High-affinity Nanobody Targeting B7H3 (CD276) for Treating Diverse Solid Tumors

Abstract

Single domain monoclonal antibodies that specifically bind B7H3 (also known as CD276) are described. The single domain antibodies are derived from camel V cells selected from a phage display library. H The present invention also describes single-domain antibodies and their conjugates that target B7H3, including H and rabbit VH domain nanobodies. Chimeric antigen receptors (CARs) and other antibody conjugates that target B7H3 are also described. Single-domain antibodies and their conjugates can be used for the diagnosis and treatment of solid tumors that express B7H3.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 924,298, filed Oct. 22, 2019, which is hereby incorporated by reference in its entirety.

[0002] Field The present disclosure relates to single - chain monoclonal antibodies that bind B7 homolog 3 (B7H3) with high affinity. The present disclosure further relates to the use of monoclonal antibodies and antibody conjugates, for example, for the treatment of solid tumors.

[0003] Approval of Government Support This invention was made with government support under Project No. Z01 BC010891 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background Art

[0004] Background Nanobodies are the smallest known antigen - binding fragments of antibodies, formed from approximately 120 amino acids with a molecular weight of 12 kD - 15 kD and a size of about 4 × 2.5 nm (Khodabakhsh et al., Int Rev Immunol, 37, 316 - 322, 2018). The most well - studied nanobodies are from camelids (V H H) and cartilaginous fish (V NAR) that can occur naturally (Feng et al., Antib Ther, 2, 1-11, 2019), and can also occur in some human heavy chain diseases in vivo (Prelli and Frangione, J Immunol, 148, 949-952, 1992), and are derived from the variable regions of heavy chains (VH). Due to their small size, high solubility, excellent thermal stability, reversible refolding ability, and relatively easy tissue penetration in vivo compared to conventional whole IgG, humanized camelid V H H, the first nanobody drug, caplacizumab (CABLIVI®), as demonstrated by its approval (Elverdi and Eskazan, Drug Des Devel Ther, 13, 1251-1258, 2019; Scully et al., N Engl J Med, 380, 335-346, 2019), nanobodies can be used in medical applications or as research tools (Khodabakhsh et al., Int Rev Immunol, 37, 316-322, 2018; Wesolowski et al., Med Microbiol Immunol, 198, 157-174, 2009; Ho, Antib Ther, 1, 1-5, 2018).

[0005] Historically, camelid V HH domain antibodies have been the mainstay of research until the emergence of human VH domain antibodies (Feng et al., Proc Natl Acad Sci U S A, 110, E1083-1091, 2013; Tang et al., Mol Cancer Ther, 12, 416-426, 2013; Li et al., Proc Natl Acad Sci U S A, 114, E6623-E6631, 2017). Camelid sera contain both conventional IgG and a substantial amount of IgG consisting only of heavy chains (HCAb) that account for 45% - 75% of total serum immunoglobulins depending on the species (Khodabakhsh et al., Int Rev Immunol, 37, 316-322, 2018). HCAb consists of a V H H fragment, a single antigen-binding domain, subsequent CH2 and CH3 domains, and a light chain that pairs with the VH-CH1 domain in conventional IgG. Recombinant V H H domains are the functional entities being exploited.

[0006] There are several structural features that make naturally evolved camelid V H H domains highly soluble and stable. First, Val37 (Kabat numbering) in the human VH germline is typically Phe37 (or Tyr37) in the V H H domain, which forms a more compact and stable hydrophobic packing of the domain (Riechmann and Muyldermans, J Immunol Methods, 231, 25-38, 1999), and this is likely the driving force for making V H H particularly stable (Shinozaki et al., J Biosci Bioeng, 125, 654-661, 2018). Second, the residues contacting the light chain in the human VH germline, G44, L45 and W47, are V HE44 (or Q44), R45 (or C45), and G47 (or Ser, Leu, Phe) in H (Holt et al., Trends Biotechnol, 21, 484 - 490, 2003), which make the accessible surface area more hydrophilic and reduce aggregability. Further, V H In a part of the H domain, W103 may be replaced by R103. Thirdly, V H The H domain usually has a CDR3 longer than the human / rodent CDR3, which typically contain Cys in CDR3 that form additional disulfide bonds with Cys at the end of CDR1 (camel) or the start site of CDR2 (llama) (Wesolowski et al., Med Microbiol Immunol, 198, 157 - 174, 2009). Further, this additional CDR3 disulfide bond and the standard C22 - C92 disulfide bond make the V H H domain more stable (T values in the range of 60 - 78 °C) and enable reversible unfolding / refolding (Holt et al., Trends Biotechnol, 21, 484 - 490, 2003). m Value), enabling reversible unfolding / refolding (Holt et al., Trends Biotechnol, 21, 484 - 490, 2003).

[0007] In 1989, the first non-V H H mammalian domain antibodies were screened from a cDNA expression library prepared from the spleens of mice immunized with lysozyme and keyhole - limpet hemocyanin, and two mouse VH domains showed affinity for lysozyme in the 20 nM range (Ward et al., Nature, 341, 544 - 546, 1989), and for the first time the name "single - domain antibody (dAb)" was suggested. Subsequently, camelid V HH was discovered, and the idea of investigating human VH domain antibodies that are more attractive in medicine and other applications grew. Both camelid domain antibodies and human domain antibodies have been widely studied, but there are some technical limitations in accessing the sources, especially regarding the discovery of domain antibodies by immunization. To overcome the limitations in access to the sources, several strategies have been developed to generate human or humanized VH domain antibodies. Phage display of naive human VH domain libraries is a proven method (Feng et al., Proc Natl Acad Sci U S A, 110, E1083-1091, 2013; Tang et al., Mol Cancer Ther, 12, 416-426, 2013; Li et al., Proc Natl Acad Sci U S A, 114, E6623-E6631, 2017), but the affinity is not always as high as that of immune antibodies. Another method for generating human domain antibodies is to use transgenic animals having human VH germline genes (Schusser et al., Eur J Immunol, 46, 2137-2148, 2016; Janssens et al., Proc Natl Acad Sci U S A, 103, 15130-15135, 2006). Instead of directly isolating human domain antibodies, generating VH domain antibodies in immunized animals and then humanizing them is also one way to generate human-like antibodies with high affinity.

Prior Art Documents

Non-Patent Documents

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Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Means for Solving the Problems

[0009] Overview The present disclosure describes 10 camel single-domain V H H monoclonal antibodies and 2 rabbit VH single-domain antibodies that specifically bind B7H3 (also known as CD276). As used herein, the B7H3-specific camel antibodies designated RWB12 (“B12”), RWG8 (“G8”), RWC4 (“C4”), RWB2, RWH5, RWD5, RWC3, RWG4, RWD9, and RWH1, and the rabbit antibodies designated RFA1 and RFB1 as used herein bind B7H3 with high affinity. Also disclosed is the generation of chimeric antigen receptor (CAR) T cells composed of the disclosed nanobodies.

[0010] Monoclonal antibodies that bind, e.g., specifically bind B7H3 are provided herein. In some embodiments, the monoclonal antibody comprises the complementarity determining region (CDR) sequence of the nanobody RWB12, RWG8, RWC4, RWB2, RWH5, RWD5, RWC3, RWG4, RWD9, RWH1, RFA1, or RFB1. Conjugates comprising the disclosed monoclonal antibodies are also provided herein. In some examples, CARs (as well as T cells and natural killer cells expressing the CAR) comprising the monoclonal antibodies disclosed herein, immunoconjugates (such as immunotoxins), bispecific antibodies (such as bispecific T-cell engagers), antibody-drug conjugates (ADCs), antibody-nanoparticle conjugates, antibody-radioisotope conjugates (for cases such as cancer diagnosis and immunoPET imaging), and fusion proteins are provided.

[0011] Compositions comprising a B7H3-specific monoclonal antibody and a pharmaceutically acceptable carrier are also provided by the present disclosure.

[0012] Nucleic acid molecules and vectors encoding the B7H3-specific monoclonal antibodies, CARs, immunoconjugates (such as immunotoxins), bispecific antibodies and fusion proteins disclosed herein are also provided herein. Isolated cells comprising a nucleic acid or vector encoding a B7H3 monoclonal antibody or CAR are further provided.

[0013] Methods of treating B7H3-positive cancer in a subject and methods of inhibiting tumor growth or metastasis of B7H3-positive cancer in a subject are also provided. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a monoclonal antibody disclosed herein, or administering to the subject a therapeutically effective amount of a CAR (or CAR T cell or CAR NK cell), immunoconjugate (such as an immunotoxin), ADC, bispecific antibody, antibody-nanoparticle conjugate or fusion protein comprising a monoclonal antibody disclosed herein.

[0014] Furthermore, methods of detecting the expression of B7H3 in a sample are provided herein. In some embodiments, the method comprises contacting the sample with a monoclonal antibody disclosed herein and detecting binding of the antibody to the sample.

[0015] Methods of diagnosing a subject as having B7H3-positive cancer are also provided. In some embodiments, the method comprises contacting a sample obtained from the subject with a monoclonal antibody disclosed herein and detecting binding of the antibody to the sample.

[0016] The foregoing and other objects and features of the present disclosure will become more apparent from the following detailed description taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017]

Figure 1

[0018] Figure 1B: SDS-PAGE analysis of purified B7H3-hFc. 1 or 5 micrograms of purified B7H3-hFc was separated on an 8% SDS-PAGE gel either non-reduced (Non.) or reduced with beta-mercaptoethanol (Red.). Protein bands were visualized by Coomassie blue R-250 staining.

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[0040] Sequence Listing The nucleic acid and amino acid sequences listed in the attached Sequence Listing are shown using standard letter abbreviations for nucleotide bases as defined in 37 C.F.R. 1.822, and the three-letter codes for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by reference to either strand shown. The Sequence Listing is submitted as a 27.8 KB ASCII text file created on October 19, 2020, which is incorporated herein by reference. In the attached Sequence Listing:

[0041] SEQ ID NO:1 is the amino acid sequence of the camel antibody RWB12.

[0042] SEQ ID NO:2 is the amino acid sequence of the camel antibody RWG8.

[0043] SEQ ID NO:3 is the amino acid sequence of the camel antibody RWC4.

[0044] SEQ ID NO:4 is the amino acid sequence of the camel antibody RWB2.

[0045] SEQ ID NO:5 is the amino acid sequence of the camel antibody RWH5.

[0046] SEQ ID NO:6 is the amino acid sequence of the camel antibody RWD5.

[0047] SEQ ID NO:7 is the amino acid sequence of the camel antibody RWC3.

[0048] SEQ ID NO:8 is the amino acid sequence of the camel antibody RWG4.

[0049] SEQ ID NO:9 is the amino acid sequence of the camel antibody RWD9.

[0050] SEQ ID NO:10 is the amino acid sequence of the camel antibody RWH1.

[0051] SEQ ID NO:11 is the amino acid sequence of the rabbit antibody RFA1.

[0052] SEQ ID NO: 12 is the amino acid sequence of rabbit antibody RFB1.

[0053] SEQ ID NO: 13 is the amino acid sequence of the extracellular domain of B7H3.

[0054] SEQ ID NOS: 14 to 26 are primer sequences.

[0055] SEQ ID NO: 27 is the amino acid sequence of GMCSFRss.

[0056] SEQ ID NO: 28 is the amino acid sequence of the CD8α hinge region.

[0057] SEQ ID NO: 29 is the amino acid sequence of the CD8α transmembrane region.

[0058] SEQ ID NO: 30 is the amino acid sequence of 4-1BB.

[0059] SEQ ID NO: 31 is the amino acid sequence of CD3ζ.

[0060] SEQ ID NO: 32 is the amino acid sequence of the self-cleaving T2A peptide.

[0061] SEQ ID NO: 33 is the amino acid sequence of huEGFRt.

[0062] SEQ ID NO: 34 is the amino acid sequence of rabbit VH domain antibody 5DUB.

[0063] SEQ ID NOS: 35 to 37 are the amino acid sequences of the B7H3 peptide.

[0064] Detailed Description I. Abbreviations ADC Antibody-Drug Conjugate ADCC Antibody-Dependent Cell-Mediated Cytotoxicity B7H3 B7 Homolog 3 BBIR Biotin-Binding Immunoreceptor CAR Chimeric Antigen Receptor CDR Complementary Determining Region CTL Cytotoxic T Lymphocyte ECD extracellular domain EGF epidermal growth factor EGFR epidermal growth factor receptor ELISA enzyme-linked immunosorbent assay EM effector moiety FACS fluorescence-activated cell sorting GMCSFRss granulocyte-macrophage colony-stimulating factor receptor signal sequence hFc human Fc huEGFRt human truncated epidermal growth factor receptor IC50 inhibitory concentration 50 Ig immunoglobulin KO knockout NK natural killer PBD pyrrolobenzodiazepine PE Pseudomonas exotoxin PET positron emission tomography TM transmembrane VH variable heavy VL variable light II. Terms and Methods

[0065] Unless otherwise noted, technical terms are used according to their conventional usage. Definitions of general terms in molecular biology can be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers in 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published in 16 volumes by Wiley-VCH in 2008; and other similar references.

[0066] As used herein, the singular forms "a", "an", and "the" refer to both the singular and the plural unless the context clearly dictates otherwise. For example, the term "an antigen" includes a single antigen as well as a plurality of antigens and can be considered equivalent to the phrase "at least one antigen". As used herein, the term "comprises" means "includes". It should be further understood that any and all base sizes or amino acid sizes, and all molecular weights or molecular mass values given for nucleic acids or polypeptides are approximate values and are provided for convenience. Many methods and materials similar to or equivalent to those described herein can be used, but particularly preferred methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various embodiments, the following explanations of terms are provided:

[0067] 4-1BB: A costimulatory molecule expressed by T cell receptor (TCR)-activated lymphocytes and by other cells including natural killer cells. Ligation of 4-1BB induces a signaling cascade that results in cytokine production, expression of anti-apoptotic molecules, and enhancement of the immune response. An exemplary amino acid sequence of 4-1BB is shown herein as SEQ ID NO: 30.

[0068] Administration: Providing or giving a drug, such as a monoclonal antibody, CAR, or cell expressing a CAR provided herein, to a subject by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraprostatic, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0069] Antibody: A polypeptide ligand that recognizes and binds to an epitope of an antigen (e.g., specifically recognizes and specifically binds). Mammalian immunoglobulin molecules are composed of heavy (H) and light (L) chains, each of which has variable regions, the variable heavy (V H ) region and the variable light (V L ) region, respectively. Together, the V H region and the V L region are responsible for the binding of the antigen recognized by the antibody. There are five major classifications (or isotypes) of heavy chains in mammalian immunoglobulins, which determine the functional activities of the antibody molecules: IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is the primary 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.

[0070] An antibody variable region contains "framework" regions and hypervariable regions known as "complementary determining regions" or "CDRs". CDRs are mainly responsible for binding to an epitope of an antigen. The framework region of an antibody serves to position and align the CDRs in three-dimensional space. The boundaries of the amino acid sequence of a given CDR can be readily determined using any of several well-known numbering schemes, including those described by 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; see 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 "Paratome CDRs"), and the ImMunoGeneTics (IMGT) database (Lefranc, Nucleic Acids Res 29:207-9, 2001; see the "IMGT" numbering scheme). The databases of Kabat, Paratome, and IMGT are maintained online.

[0071] "Single domain antibody" refers to an antibody having a single domain (variable domain) that is capable of specifically binding an antigen, or an epitope of an antigen, in the absence of additional antibody domains. Single domain antibodies include, for example, V H domain antibodies, V NAR antibodies, camelid V H H antibodies, and V LIt contains domain antibodies. V NAR Antibodies are produced by cartilaginous fish such as dogfish, wobbegong, spiny dogfish, and thresher shark. Camelidae V H H antibodies are produced by several species including camels, llamas, alpacas, human camels, and guanacos, which produce naturally light-chain-deficient heavy-chain antibodies.

[0072] A "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 are produced by methods known to those skilled in the art. Monoclonal antibodies include humanized monoclonal antibodies.

[0073] A "chimeric antibody" has framework residues from one species, such as a human, and CDRs (generally conferring antigen binding) from another species.

[0074] A "humanized" antibody is an immunoglobulin that contains one or more CDRs from a non-human (e.g., mouse, rabbit, rat, shark or synthetic) immunoglobulin and a human framework region. The non-human immunoglobulin providing the CDRs is called the "donor", and the human immunoglobulin providing the framework is called the "acceptor". In one embodiment, all CDRs are derived from the donor immunoglobulin of the humanized immunoglobulin. Constant regions need not be present, but if they are present, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85 - 90%, e.g., about 95% or higher identity. Thus, perhaps except for the CDRs, all parts of the humanized immunoglobulin 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 providing the CDRs. A humanized or other monoclonal antibody may have further conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions.

[0075] Antibody-drug conjugate (ADC): A molecule comprising an antibody (or an antigen-binding fragment of an antibody) conjugated to a drug such as a cytotoxic agent. Using an ADC, a drug can be specifically targeted to cancer cells by the specific binding of the antibody to a tumor antigen expressed on the cell surface. Exemplary drugs for use with an ADC include microtubule inhibitors (such as maytansinoids, auristatin E, and auristatin F) and interstrand crosslinking agents (e.g., pyrrolobenzodiazepine; PBD). In some cases, the ADC is a bispecific ADC and is composed of two monoclonal antibodies or antigen fragments thereof, each conjugated to a drug and directed against a different antigen or epitope. In one example, the agent conjugated to the antibody is IRDye® 700 DX (IR700, Li-cor, Lincoln, NE), which can then be used with near-infrared light NIR light to kill cancer cells to which the antibody binds (photoimmunotherapy; see, e.g., US 8,524,239 and 10,538,590). For example, an amino-reactive IR700 can be conjugated to an antibody by covalent bonding using the NHS ester of IR700.

[0076] Microtubule inhibitor: A class of drugs that block cell growth by arresting mitosis. Microtubule inhibitors, also referred to as "antimitotic agents," are used to treat cancer.

[0077] B7 homolog 3 (B7H3): An immune checkpoint molecule expressed by several types of solid tumors. This protein is a member of the co-stimulatory molecule of the B7 superfamily. B7H3 is also known as CD276.

[0078] B7H3-positive cancer: Cancer that expresses or overexpresses B7H3. Examples of B7H3-positive cancers include, but are not limited to, liver cancer (such as hepatocellular carcinoma), pancreatic cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer (such as neuroblastoma or glioblastoma), pediatric cancer (such as osteosarcoma, neuroblastoma, rhabdomyosarcoma or Ewing sarcoma), melanoma and mesothelioma (see, for example, Seaman et al., Cancer Cell 31(4):501-505, 2017).

[0079] Binding affinity: The affinity of an antibody for its antigen. In one embodiment, the affinity is calculated by a modification 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" an antigen (such as B7H3) is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens.

[0080] In some examples, a monoclonal antibody (such as the anti-B7H3 single domain antibodies provided herein) has at least 10 3 M -1 higher, 10 4 M -1 higher, or 10 5 M -1It binds specifically to a target (such as B7H3) with a high binding constant. In some examples, an antibody (e.g., a monoclonal antibody) has an equilibrium dissociation constant (Kd) of 1 μM or less, e.g., 900 nM or less, 500 nM or less, 250 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less. For example, a single-domain monoclonal antibody has a binding affinity of at least about 1×10 -6 M, at least about 0.5×10 -6 M, at least about 1×10 -7 M, at least about 0.5×10 -7 M, at least about 1×10 -8 M, at least about 0.5×10 -8 M, at least about 1×10 -9 M, at least about 0.5×10 -9 M, or at least about 0.1×10 -9 and binds to a target such as B7H3. In certain embodiments, the specific binding agent that binds to the target has a dissociation constant (Kd) of ≦1000 nM, ≦750 nM, 500 nM, ≦250 nM, ≦100 nM, ≦50 nM, ≦25 nM, ≦10 nM, ≦5 nM, ≦2.5 nM, ≦1 nM, ≦0.5 nM, ≦0.25 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -6 M or less, e.g., 10 -6 M to 10 -10 M, e.g., 10 -7 M to 10 -9 M). In some examples, the binding affinity is measured using an Octet system (Creative Biolabs) based on biolayer interferometry (BLI) technology. In some examples, the Kd is measured using a surface plasmon resonance assay using a BIACORES-2000 or BIACORES-3000 (BIAcore, Inc., Piscataway, N.J.).

