Nanobodies targeting and / or specifically binding to b7-h3

Abstract

Provided are single-domain monoclonal antibodies capable of specifically binding to B7-H3, and a multivalent antibody, a multispecific antibody, a chimeric antigen receptor, an engineered cell, an immunoconjugate, an ADC, an immune nanoparticle and a pharmaceutical composition including at least one of the single-domain monoclonal antibodies. Also provided are a use method and a use, for example, for detecting or diagnosing B7-H3 positive samples, or for treating various B7-H3 positive tumors.

Description

Nanobodies that bind to and / or are specific for b7-h3 TECHNICAL FIELD

[0001] The present application relates to the field of antibodies, in particular to single domain monoclonal antibodies that can specifically bind to B7-H3. The present application further relates to multivalent / multispecific antibodies, chimeric antigen receptors, engineered cells, immunoconjugates, ADCs, immunonanoparticles, pharmaceutical compositions comprising at least one of said monoclonal antibodies, as well as methods of use and uses thereof, e.g. for detecting or diagnosing B7-H3 positive samples, or for treating various B7-H3 positive solid tumors. BACKGROUND

[0002] B7-H3 (also known as CD276), which belongs to the B7 / CD28 immunoglobulin superfamily, is a transmembrane glycoprotein, and can be divided into two subtypes, 2Ig-B7-H3 and 4Ig-B7-H3, according to the number of V-like and C-like Ig domain structures in its extracellular part. B7-H3 is abnormally expressed in various tumor cells, tumor stroma, tumor blood vessels, and tumor-infiltrating macrophages and DC cells. It has been reported that the expression rate of B7-H3 in tissue samples of diseases such as prostate cancer, pancreatic cancer, hepatocellular carcinoma, head and neck cancer, and renal cancer is more than 90%, but rarely expressed in normal tissues (see Seaman S et al., Cancer Cell, 2017, 31(4): 501-515). Although the natural receptor of B7-H3 has not been determined, there are more and more reports about its involvement in the negative regulation of tumor immunity. For example, B7-H3 can inhibit T cell proliferation and cytokine release, or inhibit the lytic activity of NK cells on human tumor cells with high expression of B7-H3 (see Suh W K et al., Nature Immunology, 2003, 4(9): 899; Castriconi R et al., Proceedings of the National Academy of Sciences, 2004, 101(34): 12640-12645). The expression of B7-H3 in tumors is associated with poor prognosis, for example, in patients with lung cancer with high expression of B7-H3, it is found that there are fewer tumor-infiltrating lymphocytes and more likely to have lymph node metastasis (see Altan M et al., Clinical Cancer Research, 2017, 23(17): 5202).

[0003] Based on the tumor-specific expression and immune regulation activity of B7-H3, several anti-B7-H3 monoclonal antibody-based candidate drugs are in clinical research and development. For example, the humanized monoclonal anti-B7-H3 antibody Enoblituzumab with Fc optimization has shown therapeutic potential in the treatment of prostate cancer in phase II clinical trials. The murine anti-B7-H3 antibody ADC drug Burtomab conjugated with radioactive iodine has shown the ability to significantly prolong the survival of children with metastatic neuroblastoma in clinical trials.

[0004] However, when using the above full-size antibodies as therapeutic drugs, the disadvantages of full-size antibodies, such as high production cost, low stability, and large size interfering with the proximity to some hidden epitopes, are inevitable. The above problems can be solved by preparing nanobodies.

