CD276-specific antibody-drug conjugates and uses thereof

Abstract

An improved antibody-drug conjugate (ADC) that targets CD276-positive tumors has been described. The ADC comprises a CD276-specific IgG1 antibody with a modified heavy chain that prevents interaction of the Fc domain with endogenous Fc receptors and introduces a cysteine ​​for site-specific conjugation of the drug. The CD276-specific ADC is capable of potently eradicating CD276-positive tumors in several animal models.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Application No. 62 / 947,135, filed on December 12, 2019, which is hereby incorporated by reference in its entirety.

[0002] Field The present disclosure relates to improved antibody - drug conjugates (ADCs) that target CD276 (B7 - H3), and to their use in the treatment of CD276 - expressing tumors.

[0003] Statement regarding government support This invention was made with government support under Project Number ZIA BC 010578 awarded by the National Institutes of Health (USA). The government has certain rights in this invention.

Background Art

[0004] Background CD276 (also known as B7 - H3) is a type I transmembrane protein expressed on the surface of many different cell types, including cells of the immune system, liver, heart, prostate, spleen, and thymus (Picarda et al., Clin Cancer Res 22(14):3425 - 3431, 2016). The CD276 protein is also overexpressed in several human malignancies, including hepatocellular carcinoma, melanoma, leukemia, breast cancer, prostate cancer, colorectal cancer, osteosarcoma, endometrial cancer, ovarian cancer, oral squamous cell carcinoma, non - small cell lung cancer, bladder cancer, and pancreatic cancer (Picarda et al.). The expression of CD276 is positively correlated with cancer severity and patient outcome for many types of cancer. CD276 has become a target of interest for cancer immunotherapy due to its expression pattern and functional activity.

[0005] An antibody-drug conjugate (ADC) is a type of molecule currently under investigation as a therapeutic agent for the treatment of cancer. An ADC consists of an antibody (antigen-binding fragment) conjugated to a cytotoxic compound. The most effective ADCs contain drugs that specifically target tumor cells or tumor-associated stromal cells, have minimal activity against normal cells, are highly toxic to tumor cells, are very stable in circulation, and can release the drug upon internalization into the target tumor cells. Radio-labeled monoclonal antibodies that specifically bind to CD276 are currently in clinical trials for the treatment of cancer (NCT01099644, NCT01502917, and NCT00089245; Picarda et al.; and Kramer et al., J Neurooncol 97:409-418, 2010).

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0007] Summary Disclosed herein are antibody-drug conjugates (ADCs) that specifically target CD276-expressing tumor cells. The use of ADCs for treating CD276-positive cancers is also disclosed.

[0008] Provided herein is an ADC comprising a drug conjugated to a monoclonal antibody that specifically binds CD276. In some embodiments, the ADC comprises a variable heavy chain (VH) domain, a variable light chain (VL) domain, and an IgG1 Fc region, wherein the VH domain comprises a complementarity determining region 1 (CDR1) sequence, a CDR2 sequence, and a CDR3 sequence of the m276 antibody VH domain (SEQ ID NO: 2), the VL domain comprises CDR1, CDR2, and CDR3 sequences of the m276 antibody VL domain (SEQ ID NO: 6), the Fc region comprises S239C, L234A, L235A, and P329G mutations; and the drug is conjugated to cysteine at residue 239 of the Fc domain by site-specific conjugation. In some examples, the drug comprises a pyrrolobenzodiazepine (PBD) (e.g., a PBD dimer). In some examples, the drug is conjugated to the monoclonal antibody by a linker comprising a maleimide group, polyethylene glycol (PEG), and a valine-alanine dipeptide.

[0009] Also provided herein is a composition comprising the ADC disclosed herein and a pharmaceutically acceptable carrier.

[0010] Further provided are methods of treating CD276-positive cancer in a subject and methods of inhibiting tumor growth or metastasis of CD276-positive cancer in a subject. In some embodiments, the methods comprise administering a therapeutically effective amount of the ADC or composition disclosed herein.

[0011] The foregoing and other objects and features of the present disclosure will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0012]

Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

BEST MODE FOR CARRYING OUT THE INVENTION

[0019] Sequence Listing The amino acid sequences shown in the attached sequence listing are represented using the standard three-letter codes for amino acids, as defined in 37 C.F.R. 1.822. This sequence listing is submitted as a 15.3 KB ASCII text file created on November 17, 2020 (which is hereby incorporated by reference into this specification). In this attached sequence listing,

[0020] SEQ ID NO: 1 is the amino acid sequence of the signal peptide.

Chemical formula

[0021] SEQ ID NO: 2 is the amino acid sequence of the m276 variable heavy chain (VH) domain. The CDR sequences are indicated by bold underlining.

Chemical formula

[0022] SEQ ID NO: 3 is the amino acid sequence of the modified m276 constant region. The modified residues are indicated by bold underlining.

Chemical formula

[0023] SEQ ID NO: 4 is the amino acid sequence of the modified m276 heavy chain. The modified residues are indicated by bold underlining.

Chemical formula

[0024] SEQ ID NO: 5 is the amino acid sequence of the modified m276 heavy chain having an N-terminal signal peptide (underlined portion).

Chemical formula

Chem.

[0025] SEQ ID NO:6 is the amino acid sequence of the m276 variable light (VL) chain domain. The CDR sequences are indicated by underlining in bold.

Chem.

[0026] SEQ ID NO:7 is the amino acid sequence of the m276 light chain.

Chem.

[0027] Detailed Description I. Abbreviations ADC Antibody-drug conjugate B7H3 B7 homolog 3 CDR Complementary determining region Ig Immunoglobulin PBD Pyrrolobenzodiazepine PEG Polyethylene glycol VH Variable heavy VL Variable light

[0028] II. Terms and Methods Unless otherwise noted, technical terms are used according to customary usage. General terms in molecular biology will be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.

[0029] As used herein, the singular forms "a," "an," and "the" refer to both the singular and plural forms unless the context clearly dictates otherwise. For example, the term "an antigen" includes one or more antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprise" means "include." Further, unless otherwise noted, all base sizes or amino acid sizes, and all molecular weight or molecular mass values given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate discussion of the various embodiments, explanations of terms are provided below.

[0030] Administration: Providing or giving a subject a drug (e.g., a composition comprising an ADC that specifically targets CD276) by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, vaginal, and inhalation routes.

[0031] Antibody: A polypeptide ligand that contains at least one variable region that recognizes and binds (e.g., specifically recognizes and specifically binds) an epitope of an antigen. Mammalian immunoglobulin molecules consist of heavy (H) and light (L) chains (each of which has a variable region and is referred to as a variable heavy chain (V H ) region and a variable light chain (V L ) region, respectively). The V H region and the V LThe regions together are responsible for the binding of the antigen recognized by the antibody. There are five major heavy-chain classes (or isotypes) of mammalian immunoglobulins, which determine the functional activity of the antibody molecules, namely IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is the major antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0032] Antibody variable regions include "framework" regions and hypervariable regions (known as "complementary determining regions" or "CDRs"). CDRs are primarily responsible for binding to epitopes of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The boundaries of the amino acid sequences of a given CDR can be readily determined using any of a number of well-known numbering schemes (see Kabat et al. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the "Kabat" numbering scheme), Chothia et al. (Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273,927-948, 1997; the "Chothia" numbering scheme), Kunik et al. (Kunik et al., PLoS Comput Biol 8:e1002388, 2012; and Kunik et al., Nucleic Acids Res 40(Web Server issue):W521-524, 2012; see the "Paratome CDR"), and the IMGT (ImMunoGeneTics) database (Lefranc, Nucleic Acids Res 29:207-9, 2001; see the "IMGT" numbering scheme)). The Kabat, Paratome, and IMGT databases are maintained online. In some embodiments herein, amino acid numbering (e.g., for determining the position of an amino acid substitution) is referenced according to the Eu numbering convention (see Edelman et al., Proc. Natl. Acad. Sci. USA 63:78-85, 1969).

[0033] "Single-domain antibody" refers to an antibody having a single domain (variable domain) that can specifically bind to an antigen or an epitope of an antigen without additional antibody domains. Examples of single-domain antibodies include V H domain antibodies, V NAR antibodies, camelid V H H antibodies, and V L domain antibodies. V NAR antibodies are produced by cartilaginous fish (e.g., elephant sharks, kitefin sharks, dogfish sharks, and bamboo sharks). Camelid V H H antibodies are produced by several species including camels, llamas, alpacas, dromedaries, and guanacos, which produce heavy-chain antibodies that are naturally lacking in light chains.

[0034] "Monoclonal antibody" is an antibody produced by a single clone of lymphocytes or by cells transfected with the coding sequence of a single antibody. Monoclonal antibodies include humanized monoclonal antibodies.

[0035] "Chimeric antibody" has framework residues derived from one species (e.g., human) and CDRs (which generally give rise to antigen binding) derived from another species.