[0081] Bispecific antibody: A recombinant protein that contains the antigen-binding fragments of two different monoclonal antibodies, thereby enabling the binding of two different antigens. In some embodiments, bispecific antibodies are used for cancer immunotherapy by simultaneously targeting, for example, CTLs (such as CTL receptor components like CD3) or effector natural killer (NK) cells and tumor antigens (such as B7H3). Similarly, multispecific antibodies are recombinant proteins that contain the antigen-binding fragments of at least two different monoclonal antibodies, such as two, three, or four different monoclonal antibodies.

[0082] Brain cancer or tumor: Types of cancer or tumor that originate from brain tissue. Brain cancers include, but are not limited to, neuroblastoma, medulloblastoma, glioma, glioblastoma, meningioma, pituitary adenoma, astrocytoma, choroid plexus carcinoma, ependymoma, and pineoblastoma.

[0083] Breast cancer: Types of cancer that form in breast tissue, usually the milk ducts and lobules. Types of breast cancer include, for example, non-invasive ductal carcinoma, invasive ductal carcinoma, triple-negative breast cancer, inflammatory breast cancer, metastatic breast cancer, medullary carcinoma, tubular carcinoma, and mucinous carcinoma. Triple-negative breast cancer refers to a type of breast cancer in which cancer cells do not express estrogen receptor, progesterone receptor, or significant levels of HER2 / neu protein. Triple-negative breast cancer is also called ER-negative PR-negative HER2 / neu-negative breast cancer.

[0084] Chemotherapeutic agent: Any chemical agent having therapeutic utility in the treatment of diseases characterized by abnormal cell growth. 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 used in treating B7H3-positive tumors. In one embodiment, the chemotherapeutic agent is a radioactive compound. One of ordinary skill in the art can readily identify the chemotherapeutic agent to be used (e.g., see 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 nd 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 for treating cancer. One example is the administration of an antibody that binds B7H3 used in combination with a radioactive or chemical compound. In one example, the chemotherapeutic agent is a biologic agent, such as a therapeutic antibody (e.g., a therapeutic monoclonal antibody) such as the anti-B7H3 antibody provided herein, and anti-PD1 or anti-PDL1 (e.g., pembrolizumab and nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-EGFR (e.g., cetuximab), anti-VEGF (e.g., bevacizumab), or combinations thereof (e.g., anti-PD-1 and anti-CTLA-4) and other anti-cancer antibodies.

[0085] Chimeric antigen receptor (CAR): A chimeric molecule comprising an antigen-binding portion (such as a scFv or single-domain antibody) and a signaling domain, for example, a signaling domain derived from a T cell receptor (such as CD3ζ). Typically, a CAR is composed of an antigen-binding portion, a transmembrane domain, and an endodomain. The endodomain typically includes a signaling chain having an immunoreceptor tyrosine-based activation motif (ITAM) such as CD3ζ or FcεRIγ. In some cases, the endodomain further includes the intracellular portion of at least one additional costimulatory domain such as CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, and / or DAP10. In some examples, the CAR is multispecific (bispecific, etc.) or bicistronic. A multispecific CAR is a single CAR molecule composed of at least two antigen-binding domains (such as scFvs and / or single-domain antibodies) that bind different antigens or different epitopes on the same antigen (see, for example, US2018 / 0230225). For example, a bispecific CAR refers to a single CAR molecule having two antigen-binding domains that bind different antigens. A bicistronic CAR refers to two complete CAR molecules each containing an antigen-binding portion that binds a different antigen. In some cases, a bicistronic CAR construct expresses two complete CAR molecules linked by a cleavage linker. T cells or NK cells expressing a bispecific or bicistronic CAR can bind cells that express both antigens to which the binding moieties are directed (see, for example, Qin et al., Blood 130:810, 2017; and WO / 2018 / 213337).

[0086] Colorectal cancer: A type of cancer that develops in the colon or rectum. The most common type of colorectal cancer (also known as "colorectal cancer") is colorectal adenocarcinoma, which accounts for approximately 95% of all colorectal cancers. Adenocarcinoma develops in the cells that line 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 Cajal interstitial cells), leiomyosarcomas (arising from smooth muscle cells), and colorectal melanomas.

[0087] Complementary determining region (CDR): A region of hypervariable amino acid sequences that define the binding affinity and specificity of an antibody. The light and heavy chains of mammalian immunoglobulins each have three CDRs, named 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.

[0088] Conjugate: In the context of the present disclosure, a "conjugate" is an antibody or antibody fragment (e.g., an antigen-binding fragment) covalently linked to an effector molecule or a second protein (such as a second antibody). The effector molecule can be, for example, a drug, toxin, therapeutic agent, detectable label, protein, nucleic acid, lipid, nanoparticle, photon absorber, carbohydrate, or recombinant virus. Antibody conjugates are often referred to as "immunoconjugates". When the conjugate includes an antibody linked to a drug (such as a cytotoxic agent), the conjugate is often referred to as an "antibody-drug conjugate" or "ADC". Other antibody conjugates include, for example, multispecific (bispecific or trispecific, etc.) antibodies and chimeric antigen receptors (CARs).

[0089] Conserved variants: Proteins containing conservative amino acid substitutions that substantially affect, but do not reduce, the affinity of a protein, such as an antibody against B7H3. For example, monoclonal antibodies that specifically bind B7H3 can contain up to about 1, up to about 2, up to about 5, and up to about 10, or up to about 15 conservative substitutions and can specifically bind the B7H3 polypeptide. The term "conserved variant" also includes the use of an amino acid substituted in place of an unsubstituted parent amino acid as long as the antibody specifically binds B7H3. Non-conservative substitutions are substitutions that reduce the activity or binding to B7H3.

[0090] Tables of conservative amino acid substitutions that provide functionally similar amino acids are well known to those of skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 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).

[0091] Contact: To place in a direct physical association state; including both solid and liquid forms.

[0092] Cytotoxic agent: Any drug or compound that kills cells.

[0093] Cytotoxicity: The toxicity of a molecule, such as an immunotoxin, to a cell that is intended to be targeted, as contrasted with the cells of the rest of the organism. In contrast, the term "toxicity" refers to the toxicity of an immunotoxin to cells other than those that are intended to be targeted by the targeting portion 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 other than those that are intended to be targeted by the immunotoxin.

[0094] Degenerate variant: A polynucleotide that encodes a polypeptide that includes a degenerate sequence as a result of the genetic code. Twenty natural amino acids exist, and most of them are specified by more than one codon. Thus, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide is not changed.

[0095] Diagnosis: Identifying the presence or nature of a condition, such as a B7H3-positive cancer. Diagnostic methods vary in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of affected individuals who test positive in the test (percent true positives). The "specificity" of a diagnostic assay is one minus the false positive rate, where the false positive rate is defined as the proportion of those without the disease who test positive. A particular diagnostic method may be sufficient if it provides a positive indicator for diagnosis, even if it cannot provide a definitive diagnosis of the condition. "Prognosis" is the probability of the development of a condition, such as cancer (e.g., severity).

[0096] Diagnostic tumor imaging: Coupling antibodies and their derivatives having positron-emitting radionuclides for positron emission tomography (PET) is a process often referred to as immunoPET. Good immunoPET agents can be made with full-length antibodies, but due to their biological half-lives, it is necessary to wait several days before imaging, resulting in an increase in non-target radiation dose. Smaller single-domain antibodies, or nanobodies, have biological half-lives suitable for same-day imaging.

[0097] Drug: Any compound used to treat, alleviate, 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., an anti-mitotic agent or a microtubule inhibitor.

[0098] Effector molecule: The portion of a chimeric molecule that is intended to have a desired effect on the cell targeted by the chimeric molecule. Effector molecules are also known as effector moieties (EM), therapeutic agents, diagnostic agents, or similar terms. Therapeutic agents (or drugs) include compounds such as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, photon absorbers, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking to single-stranded or double-stranded DNA, and triple helix-forming oligonucleotides. Alternatively, a molecule linked to a targeting moiety such as an anti-B7H3 antibody may be a liposome or micelle or other encapsulation system containing a therapeutic composition such as a drug, a nucleic acid (such as an antisense nucleic acid), or another therapeutic moiety that can be shielded from direct exposure to the circulatory system. Means for preparing liposomes conjugated to antibodies are well known to those skilled in the art (see, e.g., U.S. Patent No. 4,957,735; and Connor et al., Pharm Ther 28:341-365, 1985). Diagnostic agents or moieties include radioisotopes and other detectable labels. Detectable labels useful for such purposes are also well known in the art, 35 S, 11 C, 13 N, 15 O, 18 F, 19 F, 99m Tc, 131 I, 3 H, 14 C, 15 N, 90 Y, 99 Tc, 111 In and 125 radioisotopes such as I, fluorophores, chemiluminescent agents, and enzymes.

[0099] Epitope: An antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic (induce a specific immune response). Antibodies specifically bind to specific antigenic epitopes on polypeptides such as B7H3.

[0100] Framework region: An amino acid sequence inserted between CDRs. The framework regions of an immunoglobulin molecule include variable light and variable heavy framework regions.

[0101] Fusion protein: A protein that contains at least a portion of two different (heterologous) proteins.

[0102] Heterologous: Derived from a distinct genetic source or species.

[0103] Immune response: The response of cells of the immune system, such as B cells, T cells, or monocytes, to a stimulus. In one embodiment, the response is specific to a particular antigen ("antigen-specific response"). In one embodiment, the immune response is a T cell response such as a CD4 + response or a CD8 + response. In another embodiment, the response is a B cell response, resulting in the production of specific antibodies.

[0104] Immunoconjugate: linkage by covalent attachment of an effector molecule to an antibody or a functional fragment thereof. The effector molecule may be, for example, a detectable label, a photon absorber (such as IR700), or a toxin (such as for forming immunotoxins including Pseudomonas exotoxin or its variants). Specific non-limiting examples of toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, or modified toxins thereof, or other toxic agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that typically cause death by hepatotoxicity. However, PE and DT can be modified into a form for use as immunotoxins by removing their native targeting components (such as domain Ia of PE and the B chain of DT) and replacing them with different targeting moieties, such as antibodies. In one embodiment, an antibody is conjugated to an effector molecule. In another embodiment, the antibody conjugated to an effector molecule is further conjugated to a lipid or other molecule, for example, to increase its half-life in vivo. The conjugation may be by chemical means or by recombinant means. In one embodiment, the conjugation is chemical, and the reaction between the antibody moiety and the effector molecule results in a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the effector molecule. Since immunoconjugates are originally prepared from two molecules having separate functional groups such as an antibody and an effector molecule, they may also be referred to as "chimeric molecules". Thus, the term "chimeric molecule" as used herein refers to a targeting moiety such as a ligand or an antibody conjugated (coupled) to an effector molecule. The terms "conjugated" or "linked" refer to making two polypeptides into one continuous polypeptide molecule.

[0105] Immunoliposome: A liposome having an antibody or antibody fragment conjugated to its surface. The immunoliposome may have a cytotoxic agent or other drug for cells targeted by an antibody, such as tumor cells.

[0106] Interstrand crosslinking agent: A type of cytotoxic drug capable of covalently binding between two strands of DNA, thereby preventing DNA replication and / or transcription.

[0107] Isolated: An "isolated" biological component such as a nucleic acid, protein (including an antibody), or organelle is substantially separated or purified from other biological components in the environment (such as a cell) in which the component naturally occurs, for example, other chromosomes and extrachromosomal DNA and RNA, proteins, and organelles. "Isolated" nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0108] Label: A detectable compound or composition conjugated directly or indirectly to a molecule, such as an antibody or another protein, to facilitate the detection of the molecule. Specific non-limiting examples of labels include fluorescent tags, enzyme conjugates, and radioisotopes. In one example, a "labeled antibody" refers to the incorporation of another molecule into the antibody. For example, the label can be the incorporation of a radiolabeled amino acid, or the binding of a biotinyl moiety to a polypeptide that can be detected by a marked avidin (such as streptavidin containing a fluorescent marker or enzyme activity that can be detected by optical or colorimetric methods). Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radioactive nucleotides (e.g., 35 S, 11 C, 13 N, 15 O, 18 F,19 F, 99m Tc, 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 phosphors), enzyme labels (e.g., horseradish peroxidase, beta - galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pairing sequences, binding sites for secondary antibodies, metal - binding domains, epitope tags), or magnetic agents such as gadolinium chelates. In some embodiments, the label is attached by spacer arms of various lengths to reduce potential steric hindrance.

[0109] Linker: In some cases, the linker is a peptide within an antibody - binding fragment (such as an 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 such as an antibody to an effector molecule such as a cytotoxin or a detectable label. The terms "conjugating," "joining," "bonding," or "linking" refer to making two polypeptides into one continuous polypeptide molecule or attaching a radionuclide or other molecule to a polypeptide such as an antibody by covalent attachment. The linkage can be by chemical means or by recombinant means. "Chemical means" refers to a reaction between an antibody moiety and an effector molecule such that a covalent bond is formed between the two molecules to form one molecule.

[0110] Liver cancer: Any type of cancer that occurs in liver tissue. The most common type of liver cancer is hepatocellular carcinoma (HCC), which develops within liver cells. Other types of liver cancer include cholangiocarcinoma, which develops in the bile ducts; hepatic angiosarcoma, a rare form of liver cancer that begins in the blood vessels of the liver; and hepatoblastoma, a very rare type of liver cancer that is most often seen in children.

[0111] Lung cancer: Any cancer that forms in the lungs. Most cancers that begin 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.

[0112] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, when necessary to join two protein-coding regions, are in the same reading frame.

[0113] Ovarian cancer: Cancer that forms in the tissues of the ovaries. Most ovarian cancers are either ovarian epithelial carcinomas (cancers that begin in the cells on the surface of the ovaries) or malignant germ cell tumors (cancers that begin in the egg cells). Another type of ovarian cancer is stromal cell cancer, which originates from cells that release hormones and are connected to various structures of the ovaries.

[0114] Pancreatic cancer: A disease in which malignant cells are found in the tissues of the pancreas. Pancreatic tumors can be either exocrine tumors or neuroendocrine tumors, based on the cell origin of the cancer. The majority (about 94%) of pancreatic cancers are exocrine tumors. Examples of exocrine cancers include, for example, adenocarcinoma (the most common type of exocrine tumor), acinar cell carcinoma, intraductal papillary mucinous neoplasm (IPMN), and mucinous cystadenocarcinoma. In some examples, pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). Pancreatic neuroendocrine tumors, also referred to as islet cell tumors, are classified by the type of hormone they produce. Exemplary neuroendocrine tumors include gastrin-producing tumors, glucagon-producing tumors (glucaganoma), insulin-producing tumors, somatostatin-producing tumors, VIP-producing tumors (vasoactive intestinal peptide), and non-functional islet cell tumors.

[0115] Pediatric cancer: Cancer that develops 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 CNS tumors (such as neuroblastoma and medulloblastoma), Wilms tumor, non-Hodgkin lymphoma, and retinoblastoma.

[0116] Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers used are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 stEdition (2005) describes compositions and formulations suitable for pharmaceutical delivery of the antibodies and other compositions disclosed herein. Generally, the nature of the carrier depends on the particular mode of administration employed. For example, parenteral formulations typically include injectable fluids containing pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, etc. as a vehicle. Solid compositions (such as in powder, pill, tablet, or capsule form) can include conventional non-toxic solid carriers such as, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate.

[0117] Photoimmunotherapy: A targeted cancer therapy that utilizes an antigen-specific antibody-photoabsorber conjugate that can be activated by near-infrared light to kill targeted cells. The photoabsorber is typically based on a phthalocyanine dye such as a near-infrared (NIR) phthalocyanine dye (e.g., IRDye® 700DX, also known as IR700). The antibody (e.g., a B7H3-specific antibody) binds to an appropriate cell surface antigen (e.g., B7H3), and the photoactivatable dye induces lethal damage to the cell membrane upon exposure to NIR light. NIR light exposure (e.g., 690 nm) induces highly selective, necrotic cancer cell death within minutes without damaging adjacent cells (see, for example, U.S. Application No. 2018 / 0236076).

[0118] Preventing, treating, or alleviating a disease: "Preventing" a disease refers to inhibiting the full development of the disease. "Treating" refers to therapeutic intervention that alleviates the signs or symptoms of a disease or condition after the disease or condition has begun to develop, such as reducing the tumor burden or the number or size of metastases. "Alleviating" refers to reducing the number or severity of the signs or symptoms of a disease such as cancer.

[0119] Purified: The term "purified" does not require absolute purity, but rather is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is enriched relative to where the peptide or protein exists 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 indicates 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 specific non-limiting example, a substantially purified protein is free of 90% of other proteins or cellular components.

[0120] Pyrrolobenzodiazepine (PBD): A classification of sequence-selective DNA minor groove-binding cross-linking agents originally discovered in Streptomyces species. PBDs are significantly more potent than synthetic chemotherapeutic agents. The mechanism of action of PBDs is related to their ability to form adducts in the minor groove of DNA, thereby interfering with DNA processing. In the context of the present disclosure, PBDs include naturally produced and isolated PBDs, chemically synthesized naturally occurring PBDs, and chemically synthesized non-naturally occurring PBDs. PBDs also include monomeric, dimeric and hybrid PBDs (see Gerratana, Med Res Rev 32(2):254-293, 2012 for review).

[0121] Recombinant: A recombinant nucleic acid or protein is one that has a non-naturally occurring sequence or a sequence created by the artificial combination of two otherwise separated segments of a sequence. This artificial combination is often achieved by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.

[0122] Sample (or biological sample): A biological specimen containing genomic DNA, RNA (including mRNA), protein, or a combination thereof obtained from a subject. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, tissue biopsy, fine needle aspirate, surgical specimen, and autopsy material. In one example, the sample includes a tumor biopsy.

[0123] Sequence identity: The similarity between amino acid sequences or nucleic acid sequences is expressed in terms of the similarity between the sequences and is alternatively referred to as sequence identity. Sequence identity is often measured in terms of the percentage identity (or similarity or homology), and the higher the percentage, the higher the similarity between the two sequences. Homologs or variants of a polypeptide or nucleic acid molecule have a relatively high degree of sequence identity when aligned using standard methods.

[0124] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described below: 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 the calculation of homology.

[0125] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. An explanation of how to use this program to determine sequence identity is available on the NCBI web site on the Internet.

[0126] Antibody homologs and variants that specifically bind a B7H3 polypeptide are typically characterized by occupancy of sequence identity of at least about 75%, such as at least about 80%, 90%, 95%, 96%, 97%, 98% or 99%, counted against the full-length alignment with the amino acid sequence of the antibody, using NCBI Blast 2.0, which is gapped blastp set to default parameters. For comparisons of amino acid sequences of more than about 30 amino acids, the Blast 2 sequence function is used with the default BLOSUM62 matrix set to default parameters (gap existence cost 11, and gap cost per residue 1). When aligning short peptides (less than approximately 30 amino acids), the alignment should be performed using the Blast 2 sequence function with the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalty). Proteins having even higher similarity to the reference sequence will show an increase in percentage 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 evaluated by this method. When comparing sequences less than the full sequence 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 their similarity to their reference sequences. Methods for determining sequence identity for such short windows are available on the NCBI website on the Internet. Those skilled in the art will understand that these ranges of sequence identity are provided for guidance only, and that it is quite possible to obtain very important homologs outside the provided ranges.