[0005] Nanobodies are the smallest known antigen-binding fragments of antibodies, most of which are derived from the variable region of the heavy chain (VH) of antibodies, usually consisting of about 120 amino acids, with a molecular weight of about 12-15 kD and a size of about 4 x 2.5 nM, which is much smaller in size than conventional four-chain antibodies. In 1989, Ward, E. et al. first screened two mouse VH domains from a cDNA expression library prepared from the spleen of a mouse immunized with lysozyme and keyhole limpet hemocyanin, which still showed a certain affinity for lysozyme, thus first proposing the concept of "single-domain antibody" (see Ward, E. et al., Nature 341, 544-546 (1989)). Subsequently, a large number of natural IgG containing only heavy chains (HCAb) were found in the serum of Camelidae, accounting for 45% to 75% of total serum immunoglobulin depending on the species (see Tu Z et al., Immunology. 2020 Sep; 161(1): 53-65). HCAb naturally lacks the light chain in the conventional four-chain antibody, consisting of two heavy chains each containing a heavy chain variable region (VH), a hinge region, and two CH2 and CH3 domains. Similar natural heavy chain antibodies are also found in cartilaginous fishes (VNAR) (Feng et al., Antib Ther, 2, 1-11, 2019) and some human heavy chain diseases (Prelli and Frangione, J Immunol, 148, 949-952, 1992). H H), one hinge region, and two CH2 and CH3 domains. Similar natural heavy chain antibodies are also found in cartilaginous fishes (VNAR) (Feng et al., Antib Ther, 2, 1-11, 2019) and some human heavy chain diseases (Prelli and Frangione, J Immunol, 148, 949-952, 1992).

[0006] Nanobodies, especially nanobodies based on recombinant VH domains prepared from camelid antibodies, are the current mainstream research and development direction. Compared with human VH domains, the following structural characteristics of naturally evolved camelid VH domains make them more suitable for the development of nanobodies: H H ​H domains have better solubility and stability: V37 in V H H domains, typically F37 or Y37 according to Kabat numbering, make the hydrophobic packing of the domain more compact and stable (Riechmann and Muyldermans, J Immunol Methods, 231, 25-38, 1999; Shinozaki et al., J Biosci Bioeng, 125, 654-661, 2018); light chain-touched residues G44, L45, and W47 in V H H, corresponding to E44 (or Q44), R45 (or C45), and G47 (or Ser, Leu, Phe) (Holt et al., Trends Biotechnol, 21, 484-490, 2003), make the accessible surface more hydrophilic and less aggregated; some V H H domains, W103 can be replaced by R103; V H H domains typically have longer CDR3 than human / rodent VH, and often contain a Cys in their CDR3, which can form an additional disulfide bond besides the canonical C22-C92 disulfide bond with the Cys at the end of CDR1 (camel) or the beginning of CDR2 (llama) (Wesolowsk et al., Med Microbiol Immunol, 198, 157-174, 2009), making V H H domains more stable (Tm values ranging from 60-78°C) and enabling reversible unfolding / refolding (Holt et al., Trends Biotechnol, 21, 484-490, 2003).

[0007] As a medical application or research tool, nanobodies have the following advantages: (1) higher permeability and tissue penetration, capable of penetrating membranes and penetrating physiological compartments, tissues and organs that are not in close proximity to each other, thus reducing the dosage and / or frequency of administration and reducing side effects; (2) improved physicochemical stability, including solubility, heat resistance, proteolytic resistance, half-life, etc., thus suitable for a wider range of administration routes including intravenous injection, oral, inhalation, subcutaneous, sustained release, etc.; (3) reduced immunogenicity; (4) increased recognition of epitopes, the small size and special CDR3 structure of single-domain antibodies make them more easily interact with epitopes that cannot be contacted by conventional antibodies. SUMMARY

[0008] The present application provides single-domain monoclonal antibodies capable of binding to and / or specifically binding to B7-H3. Also provided are a series of molecular entities formed based on the disclosed nanobodies, as well as methods of use and purposes thereof.

[0009] Accordingly, it is a first object of the present application to provide monoclonal antibodies against and / or binding (e.g., specifically binding) to B7H3. In some embodiments, the monoclonal antibodies comprise the complementarity determining region (CDR) sequences of Nanobody BP2-F12, BP1-C11, BP1-F9, BP1-C10, H-F6, BP2-A9, H-A3, BP1-H8, H-H6, H-A12, BP1-H12, BP2-A12, BP1-F1, BP2-A8, BP1-G2, BP1-C6, BP2-A4, BP1-F12.