[0036] A "humanized" antibody is an immunoglobulin that contains a human framework region and one or more CDRs derived from a non-human (mouse, rabbit, rat, shark, or synthetic) immunoglobulin. The non-human immunoglobulin that provides the CDRs is referred to as the "donor," and the human immunoglobulin that provides the framework is referred to as the "acceptor." In one embodiment, all of the CDRs in the humanized immunoglobulin are derived from the donor immunoglobulin. The constant region need not be present, but if present, it must be substantially identical to the human immunoglobulin constant region, i.e., at least about 85-90%, e.g., about 95% or more identical. Thus, all parts of the humanized immunoglobulin (except perhaps the CDRs noted above) are substantially identical to the corresponding parts of the native human immunoglobulin sequence. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. A humanized or other monoclonal antibody may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions.

[0037] Antibody-drug conjugate (ADC): A molecule that contains an antibody (or an antigen-binding fragment of an antibody) conjugated to a drug (e.g., a cytotoxic agent). ADCs can be used to specifically target a drug to cancer cells by the specific binding of the antibody to a tumor antigen expressed on the cell surface. Exemplary drugs for use with ADCs include microtubule inhibitors (e.g., maytansinoids, auristatin E, and auristatin F) and interstrand crosslinking agents (e.g., pyrrolobenzodiazepine; PBD).

[0038] Microtubule inhibitor: A type of drug that blocks cell proliferation by arresting mitosis. Microtubule inhibitors, also called "mitotic inhibitors," are used to treat cancer.

[0039] Binding affinity: The affinity of an antibody for an antigen. In one embodiment, the affinity is calculated by a variant of the Scatchard method described by Frankel et al. (Mol. Immunol., 16:101-106, 1979). In another embodiment, the binding affinity is measured by the antigen / antibody dissociation rate. In another embodiment, high binding affinity is measured by a competitive radioimmunoassay. In another embodiment, the binding affinity is measured by ELISA. In other embodiments, the antibody affinity is measured by flow cytometry or by surface plasmon resonance. An antibody that "specifically binds" to an antigen (e.g., CD276) is an antibody that binds to that antigen with high affinity but does not significantly bind to other unrelated antigens.

[0040] Breast cancer: A type of cancer that occurs in breast tissue, usually in the milk ducts and lobules. Types of breast cancer include, for example, ductal carcinoma in situ, invasive ductal carcinoma, triple-negative breast cancer, inflammatory breast cancer, metastatic breast cancer, medullary carcinoma, tubular adenocarcinoma, and mucinous carcinoma. Triple-negative breast cancer refers to a type of breast cancer in which the cancer cells do not express estrogen receptor, progesterone receptor, or significant levels of HER2 / neu protein. Triple-negative breast cancer is also referred to as ER-negative, PR-positive, HER2 / neu-negative breast cancer. Invasive (malignant) carcinomas of the breast are classified into multiple stages (I, IIA, IIB, IIIA, IIIB, and IV). See, for example, Bonadonna et al. (eds.), “Textbook of Breast cancer: A clinical Guide to Therapy,” 3 rd ; London, Tayloy & Francis, 2006.

[0041] CD276: An immune checkpoint molecule expressed by several types of solid tumors. This protein is a member of the B7 superfamily of costimulatory molecules. CD276 is also known as B7 homolog 3 (B7-H3).

[0042] CD276-positive cancer: A cancer that expresses or overexpresses CD276. Examples of CD276-positive cancers include, but are not limited to, liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), brain cancer (e.g., neuroblastoma or glioblastoma), pediatric cancer (e.g., osteosarcoma, neuroblastoma, rhabdomyosarcoma, Wilms tumor, or Ewing sarcoma), melanoma, and mesothelioma (see, e.g., Seaman et al., Cancer Cell 31(4):501-505, 2017).

[0043] Chemotherapeutic agent: Any chemical agent that has therapeutic utility in the treatment of a disease characterized by abnormal cell proliferation. Such diseases include tumors, neoplasms, and cancers, as well as diseases characterized by hyperplastic growth such as psoriasis. In one embodiment, the chemotherapeutic agent is an agent useful in the treatment of CD276-positive tumors. In one embodiment, the chemotherapeutic agent is a radioactive compound. Non-limiting examples of useful chemotherapeutic agents are described in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison’s Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2 ndIt can be found in (ed., (copyright) 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D. S., Knobf, M. F., Durivage, H. J. (eds.): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. One example is the administration of an ADC targeting CD276, used in combination with a radioactive substance or compound.

[0044] Colorectal cancer: A type of cancer that occurs in the colon or rectum. The most common type of colorectal cancer (also known as "colorectal adenocarcinoma") accounts for approximately 95% of all colorectal cancers. Adenocarcinoma occurs in the cells lining the inside of the colon and / or rectum. Other types of colorectal cancer include gastrointestinal carcinoid tumors, metastatic colorectal cancer, primary colorectal lymphoma (a type of non-Hodgkin lymphoma), gastrointestinal stromal tumors (classified as sarcomas and arising from interstitial cells of Cajal), leiomyosarcoma (arising from smooth muscle cells), and colorectal melanoma.

[0045] Complementary determining region (CDR): A region of hypervariable amino acid sequences that determines the binding affinity and specificity of an antibody. The light and heavy chains of mammalian immunoglobulins each have three CDRs (designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively). Single-domain antibodies contain three CDRs (referred to herein as CDR1, CDR2, and CDR3).

[0046] Conservative variants: Proteins that contain conservative amino acid substitutions that do not substantially affect or reduce the affinity of a protein (e.g., an antibody against CD276). For example, a monoclonal antibody that specifically binds to CD276 may contain up to about 1, up to about 2, up to about 5, up to about 10, or up to about 15 conservative substitutions and may specifically bind to the CD276 polypeptide. The term "conservative variant" also includes using the substituted amino acid in place of the original unsubstituted amino acid, provided that the antibody specifically binds to CD276. Non-conservative substitutions are substitutions that reduce the activity or binding to CD276.

[0047] The conservative amino acid substitution table presents amino acids that are functionally similar. The following six groups are examples of amino acids that are considered to be conservative substitutions for each other. 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0048] Contact: An arrangement in direct physical association, including both solid and liquid forms.

[0049] Cytotoxic agent: Any drug or compound that kills cells.

[0050] Cytotoxicity: Toxicity of a molecule (e.g., an immunotoxin) to cells intended to be targeted (as contrasted with the remaining cells of the organism). In contrast, the term "toxicity" refers to the toxicity of an immunotoxin to cells different from the cells intended to be targeted by the targeting moiety of the immunotoxin, and the term "animal toxicity" refers to the toxicity of an immunotoxin to an animal due to the toxicity of the immunotoxin to cells different from the cells intended to be targeted by the immunotoxin.

[0051] Drug: Any compound used to treat, ameliorate, or prevent a disease or condition in a subject. In some embodiments herein, the drug is an anti-cancer agent, e.g., a cytotoxic agent (e.g., a mitotic inhibitor or a microtubule inhibitor).

[0052] Epitope: An antigenic determinant. This is a specific chemical group or peptide sequence on a molecule that is antigenic (inducing a specific immune response). An antibody specifically binds to a specific antigenic epitope on a polypeptide (e.g., CD276, etc.).

[0053] Framework region: The amino acid sequence intervening between CDRs. The framework regions of an immunoglobulin molecule include the variable light chain framework region and the variable heavy chain framework region.

[0054] Fusion protein: A protein comprising at least a portion of two different (heterologous) proteins.

[0055] Heterologous: Having separate genetic origins or originating from separate species.

[0056] IgG: A polypeptide belonging to the class or isotype of antibodies substantially encoded by the recognized immunoglobulin gamma genes. In humans, this class includes IgG 1 , IgG 2 , IgG 3 , and IgG 4 . In mice, this class is IgG1 、IgG 2a 、IgG 2b 、 and IgG 3 are included.

[0057] Immune response: The response of cells of the immune system (e.g., B cells, T cells, or monocytes) to a stimulus. In one embodiment, this response is specific to a particular antigen (“antigen-specific response”). In one embodiment, the immune response is a T cell response (e.g., CD4 + response or CD8 + response). In another embodiment, this response is a B cell response, resulting in the production of a specific antibody.

[0058] Interstrand crosslinking agent: A type of cytotoxic drug that can covalently bond between two strands of DNA, thereby inhibiting DNA replication and / or transcription.

[0059] Isolated: An “isolated” biological component (e.g., a nucleic acid, a protein (including an antibody), or an organelle) is substantially separated or purified from other biological components (e.g., other chromosomes and extrachromosomal DNA and RNA, proteins, and organelles) in the environment in which the component naturally occurs (e.g., a cell). “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. This term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0060] Label: A detectable compound or composition that is directly or indirectly conjugated to another molecule (e.g., an antibody or protein) to facilitate the detection of that molecule. Specific non-limiting examples of labels include fluorescent tags, enzymatic conjugates, and radioisotopes. In one example, a "labeled antibody" refers to incorporating another molecule into the antibody. For example, a label can be a detectable marker (e.g., incorporation of a radiolabeled amino acid for a polypeptide, or binding of a biotinyl moiety that can be detected by a labeled avidin (e.g., a fluorescent marker or streptavidin with enzymatic activity that can be detected by optical or colorimetric analysis methods)). Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Labels for polypeptides include, but are not limited to, for example, the following: radioactive isotopes or nucleotides (e.g., 35 S, 11 C, 13 N, 15 O, 18 F, 19 F, 99 mTc, 131 I, 3 H, 14 C, 15 N, 90 Y, 99 Tc, 111 In, and 125 I), fluorescent labels (e.g., fluorescein isothiocyanate (FITC), rhodamine, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope tags), or magnetic agents (e.g., gadolinium chelates). In some embodiments, the label is attached by spacer arms of various lengths to reduce possible steric hindrance.