[0127] Small molecule: A molecule typically having a molecular weight of less than about 1000 daltons, or in some embodiments less than about 500 daltons, that is capable of modulating, to some measurable extent, the activity of a target molecule.

[0128] Subject: Living multicellular vertebrates in a category that includes both human and veterinary subjects, including humans and non-human mammals.

[0129] Synthetic: Generated by artificial means in a laboratory; for example, synthetic nucleic acids or proteins (e.g., antibodies) can be chemically synthesized in a laboratory.

[0130] Therapeutically effective amount: The amount of a particular substance sufficient to achieve a desired effect in the subject being treated. For example, this may be the amount necessary to inhibit or suppress tumor growth. In one embodiment, a therapeutically effective amount is an amount that eliminates a tumor, reduces the size of a tumor, or prevents metastasis of a tumor, e.g., reduces tumor size and / or volume by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even up to 100%, and / or reduces the number and / or size / volume of metastases by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even up to 100% compared to the size / volume / number prior to treatment. When administered to a subject, a dosage that achieves a target tissue concentration (e.g., in a tumor) shown to achieve the desired in vitro effect is generally used.

[0131] Toxin: A molecule that is cytotoxic to cells. Toxins include abrin, ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinum toxin, saporin, restrictocin or gelonin, or modified toxins thereof. For example, PE and DT are highly toxic compounds that typically cause death by hepatotoxicity. However, PE and DT can be modified into a form for use as an immunotoxin by removing the native target component of the toxin (such as domain Ia of PE or the B chain of DT) and replacing it with a different targeting moiety such as an antibody.

[0132] Vector: A nucleic acid molecule that is introduced into a host cell and thereby generates a transformed host cell. The vector may contain nucleic acid sequences that enable replication in the host cell, such as an origin of replication. The vector may also contain one or more selectable marker genes and other genetic elements known in the art. In some embodiments, the vector is a viral vector, such as a lentiviral vector. III. B7H3-specific single-domain monoclonal antibody ( "nanobody")

[0133] Nanobodies include camelid V H H, chondrichthyan V NAR , and human VH single-domain antibodies. Rabbit monoclonal antibodies can recognize a diverse range of epitopes, including those with low immunogenicity in mice and humans. This disclosure describes the immunization of rabbits with recombinant B7H3 protein and the generation of a phage-displayed VH single-domain library. After three rounds of phage panning, two binders (designated RFA1 and RFB1) were selected. Both binders were expressed well in E. coli, with yields of 2 mg / L (RFA1) and 10 mg / L (RFB1). The rabbit nanobodies showed antigen-dependent binding to B7H3-positive tumor cell lines (IMR32, MC38-B7H3+, A431, and NBEB), but not to B7H3 knockout cell lines (IMR32-B7H3 KO, MC38-B7H3 KO). This disclosure also describes 10 B7H3-specific camelid V H H nanobodies isolated from 8 different camelid V H H libraries. The selected nanobodies are capable of binding to B7H3-expressing cells, such as neuroblastoma cells, epidermoid carcinoma cells, and pancreatic tumor cells.

[0134] The amino acid sequences of 10 camel single-domain antibodies and 2 rabbit single-domain antibodies are provided below. The CDR sequences determined using the Kabat, IMGT, and Paratome methods are indicated by underlining, boldface, and italics, respectively. The table lists the amino acid positions of CDR1, CDR2, and CDR3 of each antibody determined using either Kabat, IMGT, or Paratome. One skilled in the art can readily determine the CDR boundaries using alternative numbering schemes such as the Chothia numbering scheme.

[0135] RWB12 (SEQ ID NO: 1)

Chemical formula

Table A-1

[0136] RWG8 (SEQ ID NO: 2)

Chemical formula

Table A-2

[0137] RWC4 (SEQ ID NO: 3)

Chemical formula

Table A-3

[0138] RWB2 (SEQ ID NO: 4)

Chemical formula

Table A-4

[0139] RWH5 (SEQ ID NO: 5) [Chemical] [Table A-5]

[0140] RWD5 (SEQ ID NO: 6) [Chemical] [Table A-6]

[0141] RWC3 (SEQ ID NO: 7) [Chemical] [Table A-7]

[0142] RWG4 (SEQ ID NO: 8) [Chemical] [Table A-8]

[0143] RWD9 (SEQ ID NO: 9) [Chemical] [Table A-9]

[0144] RWH1 (SEQ ID NO: 10) [Chemical] [Table A-10]

[0145] RFA1 (Accession No. 11)

Chem.

Table A-11

[0146] RFB1 (Accession No. 12)

Chem.

Table A-12

[0147] Monoclonal antibodies that bind (e.g., specifically bind) to B7H3, such as cell surface or soluble B7H3, are provided herein. In some embodiments, the monoclonal antibody is a single domain antibody, such as a VH single domain antibody.

[0148] In some embodiments, the single-domain monoclonal antibody comprises at least a portion of the amino acid sequence as set forth herein as any one of the CDR sequences of any one of antibody RWB12 (SEQ ID NO: 1), RWG8 (SEQ ID NO: 2), RWC4 (SEQ ID NO: 3), RWB2 (SEQ ID NO: 4), RWH5 (SEQ ID NO: 5), RWD5 (SEQ ID NO: 6), RWC3 (SEQ ID NO: 7), RWG4 (SEQ ID NO: 8), RWD9 (SEQ ID NO: 9), RWH1 (SEQ ID NO: 10), RFA1 (SEQ ID NO: 11) and RFB1 (SEQ ID NO: 12), determined by any numbering scheme such as IMGT, Kabat, Paratome or Chothia, or any combination thereof. In some examples, the single-domain antibody comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In certain examples, the CDR sequences are determined using the Kabat, IMGT or Paratome numbering scheme, or a combination of the Kabat, IMGT and Paratome numbering schemes.

[0149] In some embodiments, the CDR1, CDR2 and CD3 sequences of the antibody comprise residues 31-35, 50-66 and 97-118 of SEQ ID NO: 1; residues 26-33, 51-58 and 97-119 of SEQ ID NO: 1; or residues 27-35, 47-62 and 98-118 of SEQ ID NO: 1, respectively. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1. In a specific example, the amino acid sequence of the antibody comprises or consists of SEQ ID NO: 1.

[0150] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the antibody comprise, respectively, residues 31-35, 50-66, and 97-105 of SEQ ID NO: 2; residues 26-33, 51-58, and 97-106 of SEQ ID NO: 2; or residues 27-35, 47-61, and 97-106 of SEQ ID NO: 2. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. In a specific example, the amino acid sequence of the antibody comprises or consists of SEQ ID NO: 2.

[0151] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the antibody comprise, respectively, residues 31-35, 50-66, and 97-114 of SEQ ID NO: 3; residues 26-33, 51-58, and 97-115 of SEQ ID NO: 3; or residues 26-35, 50-61, and 98-114 of SEQ ID NO: 3. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3. In a specific example, the amino acid sequence of the antibody comprises or consists of SEQ ID NO: 3.

[0152] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the antibody comprise, respectively, residues 31-35, 50-65, and 96-110 of SEQ ID NO: 4; residues 26-33, 51-57, and 96-110 of SEQ ID NO: 4; or residues 27-35, 47-60, and 96-109 of SEQ ID NO: 4. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In a specific example, the amino acid sequence of the antibody comprises or consists of SEQ ID NO: 4.

[0153] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the antibody comprise, respectively, residues 27-30, 45-61, and 90-109 of SEQ ID NO: 5; residues 26-28, 46-53, and 90-110 of SEQ ID NO: 5; or residues 27-30, 43-55, and 90-110 of SEQ ID NO: 5. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5. In a specific example, the amino acid sequence of the antibody comprises or consists of SEQ ID NO: 5.

[0154] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the antibody comprise, respectively, residues 31-35, 50-65, and 96-111 of SEQ ID NO: 6; residues 26-33, 51-57, and 96-112 of SEQ ID NO: 6; or residues 27-35, 47-60, and 96-111 of SEQ ID NO: 6. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6. In a specific example, the amino acid sequence of the antibody comprises or consists of SEQ ID NO: 6.

[0155] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the antibody comprise, respectively, residues 31-35, 50-65, and 96-111 of SEQ ID NO: 7; residues 26-33, 51-57, and 96-112 of SEQ ID NO: 7; or residues 27-35, 47-60, and 96-111 of SEQ ID NO: 7. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7. In a specific example, the amino acid sequence of the antibody comprises or consists of SEQ ID NO: 7.

[0156] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the antibody comprise residues 31-35, 50-65, and 96-111 of SEQ ID NO: 8; residues 26-33, 51-57, and 96-112 of SEQ ID NO: 8; or residues 27-35, 47-60, and 96-111 of SEQ ID NO: 8, respectively. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8. In a specific example, the amino acid sequence of the antibody comprises or consists of SEQ ID NO: 8.

[0157] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the antibody comprise residues 31-35, 50-65, and 96-111 of SEQ ID NO: 9; residues 26-33, 51-57, and 96-112 of SEQ ID NO: 9; or residues 27-35, 47-60, and 96-111 of SEQ ID NO: 9, respectively. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9. In a specific example, the amino acid sequence of the antibody comprises or consists of SEQ ID NO: 9.

[0158] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the antibody comprise residues 31-35, 50-65, and 96-113 of SEQ ID NO: 10; residues 26-33, 51-57, and 96-114 of SEQ ID NO: 10; or residues 27-35, 47-60, and 97-114 of SEQ ID NO: 10, respectively. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10. In a specific example, the amino acid sequence of the antibody comprises or consists of SEQ ID NO: 10.

[0159] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the antibody comprise, respectively, residues 30-34, 50-64, and 93-105 of SEQ ID NO: 11; residues 25-32, 50-56, and 93-104 of SEQ ID NO: 11; or residues 26-34, 46-59, and 93-105 of SEQ ID NO: 11. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 11 or residues 1-113 of SEQ ID NO: 11. In a specific example, the amino acid sequence of the antibody comprises or consists of SEQ ID NO: 11. In another specific example, the amino acid sequence of the antibody comprises or consists of residues 1-113 of SEQ ID NO: 11.

[0160] In some embodiments, the CDR1, CDR2, and CDR3 sequences of the antibody comprise, respectively, residues 32-35, 51-65, and 94-107 of SEQ ID NO: 12; residues 26-33, 51-57, and 94-107 of SEQ ID NO: 12; or residues 27-35, 47-60, and 94-108 of SEQ ID NO: 12. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 12 or residues 1-116 of SEQ ID NO: 12. In a specific example, the amino acid sequence of the antibody comprises or consists of SEQ ID NO: 12. In another specific example, the amino acid sequence of the antibody comprises or consists of residues 1-116 of SEQ ID NO: 12.

[0161] In some embodiments, the antibody is a humanized antibody or a chimeric antibody.

[0162] Chimeric antigen receptors (CARs) comprising the single domain monoclonal antibodies disclosed herein are also provided herein. In some embodiments, the CAR further comprises a hinge region, a transmembrane domain, a co-stimulatory signaling moiety, a signaling domain, or any combination thereof. In specific non-limiting examples, the hinge region comprises a CD8α hinge region, the transmembrane domain comprises a CD8α transmembrane domain, the co-stimulatory signaling moiety comprises a 4-1BB signaling moiety, and / or the signaling domain comprises a CD3ζ signaling domain.

[0163] Also provided herein are B7H3-specific antibodies modified to enable their use in universal CAR systems. In some embodiments, the B7H3-specific antibody is fused to one component of a specific binding pair. In some examples, the antibody is fused to a leucine zipper or biotin.

[0164] Cells expressing a B7H3-specific CAR are further provided. In some examples, the cells are T lymphocytes, such as CTLs, or natural killer cells. The CARs and CAR-expressing cells are further described in Item IV.

[0165] Also provided herein are immunoconjugates comprising the single domain antibodies and effector molecules disclosed herein. In some embodiments, the effector molecule is a toxin such as, but not limited to, Pseudomonas exotoxin or a variant thereof, such as PE38. In other embodiments, the effector molecule is a detectable label such as, but not limited to, a fluorophore, an enzyme, or a radioisotope. In other embodiments, the effector molecule is a photon absorber such as IR700. Immunoconjugates comprising a photon absorber can be used for photoimmunotherapy. The immunoconjugates are further described in Item V.

[0166] Also provided herein are antibody-drug conjugates (ADCs) comprising a drug conjugated to a single domain antibody disclosed herein. In some embodiments, the drug is a small molecule, such as a microtubule inhibitor, an anti-mitotic agent, and / or a cytotoxic agent. The ADCs are further described in Item VI.

[0167] Also provided herein are multispecific antibodies comprising a single domain antibody disclosed herein and at least one additional monoclonal antibody or antigen-binding fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody. In other embodiments, the multispecific antibody is a trispecific antibody. In some embodiments, the at least one additional monoclonal antibody or antigen-binding fragment thereof specifically binds a component of a T cell receptor or a natural killer (NK) cell activating receptor. The multispecific antibodies are further described in Item VII.

[0168] Also provided herein are antibody-nanoparticle conjugates comprising a nanoparticle conjugated to a single domain antibody disclosed herein. In some embodiments, the nanoparticle comprises a polymeric nanoparticle, a nanosphere, a nanocapsule, a liposome, a dendrimer, a polymeric micelle, or a niosome. In some embodiments, the nanoparticle comprises a cytotoxic agent. The antibody-nanoparticle conjugates are further described in Item VIII.

[0169] Also provided herein are fusion proteins comprising a single domain antibody disclosed herein and a heterologous protein or peptide. In some embodiments, the heterologous protein is an Fc protein or a leucine zipper.

[0170] Also provided herein are nucleic acid molecules encoding the antibodies, CARs, immunoconjugates, bispecific antibodies or fusion proteins disclosed herein. In some embodiments, the nucleic acid molecule is operably linked to a promoter. Vectors comprising the disclosed nucleic acid molecules are also provided. Isolated cells comprising the nucleic acid molecules or vectors disclosed herein are further provided.

[0171] Nucleic acid constructs expressing CAR and truncated human EGFR (huEGFRt) are also provided herein. In some embodiments, the nucleic acid, in the 5' to 3' direction, comprises: a nucleic acid encoding a first granulocyte-macrophage colony-stimulating factor receptor signal sequence (GMCSFRss); a nucleic acid encoding a B7H3-specific single-domain monoclonal antibody disclosed herein; a nucleic acid encoding an extracellular hinge region; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding an intracellular co-stimulatory domain; a nucleic acid encoding an intracellular signaling domain; a nucleic acid encoding a self-cleaving 2A peptide; a nucleic acid encoding a second GMCSFRss; and a nucleic acid encoding truncated human epidermal growth factor receptor (huEGFRt). In some examples, the nucleic acid further comprises a human elongation factor 1α (EF1α) promoter sequence 5' to the nucleic acid encoding the first GMCSFRss. In some examples, the hinge region comprises a CD8α hinge region. In some examples, the transmembrane domain comprises a CD8α transmembrane domain. In some examples, the co-stimulatory signaling moiety comprises a 4-1BB signaling moiety. In some examples, the signaling domain comprises a CD3ζ signaling domain. In some examples, the amino acid sequence of the B7H3-specific antibody comprises any one of SEQ ID NOs: 1-12. Vectors comprising the nucleic acid construct are also provided. In some embodiments, the vector is a lentiviral vector.

[0172] Also provided herein are isolated cells co-expressing the B7H3-specific CAR and huEGFRt disclosed herein. In some examples, the cells are CTL or NK cells.

[0173] Pharmaceutically acceptable carriers, and compositions comprising the single domain monoclonal antibodies, CARs, isolated cells (cells expressing a CAR, such as CAR T cells or CAR NK cells, etc.), immunoconjugates, ADCs, multispecific antibodies, antibody-nanoparticle conjugates, or fusion proteins disclosed herein are further provided by the present disclosure. The compositions and their uses are further described in item IX. IV. Chimeric Antigen Receptor (CAR)

[0174] The disclosed nanobodies can also be used to generate CARs (also known as chimeric T cell receptors, artificial T cell receptors or chimeric immune receptors) engineered to express a CAR and / or cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells. Generally, a CAR comprises a binding portion, an extracellular hinge and spacer element, a transmembrane region and an endodomain that performs a signaling function (Cartellieri et al., J Biomed Biotechnol 2010:956304, 2010; Dai et al., J Natl Cancer Inst 108(7):djv439, 2016). In many cases, the binding portion is a monoclonal antibody such as an scFv, or an antigen-binding fragment of a single domain antibody. The spacer / hinge region typically comprises sequences derived from IgG subclasses such as IgG1, IgG4, IgD and CD8 domains. The transmembrane domain can be derived from a variety of different T cell proteins, such as CD3ζ, CD4, CD8 or CD28. Several different endodomains have been used to generate CARs. For example, the endodomain may consist of a signaling chain having an ITAM such as CD3ζ or FcεRIγ. In some cases, the endodomain further comprises the intracellular portion of at least one additional co-stimulatory domain, such as CD28, 4-1BB (CD137, TNFRSF9), OX-40 (CD134), ICOS, CD27 and / or DAP10.

[0175] CTLs, NK cells (or other immune cells) that express CARs can be used to target specific cell types such as B7H3-positive tumor cells. Thus, using the nanobodies disclosed herein, CTLs or NK cells that express a CAR containing a B7H3-specific monoclonal antibody can be engineered, whereby the engineered CTLs or NK cells can target tumor cells that express B7H3. Engineered T cells have been previously used for adoptive therapy for several types of cancer (see, e.g., Park et al., Mol Ther 15(4):825-833, 2007). The use of T cells that express CARs is more common than standard CTL-based immunotherapies because CAR-expressing CTLs are not restricted by HLA and can thus be used for any patient having a tumor that expresses the target antigen.

[0176] Bispecific (such as bispecific) or bicistronic CARs are also contemplated by the present disclosure. In some embodiments, the bispecific or bispecific CARs comprise a nanobody specific for B7H3 (such as any one of RWB12, RWG8, RWC4, RWB2, RWH5, RWD5, RWC3, RWG4, RWD9, RWH1, RFA1, and RFB1) and a monoclonal antibody specific for a different antigen, such as a T cell antigen. Similarly, a bicistronic CAR comprises two CAR molecules expressed from the same construct, where one CAR molecule is a B7H3-targeting CAR and the second CAR targets a second antigen. See, e.g., Qin et al., Blood 130:810, 2017; and WO / 2018 / 213337.

[0177] Accordingly, provided herein is a CAR comprising an antibody specific for B7H3, such as any one of the nanobodies disclosed herein. Also provided are isolated nucleic acid molecules and vectors encoding a CAR (including bispecific and bicistronic CARs), and host cells such as CTL or NK cells that express the CAR, bispecific CAR or bicistronic CAR. CTL or NK cells expressing a CAR composed of a B7H3-specific monoclonal antibody can be used for the treatment of cancers that express B7H3. In some embodiments herein, the CAR is a bispecific CAR. In other embodiments herein, the CAR is a bicistronic CAR.

[0178] In some embodiments, the CAR comprises, for example, a signal peptide sequence at the N-terminus relative to the antigen-binding domain. The signal peptide sequence can be any suitable signal peptide sequence, such as a signal sequence derived from granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), immunoglobulin light chain kappa, or IL-2. The signal peptide sequence can facilitate the expression of the CAR on the cell surface, although the presence of the signal peptide sequence in the expressed CAR is not necessarily required for the CAR to function. Upon expression of the CAR on the cell surface, the signal peptide sequence can be cleaved and removed from the CAR. Thus, in some embodiments, the CAR lacks a signal peptide sequence.