[0010] It is a second object of the present application to provide covalent conjugates comprising the monoclonal antibodies disclosed herein. In some embodiments, the present application provides chimeric antigen receptors (CARs), immunoconjugates (e.g., Nanobody-immunotoxin conjugates, Nanobody-drug conjugates (ADCs)), multivalent antibodies (e.g., bivalent or trivalent antibodies), multispecific antibodies (e.g., bispecific T-cell engagers), antibody-nanoparticle conjugates, antibody-radioisotope conjugates (e.g., for cancer diagnosis and immunoPET imaging), and antibody fusion proteins (e.g., Nanobody-FC proteins) comprising the monoclonal antibodies of the present application.

[0011] It is a third object of the present application to provide engineered cells expressing the single-domain monoclonal antibodies and / or covalent conjugates thereof disclosed herein, in particular immune effector cells (e.g., CAR-T cells and CAR-NK (natural killer) cells) expressing the CARs of the present application.

[0012] It is a fourth aspect of the present application to provide nucleic acid molecules and vectors encoding the single-domain monoclonal antibodies against and / or specifically binding to B7-H3 disclosed herein or covalent conjugates thereof, including CARs, immunoconjugates, multivalent antibodies, multispecific antibodies, antibody-nanoparticles, antibody fusion proteins.

[0013] It is a fifth aspect of the present application to provide engineered cells comprising the encoding nucleic acids or vectors disclosed herein.

[0014] It is a sixth aspect of the present application to provide compositions comprising the single-domain monoclonal antibodies or covalent conjugates thereof disclosed herein and optionally pharmaceutically acceptable excipients.

[0015] A seventh aspect of the present application is to provide a method of detecting B7-H3 expression in a sample. In some embodiments, the method comprises contacting a sample to be tested with an antibody disclosed herein (e.g., a single domain monoclonal antibody, a multivalent antibody (e.g., a bivalent or trivalent antibody), a multispecific antibody (e.g., a bispecific T-cell engager), or a covalent conjugate thereof (e.g., an immunoconjugate comprising a monoclonal antibody of the present application (e.g., a nanobody-immunoconjugate, a nanobody-drug conjugate (ADC)), an antibody-nanoparticle, an antibody-radioisotope conjugate (e.g., for cancer diagnosis and immunoPET imaging), and an antibody fusion protein (e.g., a nanobody-FC protein)), and detecting the binding of the antibody or the covalent conjugate thereof to the sample. In some embodiments, the present application also provides use of an antibody disclosed herein or a covalent conjugate thereof in the preparation of a reagent for detecting B7-H3 expression in a sample.

[0016] An eighth aspect of the present application is to provide a method of diagnosing a subject with a B7-H3 positive cancer. In some embodiments, the method comprises contacting a biological sample obtained from the subject with an antibody disclosed herein or a covalent conjugate thereof, and detecting the binding of the antibody or the covalent conjugate thereof to the biological sample. In some embodiments, the present application also provides use of an antibody disclosed herein or a covalent conjugate thereof in the preparation of a reagent for diagnosing a subject with a B7-H3 positive cancer.

[0017] A ninth aspect of the present application is to provide a method of treating a B7-H3 positive cancer. In some embodiments, the method comprises administering to a patient in need thereof a therapeutically effective amount of an antibody disclosed herein or a covalent conjugate thereof, including a chimeric antigen receptor (CAR) comprising a monoclonal antibody of the present application, an immunoconjugate (e.g., a nanobody-immunoconjugate, a nanobody-drug conjugate (ADC)), a multivalent antibody (e.g., a bivalent or trivalent antibody), a multispecific antibody (e.g., a bispecific T-cell engager), an antibody-nanoparticle conjugate, an antibody-radioisotope conjugate (e.g., for cancer diagnosis and immunoPET imaging), and an antibody fusion protein (e.g., a nanobody-FC protein). In some embodiments, the present application also provides use of an antibody disclosed herein or a covalent conjugate thereof in the preparation of a reagent for treating a B7-H3 positive cancer. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a graph showing the results of SDS-PAGE of purified B7-H3 fusion proteins prepared in Example 1. Wherein lanes 1-4 are B7H3-his (reduced), B7H3-his (non-reduced), B7H3-FC (reduced), B7H3-FC (non-reduced), respectively;