[0061] Linker: In some cases, the linker is a peptide within an antibody binding fragment (e.g., Fv fragment) that serves to indirectly link the variable heavy chain to the variable light chain. "Linker" can also refer to a peptide that serves to link a targeting moiety (e.g., an antibody, etc.) to an effector molecule (e.g., a cytotoxin or a detectable label, etc.). The terms "conjugate", "join", "bond", or "link" refer to making two polypeptides into one continuous polypeptide molecule, or covalently binding a radionuclide or other molecule to a polypeptide (e.g., an antibody, etc.). This binding can be by either chemical means or recombinant means. "Chemical means" refers to a reaction between an antibody moiety and an effector molecule in which a covalent bond is formed between these two molecules, thereby forming one molecule. The ADCs disclosed herein include a linker for conjugating an antibody to a drug. In some embodiments, the linker includes a maleimide group, PEG (e.g., PEG8), and a valine-alanine dipeptide.

[0062] Liver cancer: Any type of cancer that occurs in liver tissue. The most common type of liver cancer is hepatocellular carcinoma (HCC) that occurs in hepatocytes. Other types of liver cancer include cholangiocarcinoma (which occurs in the bile ducts); hepatic angiosarcoma (a rare form of liver cancer that starts in the blood vessels of the liver); and hepatoblastoma (a very rare type of liver cancer that is most commonly seen in children).

[0063] Lung cancer: Any cancer that forms in the lungs. Most cancers that start in the lungs are carcinomas. The two main types of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Subclasses of NSCLC include adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Lung cancer is typically divided into stages I-IV, although other classifications are also used. For example, small cell lung cancer can be classified as limited stage if it is confined within one half of the chest and within the scope of a single radiation treatment field, and advanced stage if not. See, for example, Hansen (ed.), Textbook of Lung Cancer,2 nd , London: Informa Healthcare, 2008.

[0064] Maleimide: A compound of the formula H 2 C 2 (CO) 2 NH. Maleimide groups are commonly used for bioconjugation, for example, for the conjugation of drugs to antibodies (see, for example, Ravasco et al., Chem Eur J 25:43-49, 2019). Maleimide linked to a polyethylene glycol (PEG) chain is often used as a flexible linker molecule (see Figure 4).

[0065] Neuroblastoma: A solid tumor that arises from embryonic neural crest cells. Neuroblastomas generally occur inside and around the adrenal glands, but can occur anywhere sympathetic nervous tissue is found (e.g., within nerve tissue near the abdomen, chest, neck, or spine). Neuroblastomas typically occur in children under the age of 5.

[0066] Operably linked: When a first nucleic acid sequence is arranged in functional association with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Generally, operably linked DNA sequences are contiguous and, where it is necessary to join two protein-coding regions, are in the same reading frame.

[0067] Ovarian cancer: Cancer that occurs within the tissues of the ovary. Most ovarian cancers are either ovarian epithelial cancers (cancers that begin in the cells on the surface of the ovary) or malignant germ cell tumors (cancers that begin in the egg cells). Another type of ovarian cancer is stromal cell cancer, which arises from cells that release hormones and connect different structures of the ovary.

[0068] Pancreatic cancer: A disease in which malignant cells are found within the tissues of the pancreas. Pancreatic tumors can be either exocrine tumors or neuroendocrine tumors, based on the type of cells from which the cancer originates. The majority (about 94%) of pancreatic cancers are exocrine tumors. Examples of exocrine cancers include, for instance, adenocarcinoma (the most common type of exocrine tumor), acinar cell carcinoma, intraductal papillary-mucinous neoplasm (IPMN), and mucinous cystadenocarcinoma. In some instances, pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). Pancreatic neuroendocrine tumors (also called islet cell tumors) are classified by the type of hormone they produce. Exemplary neuroendocrine tumors include gastrinoma, glucaganoma, insulinoma, somatostatinoma, VIPoma (vasoactive intestinal peptide), and non-functional islet cell tumors.

[0069] Pediatric cancer: Cancer that occurs in children aged 0 to 14 years. Major types of pediatric cancer include, for example, neuroblastoma, acute lymphoblastic leukemia (ALL), embryonal rhabdomyosarcoma (ERMS), alveolar rhabdomyosarcoma (ARMS), Ewing sarcoma, desmoplastic small round cell tumor (DRCT), osteosarcoma, brain and other central nervous system (CNS) tumors (such as neuroblastoma and medulloblastoma), Wilms tumor, non-Hodgkin lymphoma, and retinoblastoma.

[0070] Pharmaceutically acceptable carrier: Useful pharmaceutically acceptable carriers are conventional ones. Remington, The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition (2005) describes compositions and formulations suitable for the pharmaceutical delivery of the antibodies and other compositions disclosed herein. Generally, the nature of the carrier will depend on the particular mode of administration to be employed. For example, parenteral formulations usually contain injectable fluids that contain pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc. as a vehicle. For solid compositions (such as powders, pills, tablets, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain small amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents (such as sodium acetate or sorbitan monolaurate).

[0071] Polyethylene glycol (PEG): Chemical formula H-(O-CH 2 -CH 2 ) nA compound consisting of repeating ethylene oxide units of -OH. PEG molecules are often used as linkers for ADCs due to their water solubility, non-toxicity, low immunogenicity, and well-defined chain length. PEG-containing linkers promote a reduction in protein aggregation and improve the solubility of the conjugate. In some embodiments, the PEG of the ADC linker contains 4, 5, 6, 7, 8, 9, or 10 ethylene oxide units. In certain embodiments, the PEG of the ADC linker contains 8 ethylene oxide units (PEG8).

[0072] Preventing, treating, or ameliorating a disease: "Preventing" a disease refers to preventing the full onset of the disease. "Treating" refers to a therapeutic intervention that improves (e.g., reduces tumor burden, or the number or size of metastases) the signs or symptoms of a disease or pathological condition after it has begun to develop. "Ameliorating" refers to a reduction in the number or severity of the signs or symptoms of a disease (e.g., cancer, etc.).

[0073] Purified: The term "purified" is not intended to require absolute purity and is a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched compared to that peptide or protein in its natural intracellular environment. In one embodiment, the preparation is purified such that the protein or peptide corresponds to at least 50% of the total peptide or protein content of the preparation. Substantial purification means purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95%, or 98% pure. Thus, in one particular non-limiting example, a substantially purified protein contains no 90% of other proteins or cellular components.

[0074] Pyrrolobenzodiazepine (PBD): A class of sequence-selective DNA minor groove-binding crosslinking agents first discovered in Streptomyces species. PBDs are significantly more potent than systemic chemotherapeutic agents. The mechanism of action of PBDs is related to their ability to form adducts in the minor groove of DNA, thereby inhibiting DNA processing. In the context of the present disclosure, PBDs include naturally occurring and isolated PBDs, chemically synthesized native PBDs, and chemically synthesized non-native PBDs. PBDs also include monomeric PBDs, dimeric PBDs, and hybrid PBDs (see, for review, Gerratana, Med Res Rev 32(2):254-293,2012).

[0075] Recombinant: A recombinant nucleic acid or protein is one that has a sequence not found in nature or has a sequence formed by artificially combining two originally separate segments. This artificial combination is often achieved by chemical synthesis or by artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.

[0076] Sample (or biological sample): A biological sample obtained from a subject and containing genomic DNA, RNA (including mRNA), protein, or a combination thereof. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, tissue biopsy material, fine needle aspirates, surgical specimens, and autopsy material.

[0077] Sequence identity: The similarity between amino acid or nucleic acid sequences is expressed by the similarity between the sequences and is otherwise referred to as sequence identity. Sequence identity is often measured by percent identity (or similarity or homology), and the higher the percentage, the more similar the two sequences. Homologs or variants of a polypeptide or nucleic acid molecule will have relatively high sequence identity when aligned by standard methods.

[0078] Methods for aligning arrays for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994 presents a detailed discussion of sequence alignment methods and homology calculations.

[0079] The BLAST (NCBI Basic Local Alignment Search Tool) (Altschul et al., J. Mol. Biol. 215:403, 1990), which is used with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx, is available from various sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet. Instructions for methods for determining sequence identity using this program are available on the NCBI website on the Internet.

[0080] Homologs and variants of antibodies that specifically bind to a CD276 polypeptide are typically characterized by having at least about 75%, such as at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity when counted over the full-length alignment against the amino acid sequence of the antibody, using NCBI BLAST2.0, gapped blastp (with initial parameter settings). For comparison of amino acid sequences longer than about 30 amino acids, the BLAST2 sequence function is utilized using the initial BLOSUM62 matrix set to initial parameters (gap extension cost of 11 and gap cost per residue of 1). When aligning short peptides (less than approximately 30 amino acids), the alignment should be performed using the BLAST2 sequence function with the PAM30 matrix set to initial parameters (starting gap penalty of 9 and extension gap penalty of 1). Proteins having greater similarity to the reference sequence will, when analyzed by this method, exhibit increasing percent identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When sequences shorter than the full-length sequence are compared for sequence identity, homologs and variants typically have at least 80% sequence identity over a short window of 10 - 20 amino acids and may have at least 85% or at least 90% or 95% sequence identity depending on the similarity to the reference sequence. Methods for determining sequence identity over such short windows are available on the NCBI website on the Internet. These ranges of sequence identity are provided only for guidance, and it is entirely possible that very important homologs outside the indicated ranges may be obtained.