[0179] In some embodiments disclosed herein, the CAR is expressed from a construct (e.g., from a lentiviral vector) that also expresses a truncated version of human EGFR (huEGFRt). The CAR and huEGFRt are separated by a self-cleaving peptide sequence (such as T2A), such that upon expression in the transduced cell, the CAR is cleaved from the huEGFRt.

[0180] In some embodiments disclosed herein, the CAR construct encodes the following amino acid sequence in the direction from the N-terminus to the C-terminus. [Chemistry]

[0181] The human epidermal growth factor receptor is composed of four extracellular domains, one transmembrane domain, and three intracellular domains. The EGFR domains are found in the following order from the N-terminus to the C-terminus: Domain I - Domain II - Domain III - Domain IV - Transmembrane (TM) domain - juxtamembrane domain - tyrosine kinase domain - C-terminal tail. Domains I and III are leucine-rich domains involved in ligand binding. Domains II and IV are cysteine-rich domains that do not contact EGFR ligands. Domain II mediates the formation of homo- or heterodimers with similar domains from other members of the EGFR family, and Domain IV can form disulfide bonds with Domain II. The EGFR TM domain makes a single pass through the cell membrane and can play a role in protein dimerization. The intracellular domain contains the juxtamembrane domain, tyrosine kinase domain, and C-terminal tail, which mediate EGFR signal transduction (Wee and Wang, Cancers 9(52), doi:10.3390 / cancers9050052; Ferguson, Annu Rev Biophys 37:353 - 373, 2008; Wang et al., Blood 118(5):1255 - 1263, 2011).

[0182] A truncated version of human EGFR, referred to herein as "huEGFRt", contains only Domain III, Domain IV, and the TM domain. Thus, huEGFRt lacks Domain I, Domain II, and all three intracellular domains. huEGFRt is unable to bind EGF and lacks signaling activity. However, this molecule retains the ability to bind certain EGFR-specific monoclonal antibodies such as cetuximab, which is approved by the FDA (PCT Publication No. WO2011 / 056894, which is incorporated herein by reference).

[0183] By transducing T cells (or NK cells) with a construct (such as a lentiviral vector) encoding both huEGFRt and a tumor antigen-specific CAR disclosed herein, it becomes possible to select transduced T cells using cetuximab (ERBITUX™), a labeled EGFR monoclonal antibody. For example, cetuximab can be labeled with biotin, and transduced T cells can be selected using commercially available (e.g., from Miltenyi Biotec) anti-biotin magnetic beads. Co-expression of huEGFRt also enables in vivo tracking of T cells (or NK cells) expressing the adoptively transferred CAR. Furthermore, binding of cetuximab to T cells expressing huEGFRt induces the cytotoxicity of ADCC effector cells, thereby providing a mechanism for eliminating transduced T cells in vivo, such as at the end of treatment (Wang et al., Blood 118(5):1255-1263, 2011).

[0184] Also provided herein are B7H3-specific monoclonal antibodies (such as the nanobodies disclosed herein) that have been modified to be usable in universal CAR systems. Universal CAR systems have been developed to increase the flexibility of CARs and expand their use to additional antigens. Currently, for each patient undergoing CAR T cell therapy, autologous T cells must be cultured, expanded, and modified to express an antigen-specific CAR. This process is time-consuming and costly and its use is limited. Universal CARs are based on systems in which the signaling components of the CAR are split from the antigen-binding portion of the molecule but are joined using a "lock-and-key" system. For example, a biotin-binding immunoreceptor (BBIR) CAR is composed of an intracellular T cell signaling domain fused to an extracellular domain that includes avidin. A biotinylated antigen-specific (such as B7H3-specific) monoclonal antibody can then bind to the BBIR and direct the T cell to tumor antigen-expressing cells. Another example is the split, universal and programmable (SUPRA) CAR system. In the SUPRA system, the CAR includes an intracellular signaling domain fused to an extracellular leucine zipper that pairs with an antigen-specific monoclonal antibody fused to a cognate leucine zipper. For a review of universal CAR systems, see, for example, Zhao et al., J Hematol Oncol 11(1):132, 2018; and Cho et al., Cell 173:1426-1438, 2018. In some embodiments herein, the B7H3-specific monoclonal antibody is fused to one component of a specific binding pair. In some examples, the monoclonal antibody is fused to a leucine zipper or biotin.

[0185] Another type of universal CAR can be generated using a sortase enzyme. Sortase is a prokaryotic enzyme that modifies surface proteins by recognizing and cleaving a carboxyl-terminal sorting signal. Sortase catalyzes peptide transfer between a sortase recognition motif and a sortase acceptor motif. Thus, an antigen-specific CAR can be generated by contacting an antigen-specific antibody fused to a sortase recognition motif with a portion of a CAR molecule that includes an intracellular signaling domain, a transmembrane region, and an extracellular portion that includes a sortase acceptor motif. In the presence of the sortase enzyme, the two components attach by a covalent bond to form a complete antigen-specific CAR. Accordingly, in some embodiments herein, a B7H3-specific monoclonal antibody is modified to include a sortase recognition motif (see, e.g., PCT Publication No. WO2016 / 014553). V. Immunoconjugates

[0186] The disclosed single-domain monoclonal antibodies can be conjugated to a therapeutic agent or an effector molecule. Immunoconjugates include molecules that have, but are not limited to, a covalent attachment of a therapeutic agent to an antibody. A therapeutic agent is a drug having a specific biological activity directed against a particular target molecule or a cell having the target molecule. One of ordinary skill in the art will recognize that therapeutic agents can include various drugs such as vinblastine, daunomycin, cytotoxins such as native or modified Pseudomonas exotoxin or diphtheria toxin, encapsulating agents (such as liposomes) containing a pharmaceutical composition, 125 I, 32 P, 14 C, 3 H and 35 S, radiolagents such as IR700, photon absorbers such as IR700, and other labels, targeting moieties, and ligands.

[0187] The selection of a particular therapeutic agent depends on a particular target molecule or cell and the desired biological effect. Thus, for example, the therapeutic agent may be a cytotoxin used to effect the death of a particular target cell (such as a tumor cell). Conversely, if it is desired not to elicit a lethal biological response, the therapeutic agent may be conjugated to a substance having a non-lethal pharmacological effect or to liposomes containing a substance having a non-lethal pharmacological effect.

[0188] With the therapeutic agents and antibodies described herein, one of ordinary skill in the art can readily construct various clones containing nucleic acids that are functionally equivalent, such as nucleic acids encoding the same effector portion or antibody sequence but having different sequences. Thus, the present disclosure provides nucleic acids encoding antibodies and their conjugates and fusion proteins.

[0189] The effector molecule can be linked to the antibody of interest using any number of means known to those of skill in the art. Both covalent and non-covalent attachment means may be used. The procedure for binding the effector molecule to the antibody varies depending on the chemical structure of the effector. A polypeptide typically has; a carboxylic acid (COOH), a free amine (-NH 2Alternatively, it contains various functional groups such as a sulfhydryl (-SH) group, etc., which can be utilized for reaction with suitable functional groups in the antibody, resulting in the binding of effector molecules. Alternatively, the antibody is derivatized to expose or bind to additional reactive functional groups. Derivatization can involve the binding of any of several known linker molecules. The linker can be any molecule used to bind the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody and the effector molecule are polypeptides, the linker can be attached to the constituent amino acids by their side chain groups (e.g., to cysteine by a disulfide linkage), or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0190] In some situations, it is desirable to release the effector molecule from the antibody when the immunoconjugate reaches its target site. Thus, in these situations, the immunoconjugate contains a linkage that is cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the antibody may be facilitated by the enzymatic activity or conditions provided either within the target cell or in the vicinity of the target site.

[0191] Considering the numerous methods that have been reported for conjugating various radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, one of ordinary skill in the art can determine a suitable method for conjugating a given agent to an antibody or other polypeptide.

[0192] The antibodies disclosed herein may be derivatized or linked to other molecules (such as another peptide or protein). Generally, the antibody or a portion thereof is derivatized such that binding to the target antigen is not adversely affected by the derivatization or labeling. For example, the antibody may be functionally linked (by chemical coupling, genetic fusion, association by non-covalent binding or otherwise) to a protein or peptide that can mediate the association of the antibody or antibody portion with one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a photon absorber, a pharmaceutical agent, and / or another molecule of the antibody or antibody portion (e.g., a streptavidin core region or polyhistidine tag).

[0193] One type of derivatized antibody is produced by cross-linking two or more antibodies (e.g., of the same or different types to create a bispecific antibody). Suitable cross-linking agents include hetero-bifunctional or homo-bifunctional agents having two different reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester). Such linkers are commercially available.

[0194] Antibodies can be conjugated to a detectable marker, such as a detectable marker detectable by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (computed tomography (CT), computerized axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic examination, and laparoscopy, etc.). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also used. Antibodies or antigen-binding fragments can also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When an antibody or antigen-binding fragment is conjugated to a detectable enzyme, it can be detected by adding additional reagents used by the enzyme to produce a distinguishable reaction product. For example, in the presence of the enzyme horseradish peroxidase as a substrate, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that is visually detectable. Antibodies or antigen-binding fragments can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or a fluorescent label.

[0195] The antibody may be labeled with a magnetic agent such as gadolinium. The antibody may also be labeled with lanthanides (europium and dysprosium), and manganese. Paramagnetic particles such as superparamagnetic iron oxide are also used as labels. The antibody may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequence, binding site of a secondary antibody, metal binding domain, epitope tag). In some embodiments, the label is attached by spacer arms of various lengths to reduce potential steric hindrance.

[0196] The antibody may also be labeled with radiolabeled amino acids. The radiolabel can be used for both diagnostic and therapeutic purposes. For example, radiolabels can be used to detect the expression of a target antigen by x-ray, luminescence spectrum, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radiolabeled nucleotides: 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I.

[0197] The antibodies disclosed herein may also be conjugated to a photon absorber. In some embodiments, the photon absorber is a phthalocyanine dye, e.g., but not limited to, IRDye® 700DX (also known as "IR700"). The antibody-photon absorber conjugate can be used in photoimmunotherapy.

[0198] The antibody may also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups can be useful for improving the biological characteristics of the antibody, such as increasing the serum half-life or increasing binding to tissues.

[0199] Toxins can be used with the monoclonal antibodies described herein to generate immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxin and their subunits, as well as botulinum toxins A through F. These toxins are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Envisioned toxins also include variants of the toxins described herein (see, e.g., U.S. Patent Nos. 5,079,163 and 4,689,401, which are incorporated by reference). In one embodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095). As used herein, "Pseudomonas exotoxin" refers to full-length native (naturally occurring) PE or modified PE. Such modifications can include, but are not limited to, the elimination of domain Ia, various amino acid deletions in domains Ib, II and III, single amino acid substitutions, and the addition of one or more sequences at the carboxyl terminus (see, e.g., Siegall et al., J. Biol. Chem. 264:14256-14261, 1989).

[0200] The PE used in conjunction with the monoclonal antibodies described herein can include native sequences, cytotoxic fragments of native sequences, as well as conservatively modified variants of native PE and their cytotoxic fragments. Cytotoxic fragments of PE can include those that are cytotoxic by proteolysis or other processing in the target cell, or without such processing. Cytotoxic fragments of PE include PE40, PE38, and PE35. For further description of PE and its variants, see, for example, U.S. Patent Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; and 5,854,044; U.S. Patent Application Publication No. 2015 / 0099707; PCT Publications WO99 / 51643 and WO2014 / 052064; Pai et al., Proc. Natl. Acad. Sci. USA 88:3358-3362, 1991; Kondo et al., J. Biol. Chem. 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta 1333:C1-C6, 1997.

[0201] Protease-resistant PE variants, as well as PE variants with reduced immunogenicity such as, but not limited to, PE-LR, PE-6X, PE-8X, PE-LR / 6X, and PE-LR / 8X are also contemplated herein (see, for example, Weldon et al., Blood 113(16):3792-3800, 2009; Onda et al., Proc Natl Acad Sci USA 105(32):11311-11316, 2008; and PCT Publications WO2007 / 016150, WO2009 / 032954, and WO2011 / 032022, which are incorporated herein by reference).

[0202] In some examples, the PE is a variant that is resistant to lysosomal degradation such as PE-LR (Weldon et al., Blood 113(16):3792-3800, 2009; PCT Publication No. WO2009 / 032954). In other examples, the PE is a variant named PE-LR / 6X (PCT Publication No. WO2011 / 032022). In other examples, the PE variant is a PE with reduced immunogenicity. In still other examples, the PE is a variant named PE-LR / 8M (PCT Publication No. WO2011 / 032022).

[0203] Modifications of the PE can occur in any of the previously described variants, including the cytotoxic fragments of the PE (e.g., PE38, PE-LR, and PE-LR / 8M). The modified PE may include, for example, any substitution(s) of one or more amino acid residues within one or more T-cell epitopes and / or B-cell epitopes of the PE, or deletion of one or more T-cell and / or B-cell epitopes (see, e.g., US Patent Application Publication No. 2015 / 0099707).

[0204] The contemplated forms of the PE also include a deimmunized form of the PE, such as a version lacking domain II (e.g., PE24). Deimmunized forms of the PE are described, for example, in PCT Publications No. WO2005 / 052006, WO2007 / 016150, WO2007 / 014743, WO2007 / 031741, WO2009 / 32954, WO2011 / 32022, WO2012 / 154530, and WO2012 / 170617.

[0205] Using the antibodies described herein, any number of various diagnostic or therapeutic compounds can also be targeted to cells expressing B7H3 on their surface. Thus, the antibodies of the disclosure can be bound, either directly or via a linker, to drugs that are directly delivered to cells expressing B7H3 on their cell surface. This can be done for therapeutic, diagnostic, or investigative purposes. Therapeutic agents include compounds such as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, photon absorbers, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutics and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalently crosslinking to single-stranded or double-stranded DNA, and triple helix-forming oligonucleotides.

[0206] Alternatively, the molecule linked to the antibody may be an encapsulation system such as a nanoparticle, liposome, or micelle containing a therapeutic composition such as a drug, nucleic acid (e.g., antisense nucleic acid), or another therapeutic component that is preferably shielded from direct exposure to the circulatory system. Means for preparing liposomes conjugated to antibodies are well known to those of skill in the art (see, for example, U.S. Patent No. 4,957,735; Connor et al., Pharm. Ther. 28:341-365, 1985).

[0207] The antibodies described herein may be linked to a detectable label either covalently or non-covalently. Detectable labels suitable for such uses include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Useful labels include magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, etc.), radiolabels (e.g., 3 H, 125 I, 35 S, 14 C, or 32P), enzymes (such as horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA), and colorimetric labels such as colloidal gold or colored glass, or plastic (such as polystyrene, polypropylene, latex, etc.) beads.

[0208] Means for detecting such labels are well known to those skilled in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, and fluorescent markers may be detected using photodetectors to detect the irradiated illumination. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction product generated by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label. VI. Antibody - Drug Conjugates (ADCs)

[0209] An ADC is a compound composed of a tumor - antigen - specific antibody (such as a single - domain antibody or an antigen - binding fragment of an immunoglobulin) and a drug, typically a cytotoxic agent such as a microtubule inhibitor or a cross - linker. Since ADCs can specifically target cancer cells, the drugs can be much more potent than those used in standard chemotherapy. Currently, the most common cytotoxic drugs used with ADCs are 100 - 1000 times more potent than conventional chemotherapy agents in terms of IC 50 which they possess. Common cytotoxic drugs include microtubule inhibitors such as maytansinoids and auristatins (such as auristatin E and auristatin F). Other cytotoxins for use with ADCs include pyrrolobenzodiazepines (PBDs) which covalently bind to the minor groove of DNA to form interstrand cross - links. In many cases, ADCs constitute a ratio of 1:2 - 1:4 of antibody to drug (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3 - 7, 2012).

[0210] The antibody and the drug may be linked by a cleavable or non-cleavable linker. However, in some cases, it is desirable to have a linker that prevents systemic release of the cytotoxic drug, which can be stable in circulation and cause significant off-target toxicity. The non-cleavable linker prevents release of the cytotoxic agent until the ADC is internalized by the target cell. Once inside the lysosome, release of the cytotoxic agent is effected by digestion of the antibody by lysosomal proteases (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).

[0211] One method for site-specific and stable conjugation of a drug to a monoclonal antibody is by glycan engineering. Monoclonal antibodies have one conserved N-linked oligosaccharide chain at the Asn297 residue in the CH2 domain of each heavy chain (Qasba et al., Biotechnol Prog 24:520-526, 2008). By using a mutant β1,4-galactosyltransferase enzyme (Y289L-Gal-T1; U.S. Patent Application Publication Nos. 2007 / 0258986 and 2006 / 0084162, which are incorporated herein by reference), 2-keto-galactose is transferred to the free GlcNAc residue of the antibody heavy chain, providing a chemical handle for conjugation.

[0212] Oligosaccharide chains bound to monoclonal antibodies can be classified into three groups based on the terminal galactose residues - fully galactosylated (two galactose residues; IgG - G2), one galactose residue (IgG - G1), or fully degalactosylated (IgG - G0) residues. Treatment of monoclonal antibodies with β1,4 - galactosidase converts the antibody to the IgG - G0 glycoform. The mutant β1,4 - galactosyltransferase enzyme can transfer 2 - keto - galactose or 2 - azido - galactose from their respective UDP derivatives to GlcNAc residues in the IgG - G1 and IgG - G0 glycoforms. Chemical handles on the transferred sugars enable conjugation of various molecules to monoclonal antibodies by glycan residues (Qasba et al., Biotechnol Prog 24:520 - 526, 2008).

[0213] Provided herein are ADCs comprising a drug (such as a cytotoxic agent) conjugated to a monoclonal antibody that binds (e.g., specifically binds) to B7H3. In some embodiments, the drug is a small molecule. In some examples, the drug is a cross - linker, a microtubule inhibitor and / or an anti - mitotic agent, or any cytotoxic agent suitable for mediating the killing of tumor cells. Exemplary cytotoxic agents include, but are not limited to, PBD, auristatin, maytansinoid, dolastatin, calicheamicin, nemorubicin and its derivatives, PNU - 159682, anthracycline, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, eribulin, combretastain, dolastatin, duocarmycin, enediyne, geldanamycin, indolino - benzodiazepine dimer, puromycin, tubulysin, hemiasterlin, spirostatin, or pladienolide, as well as stereoisomers, homologs, analogs, and derivatives thereof having cytotoxic activity.

[0214] In some embodiments, the ADC comprises a pyrrolobenzodiazepine (PBD). The natural product anthramycin (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, several PBDs, both natural 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 an example, PBD dimers have been shown to recognize and bind to specific DNA sequences and be useful as cytotoxic agents. PBD dimers have been conjugated to antibodies and the resulting ADCs have been shown to have anti-cancer properties (see, e.g., US2010 / 0203007). Exemplary linking sites in PBD dimers include the 5-membered pyrrolo 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).

[0215] In some embodiments, the ADC comprises an antibody conjugated to one or more maytansinoid molecules. A maytansinoid is a derivative of maytansine and is a mitotic inhibitor that acts by inhibiting tubulin polymerization. Maytansine was first isolated from the African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, certain microorganisms were also found to produce maytansinoids such as 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.

[0216] In some embodiments, the ADC comprises an antibody conjugated to dolastatin or auristatin, or an analog or derivative thereof (see U.S. Patent 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 been shown to have anti-cancer (U.S. Patent No. 5,663,149) and anti-fungal 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-dolaisoleucine-dolaproine-phenylalanine), MMAE (monomethyl auristatin E or N-methylvaline-valine-dolaisoleucine-dolaproine-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other auristatins (see, e.g., U.S. Publication No. 2013 / 0129753).