[0019] Figure 2 is a graph showing the SDS-PAGE results of 12 antibody FC fusion proteins prepared in Example 4. In the figure, the antibody FC fusion proteins corresponding to each lane are as follows: 1: BP2-F12-FC; 2: BP1-C11-FC; 3: BP1-F9-FC; 4: BP1-C10-FC; 5: H-A3-FC; 6: H-H6-FC; 7: BP1-F1-FC; 8: BP2-A8-FC; 9: BP1-G2-FC; 10: BP1-C6-FC; 11: BP1-F12-FC; and 12: BP1-H8-FC.

[0020] Figure 3 is a graph showing the ELISA results of the binding activity of the FC fusion proteins of 12 B7-H3 single-domain V H H antibodies to the target antigen protein B7-H3.

[0021] Figure 4 is a graph showing the SDS-PAGE detection results of the BP2F12-HH6-FC antibody prepared in Example 5.

[0022] Figure 5 shows the binding curves of the BP2F12-HH6-FC, H-H6-FC and BP2-F12-FC antibodies to B7H3 protein as determined by ELISA.

[0023] Figure 6 shows the binding differences of the positive control antibody B7H3-PE / Cy7 antibody, the negative control antibody anti-human IgG antibody, and the exemplary B7-H3 nanobody BP2F12-HH6-FC, H-H6-FC and BP2-F12-FC of the present application to human neuroblastoma cells SH-SY5Y as determined by flow cytometry. In Figures 6A to 6D, the leftmost peak represents the binding fluorescence intensity of the anti-human IgG antibody to SH-SY5Y, and the other three peaks from left to right represent the binding fluorescence intensities of 0.0625 μg, 0.25 μg and 1 μg of the positive control antibody (Figure 6A), BP2F12-HH6-FC (Figure 6B), H-H6-FC (Figure 6C) and BP2-F12-FC (Figure 6D) to SH-SY5Y.

[0024] Figure 7 shows the difference in binding of the positive control antibody B7H3-PE / Cy7 antibody, the negative control antibody anti-human IgG antibody, and the exemplary B7-H3 Nanobodies BP2F12-HH6-FC, H-H6-FC and BP2-F12-FC of the present application to human neuroblastoma cells SK-N-AS as determined by flow cytometry; in Figures 7A to 7D, the leftmost peak in each of Figures 7A to 7D represents the binding fluorescence intensity of the anti-human IgG antibody to SK-N-AS, and the three remaining peaks from left to right represent the binding fluorescence intensity of 0.0625 pg, 0.25 pg and 1 pg of the positive control antibody (Figure 7A), BP2F12-HH6-FC (Figure 7B), H-H6-FC (Figure 7C) and BP2-F12-FC (Figure 7D) to SK-N-AS, respectively;

[0025] Figure 8 shows the difference in binding of the positive control antibody B7H3-PE / Cy7 antibody, the negative control antibody anti-human IgG antibody, and the exemplary B7-H3 Nanobodies BP2F12-HH6-FC, H-H6-FC and BP2-F12-FC of the present application to human malignant melanoma cells A375 as determined by flow cytometry; in Figures 8A to 8D, the leftmost peak in each of Figures 8A to 8D represents the binding fluorescence intensity of the anti-human IgG antibody to A375, and the three remaining peaks from left to right represent the binding fluorescence intensity of 0.0625 pg, 0.25 pg and 1 pg of the positive control antibody (Figure 8A), BP2F12-HH6-FC (Figure 8B), H-H6-FC (Figure 8C) and BP2-F12-FC (Figure 8D) to A375, respectively;