[0081] Small molecule: A molecule typically having a molecular weight of less than about 1000 daltons, or in some embodiments, having a molecular weight of less than about 500 daltons, where the molecule is capable of modifying the activity of the target molecule to some measurable extent.

[0082] Subject: A living multicellular vertebrate (a category that includes both humans and veterinary subjects (including humans and non-human mammals)).

[0083] Synthesis: Produced by artificial means in a laboratory. For example, synthetic nucleic acids or proteins (such as antibodies) can be chemically synthesized in a laboratory.

[0084] Therapeutically effective amount: The amount of an agent (e.g., an ADC targeting CD276) that, alone or in combination with one or more additional agents, causes a desired response (e.g., treatment of a tumor, etc.) in a subject. The dosage that would achieve a target tissue concentration shown to achieve the desired in vitro effect when administered to a subject would generally be used. Ideally, the therapeutically effective amount results in a therapeutic effect without causing a substantial cytotoxic effect in the subject.

[0085] In one example, the desired response is to decrease the size, volume, or number of tumors (e.g., metastases) in a subject. For example, the agent(s) can decrease the size, volume, or number of tumors by a desired amount, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, at least 75%, at least 90%, or at least 95% compared to the response without the agent.

[0086] Some of the preparations disclosed herein are administered in a therapeutically effective amount. The therapeutically effective amount of an ADC (or composition comprising an ADC) that specifically binds to CD276, when administered to a human or veterinary subject, will vary depending on a number of factors related to that subject (e.g., the overall health of the subject). The therapeutically effective amount can be determined by varying the dosage and measuring the resulting therapeutic response (e.g., tumor regression). The therapeutically effective amount can also be determined by various in vitro, in vivo, or in situ immunoassays. The agents of the present disclosure can be administered in a single dose or multiple doses, as needed to obtain the desired response. However, the therapeutically effective amount can depend on the source being applied, the subject being treated, the severity and type of the condition being treated, as well as the manner of administration.

[0087] Vector: A nucleic acid molecule that is introduced into a host cell, thereby generating a transformed host cell. The vector can contain nucleic acid sequences that enable replication in the host cell (e.g., an origin of replication, etc.). The vector can also contain one or more selectable marker genes, as well as other genetic elements known in the art. In some embodiments, the vector is a viral vector (e.g., a lentiviral vector).

[0088] III. Antibody-Drug Conjugates Targeting CD276 The CD276-specific monoclonal antibody m276 (also known as m8524) was isolated from a naive human scFv library as previously described (WO2016 / 044383, which is hereby incorporated by reference in its entirety). The m276 antibody binds to both human m276 and mouse m276. WO2016 / 044383 describes two different ADCs using the m276 antibody, one ADC containing m276 conjugated to monomethyl auristatin E (MMAE) by a linker moiety, and the second ADC consisting of m276 conjugated to pyrrolobenzodiazepine (PBD) by a glycol group (m276-PBD). The previously described ADCs are partially effective in killing CD276-expressing tumor cells, but the present disclosure provides improved CD276-specific ADCs with improved stability and efficacy and minimal off-target effects.

[0089] The improved CD276-targeted ADCs contain several mutations in the Fc region of the m276 antibody. In particular, the ADCs have the Fc region rendered non-reactive to FcγRI, FcγRII, and FcγRIII by including the L234A, L235A, and P329G substitutions (numbered according to the Eu numbering convention relative to human IgG1), which reduces off-target effects by preventing the killing of Fc receptor-expressing normal cells. The modified ADCs also include the S239C mutation (numbered according to the Eu numbering convention relative to human IgG1), which enables site-specific conjugation of a drug (e.g., PBD or a PBD dimer). As one example, the drug is conjugated to the cysteine at residue 239 using a valine-alanine dipeptide linker. Site-specific conjugation at this location improves the biophysical properties of the ADC by enabling conjugation of highly hydrophobic agents (e.g., PBD) without causing significant aggregation. The S239C mutation also prevents premature loss of the drug in circulation, thereby increasing the stability of the ADC.

[0090] An engineered ADC comprising antibody m276 and a PBD dimer as a drug component (referred to herein as "m276-PBD-SL") is disclosed herein to be highly potent in a mouse tumor xenograft model. The m276-PBD-SL ADC is capable of eradicating very large tumors (>1000 mm 3 ), which was not possible with the original unmodified m276-based ADC. Furthermore, this effect was observed with doses of m276-PBD-SL that did not cause toxicity in mice (see Examples 2 and 5).

[0091] The amino acid sequences of the m276 VH and VL domains are shown below. The CDR sequences according to IMGT are indicated by bold underlines. The amino acid residues of each CDR are listed below each sequence.

[0092] m276 variable heavy chain (VH) domain (SEQ ID NO: 2)

Chemical formula

[0093] m276 variable light chain (VL) domain (SEQ ID NO: 6)

Chemical formula

[0094] To generate a modified ADC using the m276 antibody, four amino acid substitutions were introduced into the heavy chain of m276. The sequence of the modified heavy chain is shown below and designated as SEQ ID NO: 4 herein. The four amino acid substitutions in the heavy chain (located at residues 236, 237, 241, and 331 of SEQ ID NO: 4 and corresponding to residues 234, 235, 239, and 329 of human IgG1) are indicated by bold underlining. The VH domain (residues 1-119 of SEQ ID NO: 4) is underlined. The constant region of the m276 heavy chain is shown as SEQ ID NO: 3 (and corresponds to residues 120-449 of SEQ ID NO: 4).

[0095] Modified m276 heavy chain (SEQ ID NO: 4)

Chemical formula

Chemical formula

[0096] In some embodiments, the modified m276 constant domain comprises an N-terminal signal peptide:

Chemical formula

[0097] Modified m276 heavy chain having a signal sequence (SEQ ID NO: 5)

Chemical formula

[0098] Provided herein is an ADC comprising a drug conjugated to a CD276-specific monoclonal antibody. The monoclonal antibody comprises a variable heavy chain (VH) domain, a variable light chain (VL) domain, and an IgG1 Fc region. In some embodiments, the VH domain of the monoclonal antibody comprises the complementarity-determining region 1 (CDR1) sequence, CDR2 sequence, and CDR3 sequence of the m276 VH domain (shown as SEQ ID NO: 2), the VL domain of the monoclonal antibody comprises the CDR1 sequence, CDR2 sequence, and CDR3 sequence of the m276 VL domain (shown as SEQ ID NO: 6), and the Fc region of the monoclonal antibody comprises the S239C, L234A, L235A, and P329G mutations (numbered relative to human IgG1). The drug component of the ADC is conjugated (either directly or indirectly via a linker) to the cysteine at residue 239 of the Fc domain by site-specific conjugation.

[0099] In some embodiments, the CDR sequences are determined using the Kabat, IMGT, or Chothia numbering convention.

[0100] In some embodiments, the CDR1 sequence, CDR2 sequence, and CDR3 sequence of the VH domain each comprise residues 26-33, 51-58, and 97-108 of SEQ ID NO: 2; and / or the CDR1, CDR2, and CDR3 sequences of the VL domain each comprise residues 27-32, 50-52, and 89-99 of SEQ ID NO: 6. In some examples, the VH domain (in addition to the recited CDR sequences) comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2, and / or the VL domain (in addition to the recited CDR sequences) comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6. In certain non-limiting examples, the VH domain comprises or consists of the amino acid sequence of SEQ ID NO: 2, and / or the VL domain comprises or consists of the amino acid sequence of SEQ ID NO: 6.

[0101] In some embodiments, the amino acid sequence of the Fc region comprises SEQ ID NO: 3.

[0102] In some embodiments, the monoclonal antibody is IgG1. In some examples, the monoclonal antibody is IgG1 and the amino acid sequence of the heavy chain of the IgG1 comprises SEQ ID NO: 4 or SEQ ID NO: 5. In some examples, the monoclonal antibody is IgG1 and the amino acid sequence of the light chain of the IgG1 comprises SEQ ID NO: 7.

[0103] In some embodiments, the drug of the ADC comprises a cytotoxic agent (e.g., an interstrand crosslinking agent, a mitotic inhibitor, or a microtubule inhibitor, etc.). In some examples, the interstrand crosslinking agent comprises pyrrolobenzodiazepine (PBD) (e.g., a PBD dimer, etc.).

[0104] In some embodiments, the ADC further comprises a linker that connects the drug to the monoclonal antibody. In some examples, the drug is conjugated to the monoclonal antibody by a linker comprising a valine-alanine dipeptide. In certain examples, the linker further comprises a maleimide group. In certain examples, the linker further comprises polyethylene glycol (PEG). In a particular non-limiting example, the linker comprises a valine-alanine dipeptide, PEG (e.g., PEG8), and a maleimide group.

[0105] Also provided herein is a composition comprising the ADC disclosed herein and a pharmaceutically acceptable carrier.