[0217] In some embodiments, the ADC comprises an antibody conjugated to one or more calicheamicin molecules. Antibiotics of the calicheamicin family, and analogs thereof, are capable of causing double-strand DNA breaks at concentrations below picomolar (Hinman et al., Cancer Res 53:3336-3342, 1993; Lode et al., Cancer Res 58:2925-2928, 1998). Exemplary methods for preparing ADCs that contain a calicheamicin drug moiety are described in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.

[0218] In some embodiments, the ADC comprises an anthracycline. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. Anthracyclines may act to kill cells by several different mechanisms including intercalation of the drug molecule into cellular DNA and thereby inhibition of DNA-dependent nucleic acid synthesis; induction of free radical generation that later reacts with cellular macromolecules to cause damage to the cells; and / or interaction of the drug molecule with the cell membrane. 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 at the glycosidoamino of doxorubicin (Grandi et al., Cancer Treat Rev 17:133, 1990; Ripamonti et al., Br J Cancer 65:703-707, 1992).

[0219] In some embodiments, the ADC may further include a linker. In some examples, the linker is a bifunctional or polyfunctional moiety that can be used to link one or more drug moieties to the antibody to form an ADC. In some embodiments, the ADC is prepared using a linker having reactive functional groups for covalently attaching to the drug and the antibody. For example, the cysteine thiol of the antibody can form a bond with the reactive functional group of the linker or the drug-linker intermediate to produce an ADC.

[0220] In some examples, the linker has a functional group capable of reacting 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, anhydride, acid chloride, sulfonyl chloride, isocyanate, and isothiocyanate.

[0221] In some examples, the linker has a functional group capable of reacting with an electrophilic group present on the antibody. Examples of such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some cases, the heteroatom of the reactive functional group of the linker can react with the electrophilic group on the antibody to form a covalent bond with the antibody unit. Non-limiting examples include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, carboxylic acid hydrazide, and aryl hydrazide.

[0222] In some examples, the linker is a cleavable linker that facilitates drug release. Examples of cleavable linkers include acid-labile linkers (e.g., those containing hydrazone), protease-sensitive linkers (e.g., peptidase-sensitive), photolabile linkers, and disulfide-containing linkers (Chari et al., Cancer Res 52:127-131, 1992; U.S. Patent No. 5,208,020).

[0223] The ADCs disclosed herein can be used for the treatment of B7H3-positive cancers, alone or in combination with another therapeutic agent and / or in combination with any standard treatment for the treatment of cancer (such as surgical resection of tumors, chemotherapy or radiotherapy). VII. Multispecific Antibodies

[0224] Multispecific antibodies are recombinant proteins composed of two or more monoclonal antibodies (such as single-domain antibodies) or antigen-binding fragments of two or more different monoclonal antibodies. For example, bispecific antibodies are composed of antigen-binding fragments of two different monoclonal antibodies. Thus, bispecific antibodies bind two different antigens and trispecific antibodies bind three different antigens. Multispecific antibodies can be used for cancer immunotherapy by simultaneously targeting both CTLs (such as CTL receptor components such as CD3) or effector natural killer (NK) cells and at least one tumor antigen. Using the B7H3-specific single-domain monoclonal antibodies disclosed herein, multispecific (such as bispecific or trispecific) antibodies can be generated that target both B7H3 and CTLs or both B7H3 and NK cells, thereby providing a means for treating cancers that express B7H3.

[0225] Bispecific T cell engagers (BiTEs) are a type of bispecific monoclonal antibody that are fusions of a first monoclonal antibody (such as an scFv or single-domain antibody) that targets a tumor antigen (such as B7H3) and a second antibody that binds to T cells, such as CD3 on T cells. In some embodiments herein, one of the binding moieties of the BiTE is specific for B7H3.

[0226] Bispecific killer cell engager (BiKE) is a type of bispecific monoclonal antibody that is a fusion of a first monoclonal antibody (such as scFv or single domain antibody) targeting a tumor antigen (such as B7H3) and a second scFv that binds to an NK cell activating receptor, such as CD16.

[0227] Multispecific, such as trispecific or bispecific, monoclonal antibodies that include a B7H3-specific monoclonal antibody are provided herein. In some embodiments, the multispecific monoclonal antibody further includes a monoclonal antibody that specifically binds to a component of the T cell receptor, such as CD3. In other embodiments, the multispecific monoclonal antibody further includes a monoclonal antibody that specifically binds to an NK cell activating receptor, such as CD16, Ly49, or CD94. Isolated nucleic acid molecules and vectors encoding the multispecific antibody, as well as host cells containing the nucleic acid molecule or vector, are also provided. Multispecific antibodies that include a B7H3-specific antibody can be used for the treatment of cancers that express B7H3. Thus, provided herein is a method of treating a subject having cancer by selecting a subject having cancer that expresses B7H3 and administering to the subject a therapeutically effective amount of a multispecific antibody that targets B7H3. VIII. Antibody-Nanoparticle Conjugates

[0228] The monoclonal antibodies disclosed herein can be conjugated to various different types of nanoparticles to directly deliver a cytotoxic agent or other anti-cancer agent to tumor cells by binding the antibody to B7H3 expressed on the surface of tumor cells. The use of nanoparticles reduces off-target side effects, improves the bioavailability of the drug, and can also reduce the dosage of the drug required to achieve a therapeutic effect. The nanoparticle formulation can be adapted to be suitable for the drug carried or encapsulated within the nanoparticle. For example, hydrophobic molecules may be incorporated within the core of the nanoparticle, while hydrophilic drugs may be carried within an aqueous core protected by a polymer shell or a lipid shell. Examples of nanoparticles include, but are not limited to, nanospheres, nanocapsules, liposomes, dendrimers, polymeric micelles, niosomes, and polymeric nanoparticles (Fay and Scott, Immunotherapy 3(3):381-394, 2011).

[0229] Liposomes are a common type of nanoparticles used in drug delivery. Antibodies conjugated to liposomes are often referred to as "immunoliposomes". The liposomal components of immunoliposomes are typically lipid vesicles of one or more concentric phospholipid bilayers. In some cases, the phospholipids are composed of a hydrophilic head group and two hydrophobic chains that enable the encapsulation of both hydrophobic and hydrophilic drugs. Conventional liposomes are rapidly removed from circulation by macrophages of the reticuloendothelial system (RES). To generate long-circulating liposomes, the composition, size, and charge of the liposomes can be modulated. The surface of the liposomes may also be modified, for example, with glycolipids or sialic acid. For example, by including polyethylene glycol (PEG), the circulation half-life is significantly increased. Liposomes for use as drug delivery agents, including for the preparation of immunoliposomes, have been described in the art (see, for example, Paszko and Senge, Curr Med Chem 19(31)5239-5277, 2012; Immordino et al., Int J Nanomedicine 1(3):297-315, 2006; US Patent Application Publication No. 2011 / 0268655; No. 2010 / 00329981).

[0230] Niosomes are nonionic surfactant-based vesicles having a structure similar to liposomes. The membrane of niosomes is composed only of nonionic surfactants, for example, polyglyceryl-alkyl ethers or N-palmitoyl glucosamine. Niosomes range from small unilamellar particles to large multilamellar particles. These nanoparticles are monodisperse, water-soluble, chemically stable, low-toxicity, biodegradable and non-immunogenic, and increase the bioavailability of the encapsulated drug.

[0231] Dendrimers include various branched polymer complexes. These nanoparticles are water-soluble, biocompatible, and sufficiently non-immunogenic for use in humans. Generally, dendrimers consist of an initiator core (surrounded by a layer of a selected polymer grafted to the core) that forms a branched macromolecular complex. Dendrimers are typically generated using polymers such as poly(amidoamine) or poly(L-lysine). Dendrimers are used for various therapeutic and diagnostic applications, including for the delivery of DNA, RNA, bioimaging contrast agents, and chemotherapeutic agents.

[0232] Polymer micelles are composed of aggregates of amphiphilic copolymers (consisting of both hydrophilic and hydrophobic monomer units) assembled in a hydrophobic core and surrounded by a corona of hydrophilic polymer chains exposed to an aqueous environment. In many cases, the polymers used to prepare polymer micelles are heterobifunctional copolymers composed of PEG, a hydrophilic block of poly(vinylpyrrolidone), and a hydrophobic poly(L-lactide) or poly(L-lysine) that forms the particle core. Polymer micelles can be used to carry poorly soluble drugs. These nanoparticles have been used to encapsulate several anticancer drugs, including doxorubicin and camptothecin. Cationic micelles have also been developed to carry DNA or RNA molecules.

[0233] Polymeric nanoparticles include both nanospheres and nanocapsules. Nanospheres consist of a solid matrix of polymer, while nanocapsules contain an aqueous core. The selected formulation typically depends on the solubility of the therapeutic agent to be carried / encapsulated; poorly water-soluble drugs are more easily encapsulated within nanospheres, while water-soluble and unstable drugs, such as DNA and proteins, are more easily encapsulated within nanocapsules. Polymers used to generate these nanoparticles include, for example, poly(acrylamide), poly(ester), poly(alkyl cyanoacrylate), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(D,L-lactic-co-glycolic acid) (PLGA).

[0234] Antibodies can be conjugated to suitable nanoparticles according to standard methods known in the art. For example, the conjugation can be either covalent or non-covalent. In some embodiments where the nanoparticles are liposomes, the antibody is bound to a sterically stabilized, long-circulating liposome by a PEG chain. The coupling of an antibody or antibody fragment to a liposome can also involve, for example, a thioester bond by reaction of a thiol with a maleimide group. A cross-linking agent can be used to create sulfhydryl groups for the binding of the antibody to the nanoparticles (Paszko and Senge, Curr Med Chem 19(31)5239-5277, 2012). IX. Compositions and Methods of Use

[0235] In a carrier, a composition is provided that contains one or more of the disclosed monoclonal antibodies that bind (e.g., specifically bind) B7H3. Compositions are also provided that include ADCs, CARs (and CTLs or other cells that include CARs), multispecific (such as bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes, and immunoconjugates. The composition can be prepared in a unit dosage form for administration to a subject. The amount and timing of administration are at the discretion of the physician performing the treatment to achieve the desired outcome. The antibody, ADC, CAR, cell expressing CAR, multispecific antibody, antibody-nanoparticle conjugate, immunoliposome, or immunoconjugate can be formulated for systemic or local (such as intratumoral) administration. In one example, the antibody is formulated for parenteral administration such as intravenous administration.

[0236] The composition for administration may contain a solution of an antibody, ADC, CAR, cell expressing CAR (such as a CTL), multispecific (such as bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome, or immunoconjugate in a pharmaceutically acceptable carrier such as an aqueous carrier. Various aqueous carriers, such as buffered saline, may be used. These solutions are sterile and generally free of undesirable substances. These compositions may be sterilized by conventional well-known sterilization techniques. The composition may contain pharmaceutically acceptable adjunct substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity modifiers, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antibody in these formulations may vary widely and is mainly selected based on the volume of fluid, viscosity, body weight, etc., according to the particular mode of administration selected and the needs of the subject.

[0237] Typical pharmaceutical compositions for intravenous administration contain about 0.1 to 10 mg of antibody (or ADC, CAR, bispecific antibody, antibody-nanoparticle conjugate, or immunoconjugate) per subject per day. In particular, when the agent is administered to a sequestered site and not administered into the circulation or lymphatic system, such as into a body cavity or the lumen of an organ, a dosage of 0.1 to up to about 100 mg per subject per day may be used. The actual methods for preparing the administrable compositions are known or apparent to those of ordinary skill in the art and are described in more detail in publications such as Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition (2005).

[0238] The monoclonal antibodies disclosed herein may also be administered by other routes including via inhalation, oral, topical, or intraocular. In some examples, the monoclonal antibody (or conjugate thereof) is administered by a fine needle.

[0239] Antibodies (or other therapeutic molecules) may be provided in lyophilized form and rehydrated with sterile water prior to administration, although they are also provided in sterile solutions at known concentrations. The antibody solution is then added to an infusion bag containing 0.9% sodium chloride, USP and, in some cases, administered at a dosage of 0.5 to 15 mg / kg body weight. Since the approval of RITUXAN™ in 1997, considerable experience has been gained in the administration of antibody drugs marketed in the United States. Antibodies, ADCs, CARs (or cells expressing CARs), multispecific (bispecific or trispecific, etc.) antibodies, antibody-nanoparticle conjugates, immunoliposomes or immunoconjugates may be administered by slow infusion rather than by intravenous injection or bolus. In one example, a larger loading dose is administered, followed by lower levels of maintenance doses. For example, an initial loading dose of 4 mg / kg may be infused over a period of about 90 minutes, followed by a maintenance dose of 2 mg / kg infused weekly for 4 to 8 weeks, if previous doses have been well tolerated, over a period of 30 minutes.

[0240] Controlled-release parenteral formulations may be made as implants, oily injections, or as particulate systems. For an 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). Particulate systems include, for example, microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain therapeutic proteins such as cytotoxins or drugs as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Since capillaries have a diameter of approximately 5 μm, 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).

[0241] Polymers can be used for the ion-controlled release of the antibody-based compositions disclosed herein. Various degradable 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 fluid liquid at low temperatures but forms a semi-solid gel at body temperature. This has been shown to be 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 controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous 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). A. Treatment Methods

[0242] The antibodies, compositions, CARs (and cells such as CTLs expressing CARs), ADCs, multispecific (bispecific or trispecific, etc.) antibodies, antibody-nanoparticle conjugates, immunoliposomes and immunoconjugates disclosed herein can be administered to delay or inhibit the growth of tumor cells, such as B7H3-positive solid tumors, or to inhibit the metastasis of tumor cells. In these applications, a therapeutically effective amount of the composition is administered to a subject in an amount sufficient to inhibit the growth, replication or metastasis of cancer cells or to inhibit the signs or symptoms of cancer. Suitable subjects include, but are not limited to, those diagnosed with solid tumors expressing B7H3 such as liver cancer (such as hepatocellular carcinoma), pancreatic cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer (such as neuroblastoma or glioblastoma), pediatric cancer (such as osteosarcoma, neuroblastoma, rhabdomyosarcoma or Ewing sarcoma), melanoma or mesothelioma.

[0243] Provided herein is a method of treating B7H3-positive cancer in a subject by administering a therapeutically effective amount of a B7H3-specific antibody, immunoconjugate, CAR (or cell expressing CAR), ADC, multispecific (bispecific or trispecific, etc.) antibody, antibody-nanoparticle conjugate, immunoliposome or composition disclosed herein. Also provided herein is a method of inhibiting tumor growth or metastasis of B7H3-positive cancer in a subject by administering a therapeutically effective amount of a B7H3-specific antibody, immunoconjugate, CAR (such as a cell expressing CAR), ADC, multispecific (bispecific or trispecific, etc.) antibody, antibody-nanoparticle conjugate, immunoliposome or composition disclosed herein. In some embodiments, the B7H3-positive cancer is liver cancer (such as hepatocellular carcinoma), pancreatic cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer (such as neuroblastoma or glioblastoma), pediatric cancer (such as osteosarcoma, neuroblastoma, rhabdomyosarcoma or Ewing sarcoma), melanoma or mesothelioma.

[0244] The therapeutically effective amount of the B7H3-specific monoclonal antibodies, ADCs, CARs (e.g., CTLs expressing CAR), multispecific (e.g., bispecific or trispecific) antibodies, immunoconjugates, immunoliposomes or compositions disclosed herein will vary depending on the severity of the disease, the type of disease, and the overall health of the patient. The therapeutically effective amount of an antibody-based composition is an amount that provides a subjective reduction of symptoms or an objectively identifiable improvement as referred to by a physician or other qualified observer.

[0245] In one example, the B7H3-specific antibody provided herein is conjugated to IR700 and photodynamic therapy is used to treat B7H3-positive cancer. For example, such a method can include administering to a subject having B7H3-positive cancer a therapeutically effective amount of one or more B7H3-specific antibody-IR700 conjugates, wherein the B7H3-specific antibody specifically binds to B7H3 on the cancer cells. After administration of the conjugate, the cancer is irradiated with a wavelength of 660 - 740 nm (e.g., 660 - 710 nm, e.g., 680 nm) and a dose of at least 1 J / cm -2 thereby treating the B7H3-positive cancer in the subject. In some examples, the B7H3-positive cancer is irradiated with at least 1 J / cm -2 (e.g., at least 1 J / cm -2 , at least 4 J / cm -2 , at least 10 J / cm -2 , at least 50 J / cm -2 , or at least 100 J / cm -2) at a dose of, irradiated at a wavelength of 660-740 nm (e.g., 660-710 nm, e.g., 680 nm), thereby treating tumors in a subject. In some examples, multiple treatments such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 treatment cycles are performed. In certain examples, the therapeutically effective dose of the B7H3-specific antibody-IR700 conjugate is, for example, when administered iv, at least 0.5 milligrams per kilogram (mg / kg) per 60 kilograms, at least 5 mg / 60 kg, at least 10 mg / 60 kg, at least 20 mg / 60 kg, at least 30 mg / 60 kg, at least 50 mg / 60 kg, for example, 0.5-50 mg / 60 kg, for example, 1 mg / 60 kg, 2 mg / 60 kg, 5 mg / 60 kg, 20 mg / 60 kg, or 50 mg / 60 kg. In another example, the therapeutically effective dose of the B7H3-specific antibody-IR700 conjugate is, for example, when administered intratumorally or i.p., at least 10 μg / kg, for example at least 100 μg / kg, at least 500 μg / kg, or at least 500 μg / kg, for example, 10 μg / kg to 1000 μg / kg, for example 100 μg / kg, 250 μg / kg, about 500 μg / kg, 750 μg / kg, or 1000 μg / kg. In one example, the therapeutically effective dose of the B7H3-specific antibody-IR700 conjugate, when administered in a topical solution, is at least 1 μg / ml, for example at least 500 μg / ml, for example between 20 μg / ml and 100 μg / ml, for example 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, or 100 μg / ml.

[0246] Administration of the B7H3-specific antibodies, ADCs, CARs (or cells expressing CARs), immunoconjugates, bispecific antibodies, antibody-nanoparticle conjugates, immunoliposomes, and compositions disclosed herein may also be accompanied by 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 antibodies, compositions, and immunoconjugates 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, biologic response modifiers, antihormonal agents (e.g., antiandrogens), and anti-angiogenic agents. Other anti-cancer treatments include radiation therapy and other antibodies (e.g., biologics) that specifically target cancer cells.

[0247] Non-limiting examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).

[0248] Non-limiting examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs such as mercaptopurine or thioguanine.

[0249] Non-limiting examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase).

[0250] Non-limiting examples of various agents include platinum coordination complexes (such as cis-diamine-dichloro platinum II, also known as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocortical suppressants (such as mitotane and aminoglutethimide).

[0251] Non-limiting examples of hormones and antagonists include adrenocortical steroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), anti-estrogen drugs (such as tamoxifen), and androgens (such as testosterone proprionate and fluoxymesterone). Examples of the most commonly used chemotherapeutic drugs include adriamycin, alkeran, Ara-C, BiCNU, busulfan, CCNU, carboplatinum, cisplatinum, cytoxan, daunorubicin, DTIC, 5-FU, fludarabine, hydrea, idarubicin, ifosfamide, methotrexate, mitramycin, mitomycin, mitoxantrone, nitrogen mustard, taxol (or other taxanes, such as docetaxel), velban, vincristine, VP-16. However, some newer drugs include gemcitabine (Gemzar), herceptin, irinotecan (Camptosar, CPT-11), roisastatin, navelbine, rituxan STI-571, taxotere, topotecan (Hycamtin), xeloda (capecitabine), zevelin, and calcitriol.