[0026] Figure 9 shows the difference in binding of the positive control antibody B7H3-PE / Cy7 antibody, the negative control antibody anti-human IgG antibody, and the exemplary B7-H3 Nanobodies BP2F12-HH6-FC, H-H6-FC and BP2-F12-FC of the present application to human non-small cell lung cancer cells A549 as determined by flow cytometry; in Figures 9A to 9D, the leftmost peak in each of Figures 9A to 9D represents the binding fluorescence intensity of the anti-human IgG antibody to A549, and the three remaining peaks from left to right represent the binding fluorescence intensity of 0.0625 pg, 0.25 pg and 1 pg of the positive control antibody (Figure 9A), BP2F12-HH6-FC (Figure 9B), H-H6-FC (Figure 9C) and BP2-F12-FC (Figure 9D) to A549, respectively;

[0027] Figure 10 shows the binding differences of the positive control antibody B7H3-PE / Cy7 antibody, the negative control antibody anti-human IgG antibody, and the exemplary B7-H3 Nanobodies BP2F12-HH6-FC, H-H6-FC and BP2-F12-FC of the present application to human T lymphocyte leukemia cells Jurkat as determined by flow cytometry; in Figures 10A to 10D, the leftmost peak in each of the graphs represents the binding fluorescence intensity of the anti-human IgG antibody to Jurkat, and the remaining three peaks from left to right represent the binding fluorescence intensity of 0.0625 pg, 0.25 pg and 1 pg of the positive control antibody (Figure 10A), BP2F12-HH6-FC (Figure 10B), H-H6-FC (Figure 10C) and BP2-F12-FC (Figure 10D) to Jurkat, respectively;

[0028] Figure 11 is a graph showing the flow cytometry detection results of CAR-T 001 to CAR-T 005 cells;

[0029] Figure 12 is a graph showing the quantitative evaluation of the tumor killing activity of CAR-T 001 to 005 cells and the IgG isotype negative control antibody by flow cytometry;

[0030] Figure 13 is a graph showing the surface binding of B7-H3 Nanobodies BP2F12-HH6-Fc and the IgG isotype antibody as a negative control to different B7-H3 positive tumor cells as determined by flow cytometry. DETAILED DESCRIPTION

[0031] The present application will be further described in conjunction with the specific embodiments, and the examples given are only for the purpose of illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0032] The experimental methods in the following examples, unless otherwise stated, were carried out using conventional techniques according to the techniques or procedures described in the literature or product instructions, as appropriate. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012; Ausubel et al., Current Protocols in Molecular Biology, Wiley Online Library, updated periodically. Materials, reagents, and instruments used in the following examples, unless otherwise specified, were obtained from commercial suppliers unless otherwise specified. Quantitative assays in the following examples, unless otherwise specified, were performed in triplicate and the average values are reported. In the following examples, unless otherwise specified, the nucleotide sequences in the sequence listing are written from left to right in the 5' to 3' direction, and the amino acid sequences are written from left to right in the amino-terminal to carboxy-terminal direction.

[0033] I. DEFINITIONS

[0034] Unless otherwise defined, terms or scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition.

[0035] As used herein, the terms "comprises," "comprising," "includes," "including" and the like can mean contains, but is not limited to it. "Consisting essentially of" when used in defining compositions and methods, should mean excluding other components of any essential technical effect under the patent law. "Consisting of" shall mean excluding any element not specified.

[0036] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, or a whole culture of cells. As used herein, the term "or" is understood to include.

[0037] Unless explicitly stated or evident from context, as used herein, the term "about" is understood to be within the normal tolerance range of the art, e.g., within 2 standard deviations of the mean. "About" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the stated value. Unless otherwise evident from context, all numerical values provided herein are modified by the term "about."

[0038] The term "antibody" herein refers to a polypeptide ligand comprising at least one variable region that specifically recognizes and binds to an epitope of an antigen. Mammalian antibody molecules (also known as immunoglobulins) are generally composed of heavy (H) and light (L) chains. According to the class of the heavy chain, mammalian immunoglobulins can be assigned to five major classes: IgG, IgM, IgA, IgD, and IgE. Additional antibody isotypes are found in animals other than mammals, including IgX, IgY, IgW, and IgNAR. IgX antibodies are found in amphibians. IgY antibodies are the primary antibodies produced by birds and reptiles, and are functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are found in cartilaginous fishes.