[0106] Further provided herein is a method of treating CD276-positive cancer in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of the ADC or composition disclosed herein. In some examples, the method further comprises selecting a subject diagnosed with CD276-positive cancer. In some examples, the CD276-positive cancer is hepatocellular carcinoma, melanoma, leukemia, breast cancer, neuroblastoma, prostate cancer, colorectal cancer, osteosarcoma, endometrial cancer, ovarian cancer, oral squamous cell carcinoma, non-small cell lung cancer, bladder cancer, or pancreatic cancer. In a particular non-limiting example, the CD276-positive cancer is breast cancer or neuroblastoma.

[0107] Methods for inhibiting tumor growth or metastasis of CD276-positive cancers in a subject are also provided. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an ADC or composition disclosed herein. In some examples, the method further comprises selecting a subject diagnosed with a CD276-positive cancer. In some examples, the CD276-positive cancer is hepatocellular carcinoma, melanoma, leukemia, breast cancer, neuroblastoma, prostate cancer, colorectal cancer, osteosarcoma, endometrial cancer, ovarian cancer, oral squamous cell carcinoma, non-small cell lung cancer, bladder cancer, or pancreatic cancer. In certain non-limiting examples, the CD276-positive cancer is breast cancer or neuroblastoma.

[0108] In some embodiments of the methods of the present disclosure, the method further comprises administering to the subject an additional anti-cancer agent. In some examples, the additional anti-cancer agent comprises a chemotherapeutic agent or an angiogenesis inhibitor. In some embodiments, the method further comprises surgical resection of the tumor and / or radiation therapy.

[0109] IV. DRUGS AND LINKERS An ADC is a compound consisting of a tumor antigen-specific antibody and a drug (typically a cytotoxic agent such as a microtubule inhibitor or a cross-linking agent). Since an ADC can specifically target cancer cells, the drug can be much more potent compared to drugs used in standard chemotherapy. The most common cytotoxic drugs currently used with ADCs have IC50s that are 100 to 1000 times more potent than conventional chemotherapeutic agents. Common cytotoxic drugs include pyrrolobenzodiazepine (PDB), which covalently binds to the minor groove of DNA and forms an interstrand cross-link, and microtubule inhibitors such as maytansinoids and auristatins (e.g., auristatin E and auristatin F). In some examples, the ADC comprises an antibody and a drug in a ratio of 1:2 to 1:4 (Bander, Clinical Advances in Hematology&Oncology 10(8;suppl 10):3-7,2012).

[0110] Provided herein is an ADC comprising a drug (e.g., a cytotoxic agent) conjugated to a monoclonal antibody that binds (e.g., specifically binds) to CD276. In some embodiments, the drug is a small molecule. In some examples, the drug is a cross-linking agent, a microtubule inhibitor and / or a mitotic inhibitor, or any cytotoxic agent suitable for mediating the death of tumor cells. Exemplary cytotoxic agents include, but are not limited to, PDB, auristatin, maytansinoid, dolastatin, calicheamicin, nemorubicin and its derivatives, PNU-159682, anthracycline, duocarmycin, vinca alkaloid, taxane, trichothecin, CC1065, camptothecin, elinafide, combretastatin, dolastatin, duocarmycin, enediyne, geldanamycin, indolino-benzodiazepine dimer, puromycin, tubulysin, hemiasterlin, spliceostatin, or pladienolide, as well as stereoisomers, isotears, analogs, and derivatives thereof having cytotoxic activity.

[0111] In some embodiments, the ADC comprises a pyrrolobenzodiazepine (PBD). The natural product anthramycin (a type of PBD) was first reported in 1965 (Leimgruber et al., J Am Chem Soc, 87:5793-5795, 1965; Leimgruber et al., J Am Chem Soc, 87:5791-5793, 1965). Since then, numerous PBDs (both naturally occurring analogs and synthetic analogs) have been reported (Gerratana, Med Res Rev 32(2):254-293, 2012; and U.S. Patent Nos. 6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; and 7,557,099). As one example, PDB dimers recognize and bind to specific DNA sequences and are useful as cytotoxic agents. PBD dimers have been conjugated to antibodies and the resulting ADCs have had anti-cancer properties (see, e.g., US2010 / 0203007). Exemplary linking sites on PBD dimers include the 5-membered pyrrole ring, the tether between PBD units, and the N10-C11 imine group (see WO2009 / 016516; US2009 / 304710; US2010 / 047257; US2009 / 036431; US2011 / 0256157; and WO2011 / 130598).

[0112] In some embodiments, the ADC comprises an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine and are mitotic inhibitors that function by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, certain microorganisms were also found to produce maytansinoids (e.g., maytansinol and C-3 maytansinol esters) (U.S. Patent No. 4,151,042). Synthetic maytansinoids are disclosed, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.

[0113] In some embodiments, the ADC comprises an antibody conjugated to dolastatin or auristatin, or an analog or derivative thereof (see U.S. Pat. Nos. 5,635,483; 5,780,588; 5,767,237; and 6,124,431). Auristatin is a derivative of the marine mollusk compound dolastatin 10. Dolastatin and auristatin interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al., Antimicrob Agents and Chemother 45(12):3580-3584, 2001), and have anticancer activity (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob Agents Chemother 42:2961-2965, 1998). Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin F, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (monomethyl auristatin D or monomethyl dolastatin 10), MMAF (monomethyl auristatin F or N-methylvaline-valine-drysoloyin-draproin-phenylalanine), MMAE (monomethyl auristatin E or N-methylvaline-valine-drysoloyin-draproin-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other auristatins (see, e.g., U.S. Pub. No. 2013 / 0129753).

[0114] In some embodiments, the ADC comprises an antibody conjugated to one or more calicheamicin molecules. Antibiotics of the calicheamicin family, and analogs thereof, can cause double-strand DNA breaks at subpicomolar concentrations (Hinman et al., Cancer Res 53:3336-3342, 1993; Lode et al., Cancer Res 58:2925-2928, 1998). Exemplary methods for preparing ADCs with calicheamicin drug moieties are described in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.

[0115] In some embodiments, the ADC comprises an anthracycline. Anthracyclines are antibacterial compounds that exhibit cytotoxic activity. Anthracyclines are thought to function to kill cells by a number of different mechanisms, including inhibition of DNA-dependent nucleic acid synthesis by intercalation of anthracycline molecules into cellular DNA; induction of free radical production, which then reacts with cellular macromolecules to cause damage to the cell; and / or interaction of anthracycline molecules with cell membranes. Non-limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, daunorubicin, doxorubicin, epirubicin, nemorubicin, valrubicin, and mitoxantrone, and derivatives thereof. For example, PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al., Clin Cancer Res 11(4):1608-1617, 2005). Nemorubicin is a semisynthetic analog of doxorubicin that has a 2-methoxymorpholino group on the glycoside amino of doxorubicin (Grandi et al., Cancer Treat Rev 17:133, 1990; Ripamonti et al., Br J Cancer 65:703-707, 1992).

[0116] Antibodies and drugs can be linked by cleavable or non-cleavable linkers. However, in some instances, it is desirable to have a linker that is stable in circulation to prevent systemic release of cytotoxic drugs that can cause significant off-target toxicity. Non-cleavable linkers prevent the release of cytotoxic agents until the ADC is internalized by the target cell. Once inside the lysosome, the antibody is digested by lysosomal proteases, resulting in the release of the cytotoxic agent (Bander, Clinical Advances in Hematology&Oncology 10(8;suppl 10):3-7,2012).

[0117] In some embodiments, the linker has a functional group that can react with a free cysteine present on the antibody to form a covalent bond. Exemplary linkers having such reactive functional groups include maleimide, haloacetamide, α-haloacetyl, activated esters such as succinimidyl ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0118] In some examples herein, the linker is non-cleavable and is directly conjugated to the antibody by site-specific conjugation. In some examples, the linker of the ADC comprises a valine-alanine dipeptide, a PEG molecule, and a maleimide group.

[0119] V. Compositions and Methods of Use Compositions comprising the CD276-specific ADCs disclosed herein are provided. The compositions can be formulated into unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the clinician treating to achieve the desired outcome. The ADCs can be formulated for systemic or local (e.g., intratumoral) administration. In one example, the ADCs are formulated for parenteral administration (e.g., intravenous administration, etc.).

[0120] The composition for administration may contain a solution of the ADC in a pharmaceutically acceptable carrier (such as an aqueous carrier, etc.). Various aqueous carriers, such as buffered saline, etc. can be used. These solutions are sterile and free of generally undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffering agents, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc., as necessary to approach physiological conditions. The concentration of the ADC in these formulations may vary widely and will mainly be selected according to the specific mode of administration chosen and the requirements of the subject, depending on factors such as the volume of the liquid, viscosity, body weight, etc.

[0121] The composition containing the ADC can be formulated into unit dosage forms suitable for the precise dosing of individual administrations. Further, the composition can be administered in a single dose or in a multiple-dose schedule. A multiple-dose schedule can be separate doses (e.g., 1 to 10 doses) where the main treatment process exceeds one, and then, at subsequent time intervals, other doses can be administered as necessary to maintain or enhance the action of the composition. The treatment can include doses of the compound(s) once or multiple times a day over a period of several days to several months, or even several years. Thus, the dosing regimen will also be determined, at least in part, based on the specific requirements of the subject being treated and will depend on the judgment of the physician administering it.