[0252] 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 of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0253] Non-limiting examples of biologic agents that can be used in combination with the disclosed B7H3-specific antibodies, ADCs, CARs (or cells expressing CARs), immunoconjugates, bispecific antibodies, antibody-nanoparticle conjugates, and immunoliposomes include therapeutic monoclonal antibodies such as 3F8, abagovomab, adecatumumab, afucosylated antibody, alemtuzumab, altumomab pentetate, anatumomab mafenatox,apolizumab, arcitumomab, batumiximab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, canertinib mertansine, capromab pendetide, catumaxomab, CC49, cetuximab, cixutumumab bogatox, cixutumumab, clivatuzumab tetraxetan, conatumumab, dacetuzumab, detumomab, eculizumab, eculizumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab, galiximab, gemtuzumab ozogamicin, glembatumumab vedotin, ibritumomab tiuxetan, igovomab, imciromab, inteumomab, inotuzumab ozogamicin, ipilimumab, iratumumab, labelizumab, lexatumumab, lintuzumab, lorvotuzumab mertansine, lucatumumab, lumiliximab, mapatumumab, matuzumab, mepolizumab, motavizumab, miratumumab, mitumomab, morolimumab, nacolomab tafenatox, napumomab estafenatox, nesitumumab, nimotuzumab, nofetumomab merpentan, ofatumumab, olaratumumab, oportuzumab monatox, oregovomab, panitumumab, pemtumomab, pertuzumab, pintumomab, pritumumab, ramucirumab, rilotumumab, rituximab, rovalpituzumab tesirine, satumomab pendetide, sibrotuzumab, sonelixizumab, takatuzumab tetraxetan, tabalumab paptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, trastuzumab, tremelimumab, tositumomab selmoleukin, veltuzumab, volociximab, voreloxin, and zalutumumab, one or more of which are included.In some examples, the therapeutic antibody specifically binds to and antagonizes PD-1 or PD-L1, such as one or more of atezolizumab, MPDL3280A, BNS-936558 (nivolumab), pembrolizumab, pidilizumab, CT011, AMP-224, AMP-514, MEDI-0680, BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, MSB-0010718C, MGA-271, Indoximod, Epacadostat, BMS-986016, MEDI-4736, MEDI-4737, MK-4166, BMS-663513, PF-05082566 (PF-2566), lirilumab, and durvalumab.

[0254] In some examples, the additional therapeutic agent to be administered is a T cell agonist, such as an agonist of 4-1BB (CD137), OX40, and / or GITR. In one example, the additional therapeutic agent to be administered is an OX40 agonist, such as an antibody, for example, a monoclonal antibody (mAb) (e.g., PF-04518600, MEDI-6469, MEDI-0562, MEDI-6383, MOXR-0916, BMS 986178, or GSK3174998). In some examples, the additional therapeutic agent to be administered is a 4-1BB agonist, such as a 4-1BB agonist antibody such as an mAb. Specific agonist mAbs that can be used using the disclosed methods include PF-05082566 (utomilumab), and BMS-663513 (urelumab). In one example, the 4-1BB agonist is 4-1BB ligand (4-1BBL), such as native 4-1BBL (such as human 4-1IBBL) or streptavidinylated 4-1BBL (SA-4-1BBL) complex. In some examples, the additional therapeutic agent to be administered is a GITR (glucocorticoid-induced tumor necrosis factor (TNF) receptor, or TNFRSF18) agonist, such as a GITR agonist antibody such as an mAb. Specific GITR agonist mAbs that can be used using the disclosed methods include DTA-1, TRX518, MK-4166, MK-1248, AMG 228, INCAGN01876, GWN323 (from Novartis), CK-302 (from Checkpoint Therapeutics) and BMS-986156. In one example, the GITR agonist is GITR ligand (GITRL), such as native GITRL or a multivalent GITR ligand fusion protein. In one example, the GITR agonist is MEDI1873, a hexameric GITRL molecule having a human IgG1 Fc domain. In some examples, the additional therapeutic agent to be administered is an immunotherapeutic agent.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 of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0255] In one example, the additional treatment is a surgical procedure, such as surgical resection of the cancer or a part thereof. Another example of a treatment is radiation therapy, such as the administration of a radioactive substance or energy (such as external beam therapy) to the tumor site to help eradicate or shrink the tumor prior to surgical resection. B. Methods for Diagnosis and Detection

[0256] Methods for detecting B7H3 protein in vitro or in vivo are provided herein. For example, the disclosed monoclonal antibodies can be used for in vivo tumor imaging. To use the antibodies disclosed as diagnostic reagents in vivo, the antibodies are labeled with a detectable moiety, such as a radioisotope, a fluorescent label, or a positron-emitting radionuclide. As an example, the monoclonal antibodies disclosed herein can be conjugated to a positron-emitting radionuclide for use in positron emission tomography (PET); this diagnostic process is often referred to as immunoPET. Full-length antibodies can make good immunoPET agents, but their biological half-lives require waiting several days prior to imaging, increasing the associated non-target radiation dose. Smaller single-domain antibodies / nanobodies have biological half-lives suitable for same-day imaging.

[0257] In other cases, B7H3 expression is detected in a biological sample. The sample may be any sample including, but not limited to, tissue from a biopsy, autopsy, and pathological specimen. The biological sample also includes tissue sections, for example, frozen sections taken for histological purposes. The biological sample further includes body fluids such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. In some examples, the sample is a serum sample containing exosomes. The biological sample is typically obtained from a mammal such as a human or non-human primate.

[0258] Provided herein is a method for determining whether a subject has B7H3-positive cancer by contacting a sample from the subject with a B7H3-specific monoclonal antibody disclosed herein and detecting the binding of the antibody to the sample. An increase in the binding of the antibody to the sample as compared to the binding of the antibody to a control sample identifies the subject as having B7H3-positive cancer.

[0259] In another embodiment, provided is a method for confirming the diagnosis of B7H3-positive cancer in a subject by contacting a sample from a subject diagnosed with B7H3-positive cancer with a B7H3-specific monoclonal antibody disclosed herein and detecting the binding of the antibody to the sample. An increase in the binding of the antibody to the sample as compared to the binding of the antibody to a control sample confirms the diagnosis of B7H3-positive cancer in the subject.

[0260] In some examples of the disclosed methods, the monoclonal antibody is directly labeled.

[0261] In other examples, the method further includes contacting the sample with a second antibody (detection antibody) that specifically binds the monoclonal antibody and detecting the binding of the second antibody. An increase in the binding of the second antibody to the sample as compared to the binding of the second antibody to a control sample detects B7H3-positive cancer in the subject or confirms the diagnosis of B7H3-positive cancer in the subject.

[0262] In some cases, the cancer is liver cancer (such as hepatocellular carcinoma), pancreatic cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer (such as neuroblastoma or glioblastoma), pediatric cancer (such as osteosarcoma, neuroblastoma, rhabdomyosarcoma or Ewing sarcoma), melanoma or mesothelioma.

[0263] In some examples, the control sample is a sample derived from a subject without cancer. In certain examples, the sample is a blood or tissue sample.

[0264] In some embodiments of the methods of diagnosis and detection, an antibody that binds (e.g., specifically binds) to B7H3 is directly labeled with a detectable label. In another embodiment, an antibody that binds (e.g., specifically binds) to B7H3 (the first antibody) is not labeled, and a second antibody or another molecule capable of binding an antibody that specifically binds to B7H3 is labeled. As is well known to those skilled in the art, a second antibody that can specifically bind to a particular species and classification of the first antibody is selected. For example, if the first antibody is human IgG, the secondary antibody may be anti-human IgG. Other molecules capable of binding to an antibody include, but are not limited to, protein A and protein G, both of which are commercially available.

[0265] Suitable labels for the antibody or secondary antibody include various enzymes, hapten conjugates, fluorescent substances, luminescent substances, magnetic agents and radioactive substances. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable hapten conjugate complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent substance is luminol, a non-limiting exemplary magnetic agent is gadolinium, and a non-limiting exemplary radioactive label is,125 I. 131 I. 35 contains S or 3 H.

[0266] In an alternative embodiment, B7H3 can be assayed in a biological sample by a competitive immunoassay that utilizes a B7H3 protein standard labeled with a detectable substance and an unlabeled antibody that specifically binds B7H3. In this assay, a biological sample, a labeled B7H3 protein standard, and an antibody that specifically binds B7H3 are combined, and the amount of labeled B7H3 protein standard bound to the unlabeled antibody is determined. The amount of B7H3 in the biological sample is inversely proportional to the amount of labeled B7H3 protein standard bound to the antibody that specifically binds B7H3.

[0267] The immunoassays and methods disclosed herein can be used for several purposes. In one embodiment, a specifically binding antibody can be used to detect the production of B7H3 in cells in a cell culture. In another embodiment, an antibody can be used to detect the amount of B7H3 in a biological sample, such as a tissue sample, or a blood or serum sample. In some examples, B7H3 is B7H3 on the cell surface. In other examples, the B7H3 protein is soluble (e.g., in a cell culture supernatant, or in a body fluid sample, such as a blood or serum sample).

[0268] In one embodiment, a kit for detecting B7H3 in a biological sample, such as a blood sample or a tissue sample, is provided. For example, to confirm the diagnosis of cancer in a subject, a biopsy can be performed to obtain a tissue sample for histological examination. A kit for detecting a polypeptide typically includes a monoclonal antibody that specifically binds B7H3, such as any of the monoclonal antibodies disclosed herein. In a further embodiment, the antibody is labeled (e.g., with a fluorescent, radioactive, or enzyme label).

[0269] In one embodiment, the kit includes instructions that disclose the use of an antibody that binds B7H3. The instructions may be in writing, in electronic form (such as a floppy (registered trademark) disk or a compact disk), or in video (such as a video file). The kit may further include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may further contain means for detecting a label (e.g., an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, a suitable secondary label such as a secondary antibody, etc.). The kit may further include buffers and other reagents routinely used for the practice of a particular method. Such kits and suitable contents are well known to those skilled in the art.

[0270] In one embodiment, the diagnostic kit includes an immunoassay. The details of the immunoassay can vary depending on the particular format used, but generally, a method for detecting B7H3 in a biological sample includes contacting the biological sample with an antibody that specifically reacts with B7H3 under immunologically reactive conditions. The antibody is capable of specifically binding to form an immune complex under immunologically reactive conditions, and the presence of the immune complex (the bound antibody) is detected either directly or indirectly.

[0271] The antibodies disclosed herein can also be utilized in immunoassays such as, but not limited to, radioimmunoassay (RIA), ELISA, or immunohistochemical assay. The antibodies may also be used in fluorescence-activated cell sorting (FACS). FACS separates or sorts cells using multiple color channels, low-angle and obtuse light scatter detection channels, as well as impedance channels, among other more sophisticated levels of detection (see U.S. Patent No. 5,061,620). Any of the monoclonal antibodies that bind B7H3 disclosed herein can be used in these assays. Thus, the antibodies can be used in conventional immunoassays including, but not limited to, ELISA, RIA, FACS, immunohistochemistry of tissues, Western blot, or immunoprecipitation.

[0272] The following examples are provided to illustrate certain specific features and / or embodiments. These examples should not be construed as limiting the present disclosure to the specific features or embodiments described.

Example

[0273] (Example 1) Materials and Methods This example describes the materials and experimental procedures used in the study described in Example 2. Cell Lines

[0274] Eight cancer cell lines (Hep3B, HepG2, IMR32, MC38 - B7H3+, A431, IMR32 - B7H3 KO, and MC38 - B7H3 KO) were cultured in DMEM medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 1% L - glutamine, and 1% penicillin - streptomycin (Invitrogen) in an atmosphere of 5% CO 2 in air at 37°C. The neuroblastoma cell line NBEB was cultured in RPMI - 1640 medium containing the same supplements as DMEM. The medium was refreshed twice a week. Protein Expression and Purification

[0275] The extracellular domain of B7H3 (GenBank accession number NP_001019907, amino acids 29 - 466; SEQ ID NO: 13) was fused with the hFc tag. B7H3-hFc was expressed in 293F cells. The hFc tag control, IAB-hFc (Kaneko et al., J Biol Chem, 284, 3739 - 3749, 2009), was generated in the same manner. Protein purification was achieved using a Protein A column (GE Healthcare). Rabbit VH domain antibodies were expressed in E. coli in a VH-His-FLAG fusion format. The 6×His tag was used for affinity purification by a Nickel column (GE Healthcare), and the FLAG tag was used for protein binding assays by ELISA and cell binding assays by flow cytometry. Construction of DNA Oligos and Rabbit VH Phage Library

[0276] To amplify the rabbit VH cDNA fragment, forward and reverse primers annealing to the 5’ and 3’ ends of VH cDNA were synthesized according to the literature (Peng et al., J Mol Biol, 429, 2954 - 2973, 2017). The primers are listed in Table 1, and the underlined nucleotides correspond to the SfiI restriction enzyme sites. Table 1: Primers for Construction of Rabbit VH Phage Library

Table 1-1

Table 1-2

[0277] The rabbit VH cDNA was synthesized from total RNA isolated from immunized spleens using the Invitrogen(®) SuperScript(®) IV First-Strand Synthesis Kit according to the manufacturer's instructions (ThermoFisher, catalog #18091050). Each forward primer was paired with one of two reverse primers (R1 or R2), and 22 combinations of forward / reverse primers were used to amplify VH cDNA fragments. The PCR products were gel purified, digested with the restriction enzyme SfiI (NEB, catalog #R0123S), and then ligated to the pComb3x plasmid pre-digested with the same enzyme. Using 10 micrograms of the ligation product, 0.6 ml of E. coli TG1 competent cells (Lucigen, catalog #60502-2) were transformed by electroporation according to the manufacturer's instructions. The transformed TG1 cells were harvested at 37 °C for 45 minutes while shaking at 150 rpm, then inoculated into 1 L of 2XYT medium and cultured at 37 °C for an additional 1 hour while shaking at 250 rpm. Then, 1×10 10 individual helper phage M13KO7 (NEB, catalog #N0315S) were added to the cell culture and incubation was continued at 37 °C for 4 hours. The cell culture was centrifuged at 3300 g for 30 minutes to pellet cell debris, the supernatant containing phage particles was collected, and mixed with 3 / 10 volume of PEG8000 / NaCl solution (20% PEG in 2.5 M NaCl solution autoclaved before use). The phage / PEG solution mixture was incubated on ice for 4 hours and centrifuged at 3300 g for 30 minutes. The final phage pellet was resuspended in 100 ml of PBS buffer containing 20% glycerol, aliquoted into 1 ml volumes, and stored at -80 °C. Phage panning method

[0278] Phage panning was performed using immobilized B7H3-hFc protein. To eliminate hFc tag binders, IAB-hFc control was also immobilized in parallel. ELISA plates (96-well) were coated with B7H3-hFc and IAB-hFc proteins (100 μg / mL in PBS) at 50 μl / well and incubated at 37 °C for 1 hour. After discarding the coated protein solution, the plates and phage solution were pre-blocked by mixing with PBS buffer containing 2% BSA and incubated at 37 °C for 30 minutes. After discarding the blocking buffer, the pre-blocked phage solution was added to the IAB-hFc plates and incubated at 37 °C for 1 hour to deplete hFc binders. Then, the unbound phage solution was transferred to the B7H3-hFc plates and incubated at 37 °C for 1 hour. B7H3-specific phage binders were eluted from the plates by incubation with citrate buffer at pH 2.0 and immediately neutralized with Tris-HCl buffer at pH 8.0. The eluted output phage was re-amplified by reinfection of fresh TG1 cells, and the re-amplified phage was used as input for the next round of panning. After three rounds of panning, single colonies were randomly selected from TG1 cells infected with the output phage, and monoclonal phage ELISA was performed to identify B7H3-specific binders. Phage ELISA

[0279] ELISA plates (96-well) were coated with B7H3-hFc and IAB-hFc tag controls. After blocking with PBS buffer containing 2% BSA, 50 microliters of pre-blocked phage solution was added to the plates and incubated at 37 °C for 30 minutes. The plates were washed twice with PBS buffer containing 0.05% Tween® 20, and phage binding was detected with anti-M13 antibody conjugated to HRP (Sinobiological, catalog #11973-MM05T-H).

[0280] For antibody-binding ELISA, antibodies at various concentrations (starting from 100 μg / mL and serially diluted 1:2) were incubated on plates coated with B7H3-hFc as described above, and antibody binding was detected with anti-FLAG mouse monoclonal antibody M2 (Sigma, catalog #A8592) conjugated to HRP. Flow cytometry method

[0281] Cells were harvested by detachment with trypsin-EDTA (ThermoFisher, catalog #25200114), centrifuged to form a pellet, and resuspended in ice-cold PBS. Cells at 1 million cells per ml were incubated with 10 μg / mL of the B7H3 domain antibody. Antibody binding was detected with anti-FLAG mouse monoclonal antibody conjugated to APC (Biolegend, catalog 637308). Fluorescence associated with live cells was measured using a FACS Calibur (BD Biosciences, Franklin Lakes, NJ). Statistical analysis

[0282] All statistical analyses were performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). (Example 2) Generation of rabbit nanobodies against B7H3 by protein immunization and phage display

[0283] This example describes the selection and characterization of two B7H3-specific rabbit single-domain VH monoclonal antibodies. Preparation of recombinant B7H3 protein

[0284] The extracellular domain (ECD) of B7H3 (NP_001019907, amino acids 29 - 466; SEQ ID NO: 13) was fused with human IgG1 Fc and expressed in HEK293 cells by secretion. After purification on a protein A column, the purity was checked by running on SDS-PAGE (Figure 1). The theoretical size of reduced B7H3-hFc is approximately 75 kD, and because B7H3 has six N-glycosylation sites, the apparent migration position on the gel is approximately 100 kD, presumably due to glycosylation. The transient expression level of B7H3-hFc is extremely low, approximately 0.5 mg / L. Immunization of Rabbits with Recombinant B7H3-hFc

[0285] 100 micrograms of B7H3-hFc in PBS buffer was mixed with an equal volume of Freund's adjuvant and injected intramuscularly into female New Zealand white rabbits. After three immunizations at 14-day intervals, the titer of anti-B7H3-hFc was measured by ELISA using IAB-hFc as an hFc control (Figure 2A). IAB is a fragment of mesothelin (Q13421, amino acids 296 - 359) (Kaneko et al., J Biol Chem, 284, 3739 - 3749, 2009). The sera from the second (M2) and third (M3) immunizations clearly showed increased binding to B7H3-hFc compared to the IAB-hFc tag control. The cell-binding activity of the polyclonal sera was checked by flow cytometry (Figure 2B). The polyclonal sera from the final immunization showed clear cell binding to the B7H3-positive hepatocellular carcinoma cell lines Hep3B and HepG2 (Wang et al., Cancer Invest, 32, 262 - 271, 2014; Qiu et al., Clin Chim Acta, 485, 103 - 105, 2018), while the pre-immune sera had extremely low background binding. The results of both ELISA and flow cytometry indicated that B7H3 has good immunogenicity in rabbits, despite this protein being highly conserved especially between humans and rabbits. Screening for B7H3-Specific Binding Agents

[0286] After confirmation of the success of immunization, the spleens of the immunized rabbits were harvested, VH gene fragments were cloned using degenerate primers (Peng et al., J Mol Biol, 429, 2954-2973, 2017), and then ligated with the phage display vector pComb3x. Using 10 micrograms of the ligation, TG1 competent cells were transformed by electroporation, and a VH library was generated with individual clones of 7×10 9 individual sizes. Both library phage production and subsequent antigen panning were performed at 37 °C.

[0287] Panning was performed on immobilized B7H3-hFc. Both B7-H6-hFc and IAB-hFc were coated on 96-well ELISA plates, and B7H3-specific phage particles were enriched by pre-absorbing them on plates coated with IAB-hFc and then captured on plates coated with B7H3-hFc. After three rounds of panning, monoclonal phage ELISA was performed to identify B7H3-specific binders. Of 96 randomly selected clones, 41 were B7H3-specific binders, and two representative binders named RFA1 and RFB1 were identified by sequencing analysis (Figure 3A). These two binders shared highly similar germline sequences that were also similar to the VH derived from rabbit anti-hypicin mAb (deposited in the GenBank structural database, PDB#5DUB).

