

Figure 1 shows exemplary binding results of antibodies to immobilized WT115-hPro1.ECD.his measured by ELISA. Figures 2a and 2b show exemplary binding results of antibodies to WT115-293F.hPro1.2E5 cells measured by FACS. Figures 3a and 3b show exemplary binding results of antibodies to WT115-Flpin293.cPro1.pool cells measured by FACS. Figures 4a and 4b show exemplary binding results of antibodies to immobilized WT115-MBP-mPro1.ECD.hFc measured by ELISA. Figures 5a and 5b show exemplary internalization results of antibodies by using WT115-293F.hPro1.2E5 cells. Figures 6a to 6d show exemplary epitope binning results by ELISA for antibodies to immobilized WT115-hPro1.ECD.his. Figure 6a, binning using WT1156-P3R2-1C2-uIgG1; Figure 6b, binning using WT1156-P3R2-1H6-uIgG1; Figure 6c, binning using WT1156-P8R2-1H1-uIgG1; Figure 6d, using WT115 -BMK1’s sub-position. Figure 7a shows exemplary ELISA binding results of antibodies to soluble WT115-hPro1.ECD.his or truncated proteins. Figure 7b shows ELISA binding of antibodies to immobilized WT115-hPro1.ECD.his or truncated proteins. Figure 7c shows a graph of the truncated protein. Figure 8 shows exemplary cross-family binding results of antibodies to human D1 and human D4, measured by ELISA. Figure 9 shows exemplary serum stability test results of WT1156-P3R2-1C2-z109-uIgG1. Figure 10 shows exemplary immunoglobulin single variable domains WT1156-P3R2-1C2 (1C2), WT1156-P3R2-1C2-z102 (1C2-z102), WT1156-P3R2-1C2-z109 (1C2-z109), WT1156 - Alignment of P3R2-1C9 (1C9), WT1156-P3R2-1H6 (1H6), WT1156-P3R2-1H6-z100 (1H6-z100) and WT1156-P8R2-1H1 (1H1). The boundaries of CDRs are indicated by Kabat, AbM, Chothia, Contact and IMGT numbers.

Claims (25)

A D3 binding molecule comprising an immunoglobulin single variable domain comprising the CDR1 of VHH as shown in SEQ ID NO: 12, 13, 14, 15, 16, 17, 55 or 18, CDR2 and CDR3. The D3 binding molecule of claim 1, wherein the CDR1, CDR2 and CDR3 are according to Kabat, Chothia, AbM, Contact, IMGT or any combination thereof. The D3 binding molecule of claim 1, wherein: (i) The CDR1 includes 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) The CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, 6 or 9. The D3 binding molecule as described in claim 1, wherein the D3 binding molecule contains: (A) CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2 and CDR3 as shown in SEQ ID NO: 3; (B) CDR1 as shown in SEQ ID NO: 4, CDR2 as shown in SEQ ID NO: 5 and CDR3 as shown in SEQ ID NO: 6; (C) CDR1 as shown in SEQ ID NO: 7, CDR2 as shown in SEQ ID NO: 8 and CDR3 as shown in SEQ ID NO: 9; or (D) CDR1 as shown in SEQ ID NO: 10, CDR2 as shown in SEQ ID NO: 11 and CDR3 as shown in SEQ ID NO: 6. The D3 binding molecule according to any one of claims 1 to 4, wherein the single variable domain includes: (A) The amino acid sequence shown in any one of SEQ ID NOs: 12-18 and 55; (B) An amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to the amino acid sequence shown in any one of SEQ ID NOs: 12-18 and 55, while still retaining specific binding affinity for D3 ;or (C) An amino acid sequence having one or a plurality (for example, 1, 2 or 3) of amino acids added, deleted and/or substituted compared to the amino acid sequence shown in any one of SEQ ID NOs: 12-18 and 55. The D3 binding molecule according to any one of claims 1 to 5, which contains one or a plurality of amino acids in the framework region of the single variable domain, such as in FRW1, FRW2, FRW3 and/or FRW4 additions, deletions and/or substitutions. The D3 binding molecule of any one of claims 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. The D3 binding molecule according to any one of claims 1 to 7, wherein the D3 binding molecule further comprises a human IgG constant domain. The D3 binding molecule of claim 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. The D3 binding molecule as described in any one of claims 1 to 9, has one or more of the following properties: (a) Binds human D3, cynomolgus D3 and/or mouse D3 with an EC50 in the nM range, as measured by ELISA or FACS; (b) Demonstrate dose-dependent internalization efficacy in cells expressing human D3; and (c) Binds human D3 with a KD not exceeding 0.1 nM, as measured by SPR. The D3 binding molecule according to any one of claims 1 to 10, wherein the D3 binding molecule is a chimeric antibody, a humanized antibody or a fully human antibody. The D3 binding molecule of any one of claims 1 to 11, comprising a single variable domain as shown in any one of SEQ ID NO: 12-18 and 55, and as SEQ ID NO: 19 IgG constant domains shown. The D3 binding molecule according to any one of claims 1 to 12, which is a dimer. A fusion protein comprising the D3-binding molecule as defined in any one of claims 1 to 13 fused to a heterologous peptide, such as an antigen-binding domain targeting different antigens. A nucleic acid molecule comprising a nucleic acid sequence encoding a single variable domain of a D3 binding molecule as defined in any one of claims 1 to 13. A vector comprising the nucleic acid molecule of claim 15. A host cell comprising the vector of claim 16. A pharmaceutical composition comprising the D3 binding molecule as described in any one of claims 1 to 13 and a pharmaceutically acceptable carrier. A method for preparing the D3 binding molecule as described in any one of claim 1 to claim 13, comprising the following steps: - expressing the D3 binding molecule in the host cell of claim 15; and - Isolating the D3 binding molecule from the host cell. Use of the D3-binding molecule according to any one of claims 1 to 13 or the pharmaceutical composition of claim 18 for modulating D3-related immune responses in a subject. Use of the D3 binding molecule according to any one of claims 1 to 13 or the pharmaceutical composition of claim 18 for treating or preventing a cancer in a subject, wherein the cancer is D3 positive or overexpressed D3's. The use of claim 21, wherein the cancer is selected from lung cancer and neuroendocrine cancer. Such use as claim 22, wherein the cancer is SCLC or LCNEC. Use of the D3-binding molecule as described in any one of claims 1 to 13 in the preparation of medicaments for diagnosing, treating or preventing D3-positive cancers. A kit comprising a container containing the D3 binding molecule described in any one of claim 1 to claim 13.