[0122] Typical dosages of the ADC, composition, or additional agent(s) can range from about 0.01 to about 30 mg / kg, such as from about 0.1 to about 10 mg / kg. In some examples, the dosage is at least about 0.1 mg / kg, at least about 0.2 mg / kg, at least about 0.3 mg / kg, at least about 0.4 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 4 mg / kg, at least about 3 mg / kg, at least about 5 mg / kg, at least about 6 mg / kg, at least about 7 mg / kg, at least about 8 mg / kg, at least about 9 mg / kg, at least about 10 mg / kg, at least about 11 mg / kg, at least about 12 mg / kg, at least about 13 mg / kg, at least about 14 mg / kg, at least about 15 mg / kg, at least about 16 mg / kg, at least about 17 mg / kg, at least about 18 mg / kg, at least about 19 mg / kg, at least about 20 mg / kg, at least about 21 mg / kg, at least about 22 mg / kg, at least about 23 mg / kg, at least about 24 mg / kg, at least about 25 mg / kg, at least about 26 mg / kg, at least about 27 mg / kg, at least about 28 mg / kg, at least about 29 mg / kg, or at least about 30 mg / kg.

[0123] In certain examples, the subject is administered the ADC or its composition, or additional agent(s), on a daily dosing schedule for multiple days (e.g., at least 2 consecutive days, 10 consecutive days, etc.) over a period of, for example, weeks, months, or years. In one example, the subject is administered the ADC, composition, or additional agent(s) over a period of at least 30 days (e.g., at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months).

[0124] In some embodiments, the disclosed ADC or composition is administered intravenously, subcutaneously, or by another mode, once daily or multiple times per week, over a period of time, followed by a period of no treatment, and then this cycle is repeated. In some embodiments, the initial period of treatment (e.g., administration of the therapeutic agent once daily or multiple times per week) is 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In related embodiments, the period of no treatment lasts 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In certain embodiments, the dosing regimen of the therapeutic agent is once daily for 3 days, then a 3-day break; or once daily or multiple times per week for 1 week, then a 3-day or 1-week break; or once daily or multiple times per week for 2 weeks, then a 1- or 2-week break; or once daily or multiple times per week for 3 weeks, then a 1, 2, or 3-week break; or once daily or multiple times per week for 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, then a 1, 2, 3, or 4-week break.

[0125] The ADCs disclosed herein can also be administered by other routes, including by inhalation, oral, topical, or intravitreal. In some examples, the ADC is administered by a fine needle.

[0126] The ADC may be provided as a sterile solution of known concentration, but may also be provided in lyophilized form and rehydrated with sterile water prior to administration. The ADC solution is then added to an infusion bag containing 0.9% sodium chloride (USP) and, in some cases, is administered at a dosage of 0.5 - 15 mg / kg body weight. Considerable experience in the administration of antibody drugs (which have been on the market in the United States since the approval of Rituxan™ in 1997) is available in the art. The ADC can be administered by a slow infusion rather than an intravenous push or bolus. In one example, a higher loading dose is administered, followed by a maintenance dose at a lower level.

[0127] Controlled-release parenteral formulations can be manufactured as implants, oily injections, or particulate systems. For a general overview of protein delivery systems, see Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Examples of particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein (e.g., a cytotoxin or drug, etc.) as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles smaller than about 1 μm, microspheres, and microcapsules are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm such that only nanoparticles are administered intravenously. Microparticles typically have a diameter of approximately 100 μm and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter (ed.), Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0128] Polymers can be used for the ion-controlled release of the ADC compositions disclosed herein. A variety of degradable polymer matrices and non-degradable polymer matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem.Res. 26:537-542, 1993). For example, the block copolymer polaxamer 407 exists as a viscous but mobile liquid at low temperatures, but forms a semi-solid gel at body temperature. This is an effective vehicle for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm.Res. 9:425-434, 1992; and Pec et al., J.Parent.Sci.Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int.J.Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used for the controlled release of lipid-encapsulated drugs as well as for drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). A number of additional systems for the controlled delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342; and 5,534,496).

[0129] The ADCs disclosed herein can be administered to slow or inhibit the growth of tumor cells (e.g., CD276-positive tumors (e.g., solid tumors)) or to slow or inhibit the metastasis of tumor cells. In these applications, a therapeutically effective amount of the composition is administered to the subject in an amount sufficient to inhibit the growth, replication, or metastasis of cancer cells or to suppress the signs or symptoms of cancer. Suitable subjects include those diagnosed with cancers that express CD276, such as, but not limited to, hepatocellular carcinoma, melanoma, leukemia, breast cancer, neuroblastoma, prostate cancer, colorectal cancer, osteosarcoma, endometrial cancer, ovarian cancer, oral squamous cell carcinoma, non-small cell lung cancer, bladder cancer, or pancreatic cancer.

[0130] Provided herein is a method of treating CD276-positive cancer in a subject by administering to the subject a therapeutically effective amount of an ADC or composition disclosed herein. Also provided herein is a method of inhibiting tumor growth or metastasis of CD276-positive cancer in a subject by administering to the subject a therapeutically effective amount of an ADC or composition disclosed herein. In some embodiments, the CD276-positive cancer is hepatocellular carcinoma, melanoma, leukemia, breast cancer, neuroblastoma, prostate cancer, colorectal cancer, osteosarcoma, endometrial cancer, ovarian cancer, oral squamous cell carcinoma, non-small cell lung cancer, bladder cancer, or pancreatic cancer.

[0131] The therapeutically effective amount of the CD276-specific ADC or composition disclosed herein will depend on the severity of the disease, the type of disease, and the overall health of the patient. A therapeutically effective amount of an antibody-based composition results in either a subjective reduction of symptoms or an objectively identifiable improvement as noted by a clinician or other qualified observer.

[0132] Administration of the CD276-specific ADCs and compositions disclosed herein may also be accompanied by the administration of other anti-cancer agents or therapeutic treatments (e.g., surgical resection of a tumor). Any suitable anti-cancer agent may be administered in combination with the ADCs and compositions disclosed herein. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents such as mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormonal agents (e.g., anti-androgens), and angiogenesis inhibitors. Other anti-cancer treatments include radiation therapy and other antibodies that specifically target cancer cells.

[0133] Non-limiting examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin, or dacarbazine).

[0134] Non-limiting examples of antimetabolites include folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-FU or cytarabine), and purine analogs (e.g., mercaptopurine or thioguanine).

[0135] Non-limiting examples of natural products include vinca alkaloids (e.g., vinblastine, vincristine, or vindesine), epipodophyllotoxins (e.g., etoposide or teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (e.g., L-asparaginase).

[0136] Non-limiting examples of other various agents include platinum coordination complexes (e.g., cis-diamminedichloroplatinum II, also known as cisplatin), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), and adrenocrotical suppressants (e.g., mitotane and aminoglutethimide).

[0137] Non-limiting examples of hormones and antagonists include corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (e.g., diethylstilbestrol and ethinyl estradiol), anti-estrogen agents (e.g., tamoxifen), and androgens (e.g., testosterone proprionate and fluoxymesterone). Examples of the most commonly used chemotherapeutic agents include Adriamycin, Alkeran, Ara-C, BiCNU, busulfan, CCNU, carboplatin, cisplatin, cytoxan, daunorubicin, DTIC, 5-FU, fludarabine, hydrea, idarubicin, ifosfamide, methotrexate, mitramycin, mitomycin, mitoxantrone, nitrogen mustard, taxol (or other taxanes such as docetaxel, etc.), Velban, vincristine, VP-16, while some newer agents include gemcitabine (Gemzar), Herceptin, irinotecan (Camptosar, CPT-11), roisastatin, navelbine, rituxan STI-571, Taxotere, topotecan (Hycamtin), Xeloda (capecitabine), Zevelin, and calcitriol.

[0138] Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.), broxuridine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony-stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulins (Cutter Biological), IMREG (from Imreg in New Orleans, Louisiana), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0139] Another common treatment for some types of cancer is a surgical procedure, for example, surgical resection of the cancer or a part thereof. Another example of a treatment is radiation therapy, for example, the application of radioactive substances or energy (e.g., external beam radiation therapy) to the tumor site to help eradicate the tumor or to shrink the tumor prior to surgical resection.

[0140] The following examples are provided to illustrate certain features and / or embodiments. These examples should not be construed as limiting the present disclosure to the specific features or embodiments described.

Examples

[0141] Example 1: Modification of the heavy chain of the CD276-specific antibody m276 The human CD276-specific antibody m276 (also known as "m8524") was selected from a yeast display naive human antibody library as described in PCT Publication No. WO2016 / 044383, which is hereby incorporated by reference in its entirety. Since the m276 variable domain is derived from a natural (non-synthetic) human antibody library, the m276 IgG antibody is likely to be less recognized by the immune system when administered to humans. If not all, most of the previously described CD276 antibodies were initially developed in mice using traditional hybridoma technology. As a result, those mouse antibodies still contain mouse variable domains even after humanization and are thus still more foreign (and thus more immunogenic) compared to m276, which has a fully human variable domain.