[0288] Structural modeling of A1 and B1 using online tools showed that they could have similar CDR1 and CDR2 loop conformations that were also similar to the crystal structure of rabbit anti-hypicin VH (PDB#5DUB), although their CDR3 loops appeared to be different (Figure 3B). Binding characteristics of the B7H3 binders

[0289] The VH coding sequences of binders A1 and B1 were fused with His-FLAG tags at their C-termini and cloned into an E. coli expression vector. The soluble VH domains were purified by one-step Ni-affinity chromatography according to the laboratory protocol (Feng et al., Antib Ther, 2, 1-11, 2019). The purification yields were 2 mg / L (A1) and 10 mg / L of fermentation (B1), respectively. The purity was high as separated by SDS-PAGE (Figure 4A). The protein binding affinities of RFA1 and RFB1 were measured by ELISA, and the calculated EC50 values were 403 nM (A1) and 189 nM (B1) (Figure 4B), which are relatively low but common for VH-only domain antibodies. The cell binding ability was also tested by flow cytometry (Figure 4C), showing that A1 and B1 binders had good cell binding to B7H3-positive cell lines (IMR32, MC38-B7H3+, A431, and NBEB), but not to the B7H3 knockout cell lines IMR32-B7H3 KO and MC38-B7H3 KO. Therapeutic application

[0290] Rabbits are an excellent source for generating superior monoclonal antibodies for diagnostic and therapeutic purposes as research tools (Weber et al., Exp Mol Med, 49, e305, 2017). There are several major advantages to using rabbit antibodies. First, rabbits are phylogenetically more distant from humans than mice, and thus conserved proteins that are poorly immunogenic in mice may have better immunogenicity in rabbits (Popkov et al., J Mol Biol, 325, 325-335, 2003). Second, rabbit monoclonal antibodies generally have high affinity, specifically in the affinity range of 20-200 pM (Weber et al., Exp Mol Med, 49, e305, 2017; Landry et al., J Immunol Methods, 417, 86-96, 2015). Third, rabbit monoclonal antibodies can be successfully humanized (Zhang and Ho, MAbs, 9, 419-429, 2017), and thus immunogenicity should not be a barrier to their therapeutic application. Despite many advantages, only a few rabbit monoclonal antibodies have been investigated with respect to clinical application.

[0291] Rabbit monoclonal antibodies have been widely used as excellent research reagents for many years, but the potential advantages of rabbit VH domain antibodies have not been exploited. Recently, a group demonstrated that high-affinity rabbit VH domain antibodies can be generated by a low-temperature (i.e., 16°C) phage display method (Shinozaki et al., Sci Rep, 7, 5794, 2017), which will greatly accelerate the research on rabbit VH domain antibodies. However, the low-temperature phage display method tends to enrich a significant portion of unstable and poorly expressed binders, which may require considerable efforts to improve the physicochemical properties, especially expression and thermal stability (Shinozaki et al., J Biosci Bioeng, 125, 654-661, 2018). The current study investigated the possibility of screening binders that are well-expressed with thermal stability using a high-temperature phage display method.

[0292] As a proof of concept, B7H3 was selected as a target. B7H3 is overexpressed in many cancer types and can inhibit T cell activation, so B7H3 is considered a member of an important immune checkpoint of the B7 and CD28 families (Picarda et al., Clin Cancer Res, 22, 3425-3431, 2016). B7H3 is also overexpressed extensively in many solid tumors and is targeted for treatment (Seaman et al., Cancer Cell, 31, 501-515 e508, 2017). The extracellular domain of B7H3 was expressed in 293F cells, but the expression level was very low (<0.5 mg / L). B7H3 is highly conserved among humans, mice, and rabbits, but the recombinant B7H3-hFc protein had appropriate immunogenicity in rabbits, as shown by polyclonal immune sera that could bind both the recombinant protein and B7H3-positive cells. Using a high-temperature (37°C) phage display method, rabbit VH phage library particles were prepared and panning was performed. Two representative binders with moderate affinity for B7H3 protein and B7H3-positive cancer cells were obtained. The RFA1 and RFB1 binders have good cell binding to B7H3-positive cells but not to B7H3 knockout cells. This study demonstrated that rabbit VH domain antibodies with moderate affinity and expression levels can be generated by phage display at normal temperature (37°C). Considering the important role of B7H3 in regulating T cell function, the two generated B7H3 domain antibodies can be used for cancer immunotherapy applications. (Example 3) Isolation of Camel Nanobodies (CD276) Targeting B7H3 by Phage Display

[0293] This example describes the selection of 10 camel V H H nanobodies from a phage display library and the characterization of their binding properties. Chimeric antigen receptors composed of V H H nanobodies are also described. Eight VHHs related to the B7H3 binder H Phage panning in the H library

[0294] The B7H3-Fc fusion protein was generated and selected for the B7H3 binder. The recombinant B7H3-Fc fusion protein was expressed in HEK-293 cells and purified using a Protein A column (GE Healthcare) with an AKTA Explorer (GE Healthcare). The purified B7H3-Fc fusion protein had a purity of over 99% as shown on an SDS-PAGE gel and had a molecular weight of 154 kDa under non-reducing conditions and 77 kDa under reducing conditions (Figure 5). The yield of B7H3-Fc was 2 mg / L.

[0295] Eight VHHs prepared from eight dromedary camels (Camelus dromedaries) H Using the H single-domain antibody library, phage panning with the recombinant B7H3-Fc protein was performed three times. The phage titers for each round are shown in Figure 6. The increase in phage titer in the third round of phage panning indicated the enrichment of high-affinity V H HH binders to B7H3. Phage binding to B7H3-Fc was also evaluated by ELISA; the results are shown in Table 2. All of the selected phages were able to bind to B7H3-Fc but not to the IgG control. Table 2: Selected phage binding to B7H3-Fc by ELISA

Table 2-1

Table 2-2

[0296] The binding of selected B7H3-specific phages to monkey, mouse, rat, and human B7H3 was tested by ELISA. As shown in Table 3, 9 of the selected phages were able to bind to monkey B7H3, 5 phages were able to bind to mouse B7H3, 9 phages were able to bind to rat B7H3, and 8 phages were able to bind to human B7H3 (the numbers in bold indicate positive binding). Table 3: Cross-species binding of selected B7H3 phages by ELISA

Table 3

[0297] V H H nanobodies were purified from the selected phages. The purification of RWC4, RWG8, and RWB12 nanobodies is shown in Figures 7 - 9. The V H H camelid nanobody fractions eluted from the AKTA Explorer (GE Healthcare) were subjected to SDS-PAGE (see Figures 7A, 8A, and 9A). The chromatograms of the nanobodies eluted from the nickel column (GE Healthcare) on the AKTA Explorer (GE Healthcare) are shown in Figures 7B, 8B, and 9B. The yields of RWC4, RWG8, and RWB12 were 33.8 mg / L, 50 mg / L, and 132 mg / L, respectively.

[0298] The binding of selected V H H nanobodies to hB7H3-Fc, hB7H3-His, mouse B7H3-His, monkey B7H3-His, and rat B7H3-His fusion proteins was measured by ELISA. The results are shown in Table 4 and Figure 22. Except for RWB2, V H H was able to bind to B7H3-Fc and B7H3-His fusion proteins. RWG8 showed cross-reactivity to mouse B7H3 (Figure 22). Furthermore, RWA12 showed cross-reactivity with PBS and PD-L1, and the remaining V H H did not cross-react with PDL1 (Table 5). Table 4: Binding of Selected VH to B7H3 Fusion Protein by ELISA H [Table 4] Table 5: Cross-Reactivity of Selected B7H3-Binding Agents with PD-L1 by ELISA [Table 5] Binding of Selected B7H3-Targeted VH H to B7H3-Expressing Cells

[0299] Binding of Selected B7H3-Targeted H VH Nanobodies to NBEB Neuroblastoma Cells was Evaluated by FACS Analysis (Figure 10). Six of the Test Antibodies (RWC4, RWB12, RWG8, RWA12, RWG4, and RWD5) were Able to Bind NBEB Cells.

[0300] Binding of Selected B7H3-Targeted H VH Nanobodies to A431 Epidermoid Carcinoma Cells was Evaluated by FACS Analysis (Figure 11). Six of the Test Antibodies (RWC4, RWB12, RWG8, RWA12, RWG4, and RWD5) were Able to Bind A431 Cells.

[0301] Five of the B7H3-Targeted Antibodies (RWC4, RWB12, RWG8, RWG4, and RWD5) were Also Evaluated for Their Ability to Bind MC38-CD276+ and MC38-CD276KO Cells. All of the Test Antibodies Bound to B7H3-Positive Cells but Not to B7H3-Negative Cells (See Table 6). Table 6: Summary of Cellular Binding Ability of Five VH H [Table 6] Binding Kinetics

[0302] ​​The association / dissociation characteristics of RWC4 and RWG4 were measured using either recombinant human B7H3-Fc protein or recombinant mouse B7H3-His protein in an Octet system (Creative Biolabs).

[0303] The kinetics of RWC4 binding to human B7H3-Fc are shown in Figure 12 and summarized in Table 7. The K D of RWC4 for human B7H3-Fc was 3.8×10 -9 . Table 7: Kinetics of RWC4 for human B7H3-Fc

Table 7

[0304] The kinetics of RWG4 binding to human B7H3-Fc are shown in Figure 13 and summarized in Table 8. The K D of RWG4 for human B7H3-Fc was 6.94×10 -9 . Table 8: Kinetics of RWG4 for human B7H3-Fc

Table 8

[0305] Several cancer cell lines were evaluated for B7H3 expression by FACS. Expression was detected in 14 cell lines but not in 2 cell lines in which B7H3 (CD276) was knocked out (see Table 9). Table 9: Summary of B7H3-expressing cells

Table 9-1

Table 9-2

[0306] First, PCR was performed to amplify the nanobody sequence, and the backbone of plasmid Pwpt and the PCR product were digested with NdeI and SpeI, respectively, and the digested backbone was ligated with the digested PCR product. After transformation, bacteria were selected on ampicillin plates.

[0307] The T cell transfection efficiency of the lentivirus expressing the B7H3-targeted CAR was measured by FACS. The results are shown in Figure 14 and Table 10. Table 10: Transfection efficiency of CAR-T cells

Table 10

[0308] The B7H3-targeted CAR-T cells were tested for cytotoxicity against B7H3-positive and B7H3-knockout cells. Figure 15 shows the results of a cytotoxicity assay using B7H3-positive human neuroblastoma NBEB cells (Figure 15A), human neuroblastoma LAN-1 cells (Figure 15B), human adenocarcinoma BXPC-3 cells (Figure 15C), and human pancreatic cancer Miacapa2 cells (Figure 15D). In this assay, the CAR-T cells of RWB12, RWG8, and RWC4 were the most effective in inducing specific lysis.

[0309] In a second assay, the cytotoxicity of B7H3-targeted CAR-T cells in B7H3-positive and B7H3-knockout cells was evaluated. Figure 16 shows the results of a cytotoxicity assay using human neuroblastoma IMR32 cells (Figure 16A), mouse colon adenocarcinoma MC38-CD276 + cells (Figure 16B), human neuroblastoma IMR32-CD276 - / - cells (Figure 16C), and mouse colon adenocarcinoma MC38-CD276 - / - cells (Figure 16D). Three of the CAR-T cells (RWB12, RWG8, and RWC4) showed strong cytotoxicity in B7H3-positive cells but not in B7H3-negative cells. (Example 4) B7H3-targeted CAR T cells kill pancreatic tumor cells in vitro and in vivo

[0310] The in vitro cytotoxicity of human B7H3-targeted nanobody-derived CAR T cells (abbreviated herein as RWB12, RWG8, and RWC4, which are B12, G8, and C4, respectively) was evaluated using two B7H3-positive pancreatic cancer cell lines expressing luciferase: Panc-1 GFP-Luc (GL) and BxPC-3 GL. First, flow cytometry was performed to confirm that both Panc-1 cells and BxPC-1 cells express B7H3 (Figure 17A). The transduction efficiency of lentiviral constructs expressing B7H3-targeted CAR and CAR targeting CD19, respectively, was also determined by flow cytometry. As shown in Figure 17B, the transduction efficiencies of G8, C4, and B12 CARs were 60.4%, 58.6%, and 68.4%, respectively, while the transduction efficiency of T cells with irrelevant CAR (CD19) was 32%. To evaluate cytotoxicity, B7H3-targeted (C4, G8, and B12) CAR T cells and control (CD19) CAR T cells were incubated with Panc-1 GL cells or BxPC-3 GL cells for 24 hours at various effector:target (E:T) ratios. Both Panc-1 GL cells and BxPC-3 GL cells were effectively lysed in a dose-dependent manner by all three B7H3-targeted CAR T cells, while minimal killing was observed from control CAR T cells. These results demonstrate that B7H3-targeted nanobody-based CAR T cells were able to efficiently lyse B7H3-positive pancreatic cancer cell lines in vitro.

[0311] The B7H3-targeted CAR T cells were further evaluated in a Panc-1 mouse xenograft model. One study utilized a high dose of CAR T cells (10 million), and a second study utilized a lower dose (5 million CAR T cells). A schematic of the experimental design for the high-dose study is shown in Figure 18A. 1 million Panc-1 GFP / Luc tumor cells were transplanted i.v. into NSG mice to establish a tumor model. Twenty days later (day 0), 10 million C4, G8, or B12 CAR T cells (or control CD19 CAR T cells) were injected i.v. into the mice, and imaging was performed weekly. Representative bioluminescence images of Panc-1 tumor growth are shown in Figure 18B. Mice treated with 10 million B7H3-targeted CAR T cells (C4, G8, or B12) showed a significant decrease in tumor growth compared to injection of control CAR T cells, as demonstrated by the decrease in tumor bioluminescence measured as photons per second in mice treated with CAR T cells (Figure 18C). The survival of mice treated with B7H3-targeted CAR T cells was also determined. Figure 18D shows the Kaplan-Meier survival curves of tumor-bearing mice after treatment with 10 million C4, G8, or B12 CAR T cells. The results demonstrate that C4 CAR T cells are potent in promoting mouse survival compared to G8 or B12 CAR T cells when administered at a high dose (10 million), indicating that administration of 10 million CAR T cells is safe for mice.

[0312] The second in vivo study evaluated the treatment of Panc-1 tumor-bearing mice with lower doses of B7H3-specific CAR T cells injected after tumor re-challenge. A schematic diagram of the experimental design of this study is shown in Figure 19A. Twenty days (day 0) after inoculation of 1 million Panc-1-Luc cells into Panc-1 xenograft mice, 5 million C4 CAR T cells, 5 million B12 CAR T cells, 5 million untransduced T cells (mock) or PBS were injected i.v. Mice treated with C4 CAR T cells and B12 CAR T cells that did not show detectable tumors were transplanted i.v. with 1 million Panc-1 cells on day 35. As a control, naive mice were transplanted with Panc-1 cells. Imaging was performed weekly. Representative bioluminescence images of Panc-1 tumor growth in mice treated with CAR T cells are shown in Figure 19B. Mice treated with 5 million C4 CAR T cells or 5 million B12 CAR T cells showed significantly reduced tumor growth compared to mice administered mock T cells or PBS. Tumors grew rapidly in control mice, but 100% of the mice previously treated with C4 CAR T cells remained tumor-free after re-challenge with Panc-1 tumors, and 60% of the mice previously treated with B12 CAR T cells remained tumor-free until 10 weeks after treatment. Quantification of tumor bioluminescence is shown in Figure 19C. The survival period of mice treated with B7H3-targeted CAR T cells was also determined. Figure 19D shows the Kaplan-Meier survival curves of tumor-bearing mice after treatment. Mice that received 5 million C4 or B12 CAR T cells were still alive on day 70. In contrast, mice treated with PBS or mock T cells did not survive beyond 30 days after injection. (Example 5) B7H3-targeted CAR T cells kill neuroblastoma tumor cells in vitro and in vivo

[0313] The cytotoxicity of B7H3-targeted CAR T cells against the neuroblastoma cell line IMR5 was tested in vitro. This study compared CAR T cells generated using the B7H3-targeted nanobodies disclosed herein with CAR T cells based on the commercially available anti-human B7H3 hybridoma antibody 376.96 (Du et al., Cancer Cell 35(2): 221-237, 2019). G8, B12, C4, and 376.96 CAR T cells were incubated with IMR5 GL cells at various effector:target (E:T) ratios for 24 hours. All CAR T cells effectively lysed IMR5 tumor cells in a dose-dependent manner compared to mock T cells, although B12 CAR T cells were slightly more effective than the other CAR T cells tested (Figure 20A).

[0314] Next, the CAR T cells were evaluated in an IMR5 xenograft model (see schematic in Figure 20B). IMR5 xenograft mice were injected i.v. with 5 million C4 CAR T cells, B12 CAR T cells, G8 CAR T cells, 376.96 CAR T cells, or untransduced T cells (mock) 35 days after tumor inoculation (day 0). Representative bioluminescence images of IMR5 tumor growth in the xenograft model are shown in Figure 20C, and quantification of tumor bioluminescence is shown in Figure 20D. Mice treated with 5 million B12 CAR T cells showed significantly reduced tumor growth compared to 376.96 CAR T cells and mock T cells. C4 CAR T cells also showed moderate antitumor activity. (Example 6) Cross-reactivity of G8 against mouse B7H3

[0315] The binding activities of anti-B7H3 nanobodies G8, C4, and B12 to mouse B7H3 were measured by flow cytometry. G8 showed positive binding to mouse B7H3 expressed in three KPC cell lines (CREP128096, CREP133239, and PDA95775; pancreatic ductal adenocarcinoma cells) and mouse melanoma cell line B16, while neither C4 nor B12 showed such binding. The in vitro cytotoxicity of B7H3-targeted CAR was evaluated using B16 melanoma cells and B7H3 (CD276) knockout cells. Only G8 CAR T cells showed specific killing of mouse B7H3-positive B16 cells (Figure 21B). (Example 7) Epitope mapping of B7H3 nanobodies

[0316] Epitope mapping of anti-B7H3 nanobodies and the commercial antibody 376.96 was performed. A total of 48 peptides derived from human B7H3 protein were designed and synthesized. Each peptide consisted of 18 amino acids and overlapped with adjacent peptides by 9 amino acids. The binding ability of each antibody to each peptide was tested using ELISA technology (Figure 21C). The results demonstrate that G8 and 376.96 bind to similar epitopes as they both bound to peptides 10, 11, and 15 (SEQ ID NOs: 35 - 37). C4 and B12 may have conformational epitopes that could not be predicted by a library of linearized peptides.