[0039] The amino-terminal domains of the heavy or light chains of an antibody comprise the variable region, referred to as variable region of the heavy chain (V H ) and variable region of the light chain (V L ), respectively. The variable region is usually the most variable part of an antibody (relative to other antibodies of the same class), contains the antigen binding sites, and together with the V H and V L is responsible for binding the antigen recognized by the antibody. Heavy chain-only antibodies of camelids have a single heavy chain variable region, referred to as "V H H". Thus, V H H is a special type of V H .

[0040] However, variability is not distributed evenly across the entire variable region. In fact, in both light and heavy chain variable regions, the variability is concentrated in three segments called hypervariable regions (HVRs), which are also known as "complementarity determining regions" or "CDRs". The sequences of the variable region outside of the HVRs are more conserved, and these parts are called framework regions (FRs). CDRs are the regions of the variable region primarily responsible for binding to an epitope of an antigen, and are generally thought to be responsible for defining the binding affinity and specificity of an antibody; while framework regions are primarily used to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including the numbering schemes described below: the Kabat numbering convention (Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991), the Chothia numbering convention (Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273, 927-948, 1997), the Paratome CDR (Kunik et al., PLoS Comput Biol 8:el002388, 2012; Kunik et al., Nucleic Acids Res 40 (Web Server issue):W521-524, 2012), and the IMGT numbering convention (ImMunoGeneTics (IMGT) database, see Lefranc, Nucleic Acids Res 29:207-9, 2001). The Kabat, Paratome, and IMGT databases are all available online. Of these, Kabat numbering is based on sequence variability and is the most commonly used. However, unless otherwise specified, the numbering of antibody heavy chain residues in this application is determined according to the IMGT numbering convention.

[0041] The light and heavy chains of a mammalian immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. A single domain antibody comprises three CDRs, referred to herein as CDR1, CDR2, and CDR3. In the case of a camelid V H In the case of a H domain antibody, it is generally accepted that according to Kabat numbering, V HH's FR1 contains amino acid residues from positions 1 to 30, CDR1 contains amino acid residues from positions 31 to 35, FR2 contains amino acid residues from positions 36 to 49, CDR2 contains amino acid residues from positions 50 to 65, FR3 contains amino acid residues from positions 66 to 94, CDR3 contains amino acid residues from positions 95 to 102, and FR4 contains amino acid residues from positions 103 to 113. It should be noted that, as is known in the art, V H Domain and V H As shown in the H field, the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). These variations correspond to the shortening or insertion of FR or HVR in the variable region. For example, the heavy chain variable region may include a single amino acid insertion after residue 52 (residue 52a according to Kabat) and an insertion after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). For a given antibody, those skilled in the art can determine the Kabat number of the residue by aligning it to a “standard” Kabat numbered sequence in the sequence homology region. Similarly, the IMGT number of the residue can also be determined by alignment.

[0042] The term "monoclonal antibody" refers herein to a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translation modifications (e.g., isomerization, amidation) that can occur during production. Accordingly, the term "monoclonal antibody" as used herein refers to a single species of antibody. In contrast to polyclonal antibodies that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture or recombinantly, free of other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nded., 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)), and technologies for producing human or human-like antibodies in animals that have part or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 5107-5111 (1993); Jakobovits, Curr. Opin. Biotechnol. 5: 644-653 (1994); Bruggeman et al., Year in Immunol. 7:33-40 (1993); and US Patent Nos. 5,545,806; 5,545,807; 5,569,825; 5,661,016; 5,770,429; 5,789,650; 5,814,318; 5,982,978; 5,591,669; 6,175,798; 6,786,187; 6,807,928; 6,816,314; and 7,053,556).USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Monoclonal antibodies include humanized monoclonal antibodies.