[0142] The m276 antibody binds to both human CD276 and mouse CD276. The amino acid sequences of the VH and VL domains of m276 are shown herein as SEQ ID NO: 2 and SEQ ID NO: 6, respectively.

[0143] This example describes the generation of a modified version of m276 IgG1. Specifically, four amino acid substitutions (L234A, L235A, P329G, and S239C (numbered according to the Eu numbering convention for human IgG1; Edelman et al., Proc. Natl. Acad. Sci. USA 63:78-85, 1969)) were introduced into the heavy chain constant region of m276. The sequence of the modified m276 heavy chain is shown below (also shown as SEQ ID NO: 5):

Chemical formula

[0144] The heavy chain sequence includes a 19-amino acid signal sequence (italicized above; SEQ ID NO: 1), the m276 VH domain (underlined above; SEQ ID NO: 2), and the m276 heavy chain constant domain (SEQ ID NO: 3), and the four amino acid substitutions are indicated by bold underlining.

[0145] To prevent the interaction between the Fc domain and endogenous Fc receptors present on cells of the reticuloendothelial system, L234A, L235A, and P329G ("LALAPG") mutations were introduced (Lo et al., J Biol Chem 292(9):3900-3908, 2017). These three mutations render the Fc region non-reactive to Fcγ receptor I (RI), FcγRII, and FcγRIII. Many groups have attempted to enhance Fc / Fc receptor interactions to improve the ADCC or CDC activity of non-armed antibodies (i.e., antibodies without drug conjugates), but in the case of toxic ADCs, such interactions can be disadvantageous when the internalized antibody kills phagocytic target cells. Thus, by blocking the ADC-Fc / Fc receptor interaction, inappropriate killing of normal cells with Fc receptors can be prevented and off-target toxicity can be minimized.

[0146] The cysteine introduced at position 239 is used for the binding of the drug (PBD dimer in this example). The resulting ADC is "m276-PBD-SL" (SL is

Chemical formula

[0147] Example 2: Treatment with m276-PBD-SL results in potent antitumor activity in an animal tumor model This example shows that m276-PBD-SL can eradicate large tumors in mouse models of human neuroblastoma and breast cancer.

[0148] The m276-PBD-SL ADC was tested in a human NB-EB neuroblastoma xenograft model grown subcutaneously in mice. In this study, treatment with m276-PBD-SL was initiated when the average tumor size reached approximately 1200 mm 3 . Animals began receiving 0.5 mg / kg of m276-PBD-SL (N = 4) once a week starting on day 24 after tumor cell inoculation. Untreated animals were used as controls (N = 6). These results showed that m276-PBD-SL induced strong antitumor activity against neuroblastoma xenograft tumors (Figure 1).

[0149] The m276-PBD-SL ADC was evaluated in a second model of human neuroblastoma called IMR-5. Treatment with vehicle (N = 8) or m276-PBD-SL (N = 7) was initiated when the average tumor size reached approximately 1000 mm 3 . Animals began receiving vehicle or 0.5 mg / kg of m276-PBD-SL once a week starting on day 40 after tumor cell inoculation. The results of this study showed that m276-PBD-SL induced strong antitumor activity against human neuroblastoma xenograft tumors grown subcutaneously in mice (Figure 2).

[0150] In another study, the m276-PBD-SL ADC was tested in a human MDA-MB-231 breast xenograft tumor model grown orthotopically in mice. Treatment with vehicle (N = 10) or m276-PBD-SL (N = 11) was initiated when the average tumor size reached approximately 1000 mm 3It was started when [a certain condition] was reached. The animals began to receive vehicle, 0.1 mg / kg of m276-PBD-SL, or 0.5 mg / kg of m276-PBD-SL once a week starting from the 31st day after inoculation of tumor cells. As shown in Figure 3, m276-PBD-SL induced potent antitumor activity in this human breast cancer model.

[0151] Additional studies were conducted to compare the efficacy of the previously described m276-PBD glycoconjugate (WO2016 / 044383) with that of the m276-PBD-SL ADC disclosed herein. In the first study, m276-PBD was tested in the syngeneic Py230 breast cancer model. Mice bearing Py230 tumors were administered vehicle or 1 mg / kg of m276-PBD twice a week for 4 weeks. The treatment was started when the average volume of the tumors reached 140 mm 3 As shown in Figure 5, the mammary tumors in all mice treated with the m276-PBD glycoconjugate first regressed and then recurred after treatment. As a comparison, the m276-PBD-SL ADC was evaluated in the larger syngeneic MDA-MB-231 breast cancer model. The treatment was started when the average volume of the tumors reached 1000 mm 3 Mice bearing MDA-MB-231 tumors received vehicle, 0.1 mg / kg of m276-PBD-SL, or 0.5 mg / kg of m276-PBD-SL once a week for 5 weeks. As shown in Figure 6, recurrence occurred in some of the mice treated with the lower (0.1 mg / kg) dose, but complete responses were observed in all mice treated with the higher (0.5 mg / kg) dose of m276-PBD-SL. Thus, compared with the ability of the higher dose of the m276-PBD glycoconjugate to treat smaller tumors, the lower dose of m276-PBD-SL was able to successfully treat larger tumors.

[0152] The m276-PBD-SL ADC was further tested in an orthotopic SUM519 breast cancer model. Mice bearing SUM159 tumors were administered vehicle or 0.5 mg / kg of m276-PBD-SL (right) once a week for 4 weeks. Treatment was initiated when the mean tumor volume reached 1000 mm 3 3 . Treatment with m276-PBD-SL was shown to result in complete regression of SUM159 tumors (Figure 7).

[0153] These results indicate that the m276-PBD-SL ADC is very potent and eradicates tumors at drug doses that show no signs of toxicity in mice. This drug is potent enough to eradicate tumors that are 1000 mm 3 or larger. This level of efficacy is extremely rare for any preclinical drug and has not been observed for m276-PBD glycoconjugates.

[0154] Example 3: Evaluation of m276-PBD-SL in a mouse model of UACC-62 human melanoma This example describes a study to compare the efficacy of m276-PBD-SL and m276 PBD glycoconjugate ADC in a mouse model of UACC-62 human melanoma.

[0155] Mice bearing UACC-62 melanoma tumors are administered vehicle, 0.1 mg / kg of m276-PBD, 0.5 mg / kg of m276-PBD, 0.1 mg / kg of m276-PBD-SL, or 0.5 mg / kg of m276-PBD-SL once a week for 4, 5, or 6 weeks. Treatment with m276-PBD-SL is expected to result in complete or significant eradication of the tumors. m276-PBD-SL is also expected to be significantly more effective compared to the m276-PDB glycoconjugate.

[0156] Example 4: Evaluation of m276-PBD-SL in a mouse model of HCT-116 human colon cancer This example describes a study to compare the efficacy of m276-PBD-SL and m276 PBD conjugate carbohydrate ADC in a mouse model of HCT-116 human colon cancer.

[0157] Mice bearing HCT-116 colon cancer tumors are administered vehicle, 0.1 mg / kg of m276-PBD, 0.5 mg / kg of m276-PBD, 0.1 mg / kg of m276-PBD-SL, or 0.5 mg / kg of m276-PBD-SL once a week for 4, 5, or 6 weeks. Treatment with m276-PBD-SL is expected to result in complete or significant eradication of the tumors. m276-PBD-SL is also expected to be significantly more effective compared to m276-PDB conjugate carbohydrate.

[0158] Example 5: Evaluation of m276-PBD-SL in a preclinical model of pediatric cancer This example describes the antitumor activity of m276-PBD-SL against a preclinical xenograft model of pediatric solid tumors.

[0159] Methods The antibody conjugate m276-PBD-SL was tested in subcutaneous mouse xenograft models of Ewing sarcoma, rhabdomyosarcoma, Wilms tumor, osteosarcoma, and neuroblastoma. m276-PBD-SL was administered by intraperitoneal injection at a dose of 0.5 mg / kg once a week for 3 consecutive weeks. An event was defined as a 4-fold increase in tumor volume from the first day of treatment. The Kaplan-Meier method was used for comparison between the treatment and control groups for time-to-event. The categories of objective response are as follows (see also Houghton et al., Pediatr Blood Cancer 49(7):928-940, 2007): PD = Progressive disease, <50% tumor regression throughout the study, and >25% tumor growth at the end of the study PD1 = PD and time to event in mice ≤ 200% of the KM median time to event in the control group PD2 = PD, and further, time to event > 200% of the time to event of the Kaplan-Meier (KM) median in the control group SD = stable disease, < 50% tumor regression throughout the trial, and ≤ 25% tumor growth at the end of the trial PR = partial response, ≥ 50% tumor regression at any point during the trial, however, measurable tumor throughout the trial period CR = complete response, disappearance of measurable tumor during the trial period MCR = maintained complete response, no measurable tumor mass for at least three consecutive weekly readings at any point after treatment completion

[0160] For the neuroblastoma test, two animals per model were used to evaluate tumor regression, while for other histologies, standard test procedures (n = 8 - 10) were used to evaluate tumor regression and time to event.