[0317] Considering the many possible embodiments to which the principles of the disclosed subject matter may be applied, it should be recognized that the illustrated embodiments are merely examples of the disclosure and should not be construed as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. Accordingly, the inventors claim rights to all within the scope and spirit of these claims. In certain embodiments, for example, the following items are provided. (Item 1) A single-domain monoclonal antibody that specifically binds to B7H3, the antibody comprising complementarity-determining region 1 (CDR1), CDR2, and CDR3 sequences of SEQ ID NO: 3, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. (Item 2) The antibody according to Item 1, wherein the CDR sequences are defined using the Kabat, IMGT, or Paratome numbering scheme, or a combination of the Kabat, IMGT, and Paratome numbering schemes. (Item 3) The CDR1, CDR2, and CD3 sequences are respectively residues 31-35, 50-66, and 97-114 of SEQ ID NO: 3; residues 26-33, 51-58, and 97-115 of SEQ ID NO: 3; or residues 26-35, 50-61, and 98-114 of SEQ ID NO: 3 The antibody according to Item 1 or Item 2. (Item 4) The CDR1, CDR2, and CD3 sequences are respectively residues 31-35, 50-66, and 97-118 of SEQ ID NO: 1; residues 26-33, 51-58, and 97-119 of SEQ ID NO: 1; or residues 27-35, 47-62, and 98-118 of SEQ ID NO: 1 The antibody according to Item 1 or Item 2. (Item 5) The CDR1, CDR2, and CD3 sequences are respectively residues 31-35, 50-66, and 97-105 of SEQ ID NO: 2; residues 26-33, 51-58, and 97-106 of SEQ ID NO: 2; or residues 27-35, 47-61, and 97-106 of SEQ ID NO: 2 The antibody according to Item 1 or Item 2. (Item 6) The CDR1, CDR2, and CD3 sequences are respectively residues 31-35, 50-65, and 96-110 of SEQ ID NO: 4; Residues 26 - 33, 51 - 57, and 96 - 110 of SEQ ID NO:4; or Residues 27 - 35, 47 - 60, and 96 - 109 of SEQ ID NO:4 The antibody according to item 1 or item 2, comprising the same. (Item 7) Wherein said CDR1, CDR2, and CDR3 sequences are respectively Residues 27 - 30, 45 - 61, and 90 - 109 of SEQ ID NO:5; Residues 26 - 28, 46 - 53, and 90 - 110 of SEQ ID NO:5; or Residues 27 - 30, 43 - 55, and 90 - 110 of SEQ ID NO:5 The antibody according to item 1 or item 2, comprising the same. (Item 8) Wherein said CDR1, CDR2, and CDR3 sequences are respectively Residues 31 - 35, 50 - 65, and 96 - 111 of SEQ ID NO:6; Residues 26 - 33, 51 - 57, and 96 - 112 of SEQ ID NO:6; or Residues 27 - 35, 47 - 60, and 96 - 111 of SEQ ID NO:6 The antibody according to item 1 or item 2, comprising the same. (Item 9) Wherein said CDR1, CDR2, and CDR3 sequences are respectively Residues 31 - 35, 50 - 65, and 96 - 111 of SEQ ID NO:7; Residues 26 - 33, 51 - 57, and 96 - 112 of SEQ ID NO:7; or Residues 27 - 35, 47 - 60, and 96 - 111 of SEQ ID NO:7 The antibody according to item 1 or item 2, comprising the same. (Item 10) Wherein said CDR1, CDR2, and CDR3 sequences are respectively Residues 31 - 35, 50 - 65, and 96 - 111 of SEQ ID NO:8; Residues 26 - 33, 51 - 57, and 96 - 112 of SEQ ID NO:8; or Residues 27 - 35, 47 - 60, and 96 - 111 of SEQ ID NO:8 The antibody according to item 1 or item 2, comprising the same. (Item 11) Wherein said CDR1, CDR2, and CDR3 sequences are respectively Residues 31 - 35, 50 - 65, and 96 - 111 of SEQ ID NO:9; Residues 26 - 33, 51 - 57, and 96 - 112 of SEQ ID NO:9; or Residues 27 - 35, 47 - 60, and 96 - 111 of SEQ ID NO:9 The antibody according to item 1 or item 2, comprising the same. (Item 12) Wherein said CDR1, CDR2, and CDR3 sequences are respectively Residues 31 - 35, 50 - 65, and 96 - 113 of SEQ ID NO:10; Residues 26 - 33, 51 - 57, and 96 - 114 of SEQ ID NO:10; or Residues 27 - 35, 47 - 60, and 97 - 114 of SEQ ID NO:10 The antibody according to item 1 or item 2, comprising the same. (Item 13) Wherein said CDR1, CDR2, and CDR3 sequences are respectively Residues 30 - 34, 50 - 64, and 93 - 105 of SEQ ID NO:11; Residues 25-32, 50-56 and 93-104 of SEQ ID NO: 11; or Residues 26-34, 46-59 and 93-105 of SEQ ID NO: 11 The antibody according to item 1 or item 2, comprising the same. (Item 14) The CDR1, CDR2 and CD3 sequences are respectively Residues 32-35, 51-65 and 94-107 of SEQ ID NO: 12; Residues 26-33, 51-57 and 94-107 of SEQ ID NO: 12; or Residues 27-35, 47-60 and 94-108 of SEQ ID NO: 12 The antibody according to item 1 or item 2, comprising the same. (Item 15) The amino acid sequence of the antibody is at least 90% identical to SEQ ID NO: 3, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, the antibody according to any one of items 1 to 14. (Item 16) The amino acid sequence of the antibody comprises or consists of SEQ ID NO: 3, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, the antibody according to any one of items 1 to 15. (Item 17) The antibody according to any one of items 1 to 14, which is a humanized antibody. (Item 18) The antibody according to any one of items 1 to 14, which is a chimeric antibody. (Item 19) A chimeric antigen receptor (CAR) comprising the antibody according to any one of items 1 to 18. (Item 20) The CAR according to item 19, further comprising a hinge region, a transmembrane domain, a co-stimulatory signaling moiety, a signaling domain, or any combination thereof. (Item 21) The hinge region comprises a CD8α hinge region; The transmembrane domain comprises a CD8α transmembrane domain; The co-stimulatory signaling moiety comprises a 4-1BB signaling moiety; and / or The signaling domain comprises a CD3ζ signaling domain, The CAR according to item 20. (Item 22) An isolated cell expressing the CAR according to any one of items 19 to 21. (Item 23) The isolated cell according to item 22, which is a cytotoxic T lymphocyte (CTL) or a natural killer (NK) cell. (Item 24) An immunoconjugate comprising the antibody according to any one of items 1 to 18 and an effector molecule. (Item 25) The immunoconjugate according to item 24, wherein the effector molecule is a toxin. (Item 26) The immunoconjugate according to item 25, wherein the toxin is Pseudomonas exotoxin or a variant thereof. (Item 27) The immunoconjugate according to item 26, wherein the variant of Pseudomonas exotoxin is PE38. (Item 28) The immunoconjugate according to item 24, wherein the effector molecule is a photon absorber. (Item 29) The immunoconjugate according to item 24, wherein the effector molecule is a detectable label. (Item 30) The immunoconjugate according to item 29, wherein the detectable label includes a fluorophore, an enzyme, or a radioisotope. (Item 31) An antibody-drug conjugate (ADC) comprising a drug conjugated to an antibody according to any one of items 1 to 18. (Item 32) The ADC according to item 31, wherein the drug is a small molecule. (Item 33) The ADC according to item 31 or item 32, wherein the drug is a microtubule inhibitor, an anti-mitotic agent, and / or a cytotoxic agent. (Item 34) A multispecific antibody comprising an antibody according to any one of items 1 to 18 and at least one additional monoclonal antibody or an antigen-binding fragment thereof. (Item 35) The multispecific antibody according to item 34, which is a bispecific antibody. (Item 36) The multispecific antibody according to item 34, which is a trispecific antibody. (Item 37) The multispecific antibody according to any one of items 34 to 36, wherein the at least one additional monoclonal antibody or an antigen-binding fragment thereof specifically binds to a component of a T cell receptor or a natural killer (NK) cell activation receptor. (Item 38) An antibody-nanoparticle conjugate comprising a nanoparticle conjugated to an antibody according to any one of items 1 to 18. (Item 39) The antibody-nanoparticle conjugate according to item 38, wherein the nanoparticle includes a polymeric nanoparticle, a nanosphere, a nanocapsule, a liposome, a dendrimer, a polymeric micelle, or a niosome. (Item 40) The antibody-nanoparticle conjugate according to item 38 or item 39, wherein the nanoparticle includes a cytotoxic agent. (Item 41) A fusion protein comprising an antibody according to any one of items 1 to 18 and a heterologous protein or peptide. (Item 42) The fusion protein according to item 41, wherein the heterologous protein is an Fc protein or a leucine zipper. (Item 43) An isolated nucleic acid molecule encoding an antibody according to any one of items 1 to 18, a CAR according to any one of items 19 to 21, an immunoconjugate according to any one of items 24 to 30, a multispecific antibody according to any one of items 34 to 37, or a fusion protein according to item 41 or item 42. (Item 44) The isolated nucleic acid molecule according to item 43, operably linked to a promoter. (Item 45) A vector comprising the nucleic acid molecule according to item 43 or item 44. (Item 46) An isolated host cell comprising the nucleic acid molecule according to item 44 or the vector according to item 45. (Item 47) A composition comprising a pharmaceutically acceptable carrier and an antibody according to any one of items 1 to 18, a CAR according to any one of items 19 to 21, an isolated cell according to any one of items 22, 23 and 46, an immunoconjugate according to any one of items 24 to 30, an ADC according to any one of items 31 to 33, a multispecific antibody according to any one of items 34 to 37, an antibody-nanoparticle conjugate according to any one of items 38 to 40, or a fusion protein according to item 41 or item 42. (Item 48) A method for detecting the expression of B7H3 in a sample, comprising: contacting the sample with an antibody according to any one of items 1 to 18; and detecting the binding of the antibody to the sample, thereby detecting the expression of B7H3 in the sample. A method comprising the steps of: (Item 49) A method for diagnosing a subject as having B7H3-positive cancer, comprising: contacting a sample obtained from the subject with an antibody according to any one of items 1 to 18; and detecting the binding of the antibody to the sample, thereby diagnosing the subject as having B7H3-positive cancer. A method comprising the steps of: (Item 50) The method according to item 48 or item 49, wherein the antibody is directly labeled. (Item 51) contacting the antibody with a detection antibody; and detecting the binding of the detection antibody to the antibody, thereby further detecting the expression of B7H3 in the sample or diagnosing the subject as having B7H3-positive cancer, the method according to item 48 or item 49. (Item 52) The method according to any one of items 48 to 51, wherein the sample is obtained from a subject suspected of having B7H3-positive cancer. (Item 53) The method according to any one of items 48 to 52, wherein the sample is a tumor biopsy. (Item 54) A method for treating B7H3-positive cancer in a subject, comprising administering to the subject the antibody according to any one of items 1 to 18, the CAR according to any one of items 19 to 21, the isolated cell according to any one of items 22 to 23, the immunoconjugate according to any one of items 24 to 30, the ADC according to any one of items 31 to 33, the multispecific antibody according to any one of items 34 to 37, the antibody-nanoparticle conjugate according to any one of items 38 to 40, the fusion protein according to item 41 or item 42, or the composition according to item 47. (Item 55) A method for inhibiting tumor growth or metastasis of B7H3-positive cancer in a subject, comprising administering to the subject the antibody according to any one of items 1 to 18, the CAR according to any one of items 19 to 21, the isolated cell according to any one of items 22 to 23, the immunoconjugate according to any one of items 24 to 30, the ADC according to any one of items 31 to 33, the multispecific antibody according to any one of items 34 to 37, the antibody-nanoparticle conjugate according to any one of items 38 to 40, the fusion protein according to item 41 or item 42, or the composition according to item 47. (Item 56) The method according to item 54 or item 55, wherein the B7H3-positive cancer is a solid tumor. (Item 57) The method according to item 56, wherein the solid tumor is liver cancer, pancreatic cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer, pediatric cancer, melanoma or mesothelioma. (Item 58) The method according to item 57, wherein the liver cancer is hepatocellular carcinoma. (Item 59) The method according to item 57, wherein the brain cancer is neuroblastoma or glioblastoma. (Item 60) The method according to item 57, wherein the pediatric cancer is osteosarcoma, neuroblastoma, rhabdomyosarcoma or Ewing sarcoma.

Claims

1. A single-domain monoclonal antibody that specifically binds to B7H3, comprising complementarity-determining region 1 (CDR1), CDR2, and CDR3 sequences of SEQ ID NO: 3, wherein said CDR sequences are defined using the Kabat, IMGT, Paratome, or Chothia numbering scheme, or any combination thereof, an antibody.

2. Said CDR1, CDR2, and CDR3 sequences are respectively residues 31-35, 50-66, and 97-114 of SEQ ID NO: 3; residues 26-33, 51-58, and 97-115 of SEQ ID NO: 3; or residues 26-35, 50-61, and 98-114 of SEQ ID NO: 3 The antibody according to claim 1, comprising.

3. The antibody according to claim 1 or claim 2, wherein the amino acid sequence of said antibody is at least 90% identical to SEQ ID NO:

3.

4. The antibody according to any one of claims 1 to 3, wherein the amino acid sequence of said antibody comprises or consists of SEQ ID NO:

3.

5. The antibody according to claim 1 or claim 2, which is a humanized antibody.

6. The antibody according to claim 1 or claim 2, which is a chimeric antibody.

7. A chimeric antigen receptor (CAR) comprising the antibody according to any one of claims 1 to 6.

8. The CAR according to claim 7, further comprising a hinge region, a transmembrane domain, a co-stimulatory signaling moiety, a signaling domain, or any combination thereof.

9. Said hinge region comprises a CD8α hinge region; Said transmembrane domain comprises a CD8α transmembrane domain; Said co-stimulatory signaling moiety comprises a 4-1BB signaling moiety; and / or Said signaling domain comprises a CD3ζ signaling domain, The CAR according to claim 8.

10. An isolated cell expressing the CAR according to any one of claims 7 to 9.

11. The isolated cell according to claim 10, which is a cytotoxic T lymphocyte (CTL) or a natural killer (NK) cell.

12. An immunoconjugate comprising the antibody according to any one of claims 1 to 6 and an effector molecule.

13. The immunoconjugate according to claim 12, wherein said effector molecule is a toxin.

14. The immunoconjugate according to claim 13, wherein said toxin is Pseudomonas exotoxin or a variant thereof.

15. The immunoconjugate according to claim 14, wherein the variant of the Pseudomonas exotoxin is PE38.

16. The immunoconjugate according to claim 12, wherein the effector molecule is a photon absorber.

17. The immunoconjugate according to claim 12, wherein the effector molecule is a detectable label.

18. The immunoconjugate according to claim 17, wherein the detectable label comprises a fluorophore, an enzyme, or a radioisotope.

19. An antibody-drug conjugate (ADC) comprising a drug conjugated to an antibody according to any one of claims 1 to 6.

20. The ADC according to claim 19, wherein the drug is a small molecule.

21. The ADC according to claim 19 or claim 20, wherein the drug is a microtubule inhibitor, an anti-mitotic agent, and / or a cytotoxic agent.

22. A multispecific antibody comprising an antibody according to any one of claims 1 to 6 and at least one additional monoclonal antibody or an antigen-binding fragment thereof.

23. The multispecific antibody according to claim 22, which is a bispecific antibody.

24. The multispecific antibody according to claim 22, which is a trispecific antibody.

25. The multispecific antibody according to any one of claims 22 to 24, wherein the at least one additional monoclonal antibody or an antigen-binding fragment thereof specifically binds to a component of a T cell receptor or a natural killer (NK) cell activating receptor.

26. An antibody-nanoparticle conjugate comprising a nanoparticle conjugated to an antibody according to any one of claims 1 to 6.

27. The antibody-nanoparticle conjugate according to claim 26, wherein the nanoparticle comprises a polymeric nanoparticle, a nanosphere, a nanocapsule, a liposome, a dendrimer, a polymeric micelle, or a niosome.

28. The antibody-nanoparticle conjugate according to claim 26 or claim 27, wherein the nanoparticle comprises a cytotoxic agent.

29. A fusion protein comprising an antibody according to any one of claims 1 to 6 and a heterologous protein or peptide.

30. The fusion protein according to claim 29, wherein the heterologous protein is an Fc protein or a leucine zipper.

31. An isolated nucleic acid molecule encoding an antibody according to any one of claims 1 to 6, a CAR according to any one of claims 7 to 9, an immunoconjugate according to any one of claims 12 to 18, a multispecific antibody according to any one of claims 22 to 25, or a fusion protein according to claim 29 or claim 30.

32. The isolated nucleic acid molecule according to claim 31, operably linked to a promoter.

33. A vector comprising the nucleic acid molecule according to claim 31 or claim 32.

34. An isolated host cell comprising the nucleic acid molecule according to claim 32 or the vector according to claim 33.

35. A composition comprising a pharmaceutically acceptable carrier and an antibody according to any one of claims 1 to 6, a CAR according to any one of claims 7 to 9, an isolated cell according to any one of claims 10, 11 and 34, an immunoconjugate according to any one of claims 12 to 18, an ADC according to any one of claims 19 to 21, a multispecific antibody according to any one of claims 22 to 25, an antibody-nanoparticle conjugate according to any one of claims 26 to 28, or a fusion protein according to claim 29 or claim 30.

36. A method for detecting the expression of B7H3 in a sample, comprising: contacting the sample with an antibody according to any one of claims 1 to 6; and detecting the binding of the antibody to the sample, thereby detecting the expression of B7H3 in the sample. A method comprising the steps of:

37. A composition comprising an antibody according to any one of claims 1 to 6 for use in a method of diagnosing a subject as having B7H3-positive cancer, the method comprising: contacting a sample obtained from the subject with the composition; and detecting the binding of the antibody to the sample, thereby diagnosing the subject as having B7H3-positive cancer. A composition comprising the steps of:

38. The method according to claim 36, wherein the antibody is directly labeled.

39. The method according to claim 36, further comprising: contacting the antibody with a detection antibody; and detecting the binding of the detection antibody to the antibody, thereby detecting the expression of B7H3 in the sample.

40. The method according to any one of claims 36 and 38 to 39, wherein the sample is obtained from a subject suspected of having B7H3-positive cancer.

41. The method according to any one of claims 36 and 38 to 40, wherein the sample is a tumor biopsy.

42. The composition according to claim 37, wherein the antibody is directly labeled.

43. The method comprises contacting the antibody with a detection antibody detecting the binding of the detection antibody to the antibody, thereby detecting the expression of B7H3 in the sample or diagnosing the subject as having B7H3-positive cancer. The composition according to claim 37.

44. The composition according to any one of claims 37 and 42 to 43, wherein the sample is obtained from a subject suspected of having B7H3-positive cancer.

45. The composition according to any one of claims 37 and 42 to 44, wherein the sample is a tumor biopsy.

46. For treating B7H3-positive cancer in a subject, an antibody according to any one of claims 1 to 6, a CAR according to any one of claims 7 to 9, an isolated cell according to any one of claims 10 to 11, an immunoconjugate according to any one of claims 12 to 18, an ADC according to any one of claims 19 to 21, a multispecific antibody according to any one of claims 22 to 25, an antibody-nanoparticle conjugate according to any one of claims 26 to 28, a composition comprising a fusion protein according to claim 29 or claim 30, or a composition according to claim 35.

47. For inhibiting tumor growth or metastasis of B7H3-positive cancer in a subject, an antibody according to any one of claims 1 to 6, a CAR according to any one of claims 7 to 9, an isolated cell according to any one of claims 10 to 11, an immunoconjugate according to any one of claims 12 to 18, an ADC according to any one of claims 19 to 21, a multispecific antibody according to any one of claims 22 to 25, an antibody-nanoparticle conjugate according to any one of claims 26 to 28, a composition comprising a fusion protein according to claim 29 or claim 30, or a composition according to claim 35.

48. The composition according to claim 46 or claim 47, wherein the B7H3-positive cancer is a solid tumor.

49. The composition according to claim 48, wherein the solid tumor is liver cancer, pancreatic cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer, pediatric cancer, melanoma or mesothelioma.

50. The composition according to claim 49, wherein the liver cancer is hepatocellular carcinoma.

51. The composition according to claim 49, wherein the brain cancer is neuroblastoma or glioblastoma.

52. The composition according to claim 49, wherein the pediatric cancer is osteosarcoma, neuroblastoma, rhabdomyosarcoma or Ewing's sarcoma.

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