[0043] As used herein, the term "single domain antibody" (which can be abbreviated as sdAb) refers to an antibody having a single domain (variable domain) capable of specifically binding to an antigen or an antigenic epitope in the absence of other antibody domains. By way of non-limiting example, single domain antibodies can be cited, for example, VHHantibodies, V H HHantibodies, V NAR HHantibodies, (camelid) V H HHantibodies, and V L HHantibodies. V NAR HHantibodies are produced by cartilaginous fishes, such as nurse sharks, whale shark, spiny dogfish and bamboo shark. H HHantibodies are produced by several species of camelids, including camels, llamas, alpacas, dromedaries and guanacos, which are capable of producing heavy chain antibodies naturally devoid of light chains. A classical V H HHantibody comprises, in order from N- to C-terminus, a framework region 1 (FR1), a complementarity determining region 1 (CDR1), a framework region 2 (FR2), a complementarity determining region 2 (CDR2), a framework region 3 (FR3), a complementarity determining region 3 (CDR3), a framework region 4 (FR4). The overall size of a single "single domain antibody" is in the order of nanometers, and it is therefore also used herein interchangeably with "nanobody".

[0044] As used herein, the terms "specifically binds," "specifically recognizes," or "has specificity for" refer to a measurable and reproducible interaction such as binding between a target and an antigen binding protein (such as a CAR or sdAb) that is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antigen binding protein that specifically binds a target molecule of interest (which can also be an epitope) is an antigen binding protein that binds the target molecule of interest with greater affinity, avidity, more readily, and / or with greater duration than it binds to non-target molecules. In some embodiments, the antigen binding protein binds to a non-target molecule to a lesser extent than it binds to the target molecule of interest by, for example, about 10% as determined by a radioimmunoassay (RIA). In some embodiments, an antigen binding protein of the application binds B7-H3 with a binding affinity of at least about 1 x 10 -6 M, at least about 0.5 x 10 -6 M, at least about 1 x 10 -7 M, at least about 0.5 x 10 -7 M, at least about 1 x 10 -8 M, at least about 0.5 x 10 -8 M, at least about 1 x 10 -9 M, at least about 0.5 x 10 -9 M, or at least about 0.1 x 10 -9 M. In some embodiments, an antigen binding protein of the application (such as an anti-B7-H3 single domain monoclonal antibody) specifically binds a target molecule of interest (such as a B7-H3 protein) with a binding constant that is at least 10 3 M -1 , 10 4 M -1 , or 10 5 M -1 In some embodiments, a complex formed after specific binding of an antigen binding protein of the application to a target of interest 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 nM or less, e.g., from 10 -5 M to 10 -10 M, e.g., 10 -6 M to 10 -9M). Methods of determining binding affinity are known in the art, any of which can be used for the purposes of the present application. In some embodiments, an instrument capable of measuring protein interactions (such as the Octet RED384 protein interaction instrument) is used to determine parameters that can characterize the strength of binding and dissociation. In some embodiments, the binding affinity is measured using the Octet system (Creative Biolabs) based on the Bio-Layer Interferometry (BLI) technology. In some embodiments, Kd is measured using surface plasmon resonance assays using a BIACORE S-2000 or BIACORE S-3000 (BIAcore, Inc., Piscataway, N.J.). In some embodiments, specific binding can include, but need not be, exclusive binding.

[0045] The term "bispecific" means that an antigen binding protein (such as a CAR or sdAb) comprises antigen binding fragments of two different monoclonal antibodies, thereby being able to recognize and bind to two different antigens or different epitopes on the same antigen. Similarly, "multispecific" means that an antigen binding protein comprises antigen binding fragments of at least two (such as two, three, or four) different monoclonal antibodies (see, e.g., US 2018 / 0230225).

[0046] As used herein, the term "valency" means the number of binding sites in an antigen binding protein (such as a CAR or sdAb). For example, a natural four-chain antibody has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" mean that there are two, three, four, five, and six binding sites, respectively, in an antigen binding protein.