[0161] CD276 expression in the pediatric preclinical testing consortium (PPTC) model CD276 mRNA expression was measured and evaluated in the PPTC model using RNA-Seq in fragments per kilobase million (FPKM) per million. As a result, CD276 expression was highest in solid tumors (median 41 FPKM), and the highest expression was observed in osteosarcoma (median 82 FPKM). Neuroblastoma, rhabdomyosarcoma, Wilms tumor, and embryonal brain tumor models also had high expression levels, while the expression level shown by the acute lymphoblastic leukemia (ALL) model was low. The RNA-Seq data is consistent with protein expression data from clinical specimens (Majzner et al., Clin Cancer Res 25(8):2560 - 2574, 2019).

[0162] Summary of tumor growth results m276-PBD-SL demonstrated very high levels of antitumor activity against several pediatric solid tumor preclinical models at a dose of 0.5 mg / kg administered weekly for 3 weeks. Objective responses (PR / CR / MCR) were observed in 23 of 25 models (92%), including complete responses (CR) / maintained complete responses (MCR) in 4 / 5 osteosarcoma models, 4 / 4 rhabdomyosarcoma models, 3 / 3 Ewing sarcoma models, 2 / 2 Wilms tumor models, and 6 / 11 neuroblastoma models. The duration of response continued beyond the last day of treatment (day 15), and most models that achieved CR did not show regrowth until day 56.

[0163] There was no clear relationship between CD276 mRNA expression measured by RNA-Seq and response to m276-PBD-SL, and CR and MCR were observed in models with CD276 expression ranging from 20 - 166 FPKM, while stable disease (SD) / partial response (PR) was observed at expression levels of 14 - 131 FPKM.

[0164] Furthermore, m276-PBD-SL was highly tolerable, as evidenced by a toxicity mortality of less than 2% and an average weight loss of 9.5%.

[0165] Considering the many possible embodiments to which the principles of the subject matter of this disclosure may be applied, it should be recognized that the described embodiments are only preferred examples of this disclosure and should not be construed as limiting the scope of this disclosure. Rather, the scope of this disclosure is defined by the appended claims. Accordingly, the applicants claim all that falls within the scope and spirit of those claims. The present invention provides, for example, the following items. (Item 1) An antibody-drug conjugate (ADC) comprising a drug conjugated to a monoclonal antibody that specifically binds to CD276, wherein the monoclonal antibody comprises a variable heavy chain (VH) domain, a variable light chain (VL) domain, and an IgG1 Fc region, wherein the VH domain comprises a complementarity-determining region 1 (CDR1) sequence, a CDR2 sequence, and a CDR3 sequence of SEQ ID NO: 2, the VL domain comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of SEQ ID NO: 6, and the Fc region comprises S239C, L234A, L235A, and P329G mutations according to the Eu numbering convention; and the drug is conjugated to cysteine at residue 239 of the Fc domain by site-specific conjugation, an antibody-drug conjugate. (Item 2) The ADC according to Item 1, wherein the CDR sequences are determined using the Kabat, IMGT, or Chothia numbering convention. (Item 3) The ADC according to Item 1 or Item 2, wherein the CDR1 sequence, CDR2 sequence, and CDR3 sequence of the VH domain comprise residues 26-33, 51-58, and 97-108 of SEQ ID NO: 2, respectively, and the CDR1 sequence, CDR2 sequence, and CDR3 sequence of the VL domain comprise residues 27-32, 50-52, and 89-99 of SEQ ID NO: 6, respectively. (Item 4) The ADC according to any one of Items 1 to 3, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 2 and the VL domain comprises the amino acid sequence of SEQ ID NO: 6. (Item 5) The ADC according to any one of Items 1 to 4, wherein the amino acid sequence of the Fc region comprises SEQ ID NO: 3. (Item 6) The ADC according to any one of Items 1 to 5, wherein the monoclonal antibody is IgG1. (Item 7) The ADC according to Item 6, wherein the amino acid sequence of the heavy chain of the IgG1 comprises SEQ ID NO: 4 or consists of SEQ ID NO: 4. (Item 8) The ADC according to Item 6, wherein the amino acid sequence of the light chain of the IgG1 comprises SEQ ID NO: 7 or consists of SEQ ID NO: 7. (Item 9) The ADC according to any one of Items 1 to 8, wherein the drug is a dimer of pyrrolobenzodiazepine (PBD). (Item 10) The ADC according to any one of items 1 to 9, wherein the drug is conjugated to the monoclonal antibody by a linker containing valine-alanine dipeptide. (Item 11) The ADC according to item 10, wherein the linker further contains a maleimide group. (Item 12) The ADC according to item 10 or item 11, wherein the linker further contains polyethylene glycol (PEG). (Item 13) A composition comprising the ADC according to any one of items 1 to 12 and a pharmaceutically acceptable carrier. (Item 14) A method for treating CD276-positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of the ADC according to any one of items 1 to 12 or the composition according to item 13, thereby treating the CD276-positive cancer in the subject. (Item 15) A method for inhibiting tumor growth or metastasis of CD276-positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of the ADC according to any one of items 1 to 12 or the composition according to item 13, thereby inhibiting tumor growth or metastasis of the CD276-positive cancer in the subject. (Item 16) The method according to item 14 or item 15, wherein the cancer is hepatocellular carcinoma, melanoma, leukemia, breast cancer, neuroblastoma, prostate cancer, colorectal cancer, osteosarcoma, endometrial cancer, ovarian cancer, oral squamous cell carcinoma, non-small cell lung cancer, bladder cancer, or pancreatic cancer. (Item 17) The method according to item 16, wherein the cancer is breast cancer or neuroblastoma. (Item 18) The method according to any one of items 14 to 17, further comprising administering an additional anti-cancer agent to the subject. (Item 19) The method according to item 18, wherein the additional anti-cancer agent comprises a chemotherapeutic agent or an angiogenesis inhibitor.

Claims

1. An antibody-drug conjugate (ADC) comprising a drug conjugated to a monoclonal antibody that specifically binds to CD276, the monoclonal antibody comprises a variable heavy (VH) domain, a variable light (VL) domain, and an IgG1 Fc region, wherein the VH domain comprises the complementarity determining region 1 (CDR1), CDR2, and CDR3 sequences of the amino acid sequence set forth in SEQ ID NO:2, the VL domain comprises the CDR1, CDR2, and CDR3 sequences of the amino acid sequence set forth in SEQ ID NO:6, and the Fc region comprises S239C, L234A, L235A, and P329G mutations according to the Eu numbering convention; and The drug is conjugated to the cysteine ​​at residue 239 of the Fc domain by site-specific conjugation. Antibody-drug conjugates.

2. 2. The ADC of claim 1, wherein the CDR sequences are determined using the Kabat, IMGT, or Chothia numbering conventions.

3. The ADC of claim 1 or claim 2, wherein the CDR1, CDR2, and CDR3 sequences of the VH domain comprise residues 26-33, 51-58, and 97-108, respectively, of the amino acid sequence set forth in SEQ ID NO:2, and the CDR1, CDR2, and CDR3 sequences of the VL domain comprise residues 27-32, 50-52, and 89-99, respectively, of the amino acid sequence set forth in SEQ ID NO:

6.

4. The ADC of any one of claims 1 to 3, wherein the VH domain comprises the amino acid sequence set forth in SEQ ID NO:2 and the VL domain comprises the amino acid sequence set forth in SEQ ID NO:

6.

5. The ADC according to any one of claims 1 to 4, wherein the amino acid sequence of the Fc region comprises the amino acid sequence shown in SEQ ID NO:

3.

6. The ADC of any one of claims 1 to 5, wherein the monoclonal antibody is IgG1.

7. 7. The ADC of claim 6, wherein the amino acid sequence of the IgG1 heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:

4.

8. 7. The ADC of claim 6, wherein the amino acid sequence of the light chain of IgG1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:

7.

9. 9. The ADC of any one of claims 1 to 8, wherein the drug is a dimer of pyrrolobenzodiazepine (PBD).

10. 10. The ADC of any one of claims 1 to 9, wherein the drug is conjugated to the monoclonal antibody by a linker comprising a valine-alanine dipeptide.

11. 11. The ADC of claim 10, wherein the linker further comprises a maleimide group.

12. 12. The ADC of claim 10 or claim 11, wherein the linker further comprises polyethylene glycol (PEG).

13. A composition comprising the ADC of any one of claims 1 to 12 and a pharma- ceutically acceptable carrier.

14. A composition comprising the ADC of any one of claims 1 to 12 or the composition of claim 13, for treating a CD276-positive cancer in a subject.

15. A composition comprising the ADC of any one of claims 1 to 12 or the composition of claim 13, for inhibiting tumor growth or metastasis of a CD276-positive cancer in a subject.

16. 16. The composition of claim 14 or claim 15, wherein the cancer is hepatocellular carcinoma, melanoma, leukemia, breast cancer, neuroblastoma, prostate cancer, colorectal cancer, osteosarcoma, endometrial cancer, ovarian cancer, oral squamous cell carcinoma, non-small cell lung cancer, bladder cancer, or pancreatic cancer.

17. The composition of claim 16, wherein the cancer is breast cancer or neuroblastoma.

18. The composition according to any one of claims 14 to 17, characterized in that it is administered in combination with an additional anticancer agent.

19. 20. The composition of claim 18, wherein the additional anti-cancer agent comprises a chemotherapeutic agent or an angiogenesis inhibitor.

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