[0047] A "humanized" antibody is an immunoglobulin comprising human framework regions and one or more CDRs of a non-human (e.g., mouse, rabbit, rat, shark, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor" and the human immunoglobulin providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are from the donor immunoglobulin. Constant regions are not necessarily present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90% identical, such as about 95% or more identical. Thus, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody binds the same determinant as the donor antibody providing the CDRs. In addition, a humanized antibody can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications (e.g., substitution of one or more FR residues) can further refine antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of amino acid substitutions in the FRs of the H chain is typically no more than 6. In some cases, the number of amino acid substitutions in the FRs of the H chain is 6, 5, 4, 3, 2, or 1.

[0048] The term "chimeric antibody" is used herein to refer to an immunoglobulin having framework region residues of one species (e.g., human) and CDRs (typically responsible for antigen binding) of another species.

[0049] As used herein, the term "chimeric antigen receptor" is used interchangeably with "CAR" to refer to a genetically engineered receptor that can be used to graft antigen specificity(ies) onto an immune effector cell, such as a T cell or NK cell. Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immunoreceptors." Generally, a CAR comprises an extracellular antigen-binding portion specific for one or more antigens (such as a tumor antigen), a transmembrane region, and an intracellular portion. "CAR-T" and "CAR-NK" refer to T cells and NK cells, respectively, that express a CAR. A "B7-H3 CAR" refers to a CAR having an extracellular binding region specific for B7-H3. A "bispecific CAR" refers to a CAR having an extracellular binding domain specific for two different target molecules or for two different epitopes on the same target molecule.

[0050] A "chimeric antigen receptor" as described generally comprises an antigen binding moiety (e.g., scFv or single domain antibody), a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain typically includes a signaling chain with an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3 zeta or Fc epsilon RI gamma. In some embodiments, the CAR is monospecific. In some embodiments, the CAR is monovalent. In some embodiments, the CAR is multivalent (e.g., bivalent or trivalent). In some embodiments, the CAR is multispecific (e.g., bispecific). A T cell or NK cell expressing a bispecific CAR can bind to a cell expressing two antigens to which the binding moieties are directed (see, e.g., Qin et al., Blood, 130:810, 2017 and WO / 2018 / 213337). In some embodiments, the chimeric antigen receptor further includes an intracellular portion of at least one additional costimulatory domain, such as CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, and / or DAP10.

[0051] As used herein, a "covalent conjugate" is an antibody or antibody fragment (e.g., antigen binding fragment) covalently linked to an effector molecule or second protein (e.g., a second antibody). The effector molecule can be, for example, a drug, a toxin, a therapeutic agent, a detectable label, a protein, a nucleic acid, a lipid, a nanoparticle, a photon absorber, a carbohydrate, or a recombinant virus. Antibody conjugates are often referred to as "immunoconjugates." When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is often referred to as an "antibody-drug conjugate" or "ADC." Other antibody conjugates include, for example, multispecific (e.g., bispecific or trispecific) antibodies and chimeric antigen receptors (CARs).

[0052] An "antibody-drug conjugate" or "ADC" is intended herein to include a molecule of an antibody (or antigen binding fragment thereof) conjugated to a drug (e.g., a cytotoxic agent). ADCs can be used to specifically target drugs to cancer cells through the specific binding of the antibody to a tumor antigen expressed on the surface of the cell. Exemplary drugs used with ADCs include anti-microtubule agents (e.g., maytansinoids, auristatin E, and auristatin F) and interchain cross-linking agents (e.g., pyrrolobenzodiazepine; PBD). In some cases, the ADC is a bispecific ADC, which consists of two monoclonal antibodies or antigen fragments thereof, each directed to a different antigen or different epitope on the same antigen, and conjugated to a drug.

[0053] Herein, a "cytotoxic agent" is any drug or compound that is capable of killing cells.

[0054] As used herein, an "anti-microtubule agent", also known as an "anti-mitotic agent", is a drug that stops cell growth by halting mitosis. Anti-microtubule agents can be used to treat cancer.

[0055] As used herein, an "interstrand crosslinking agent" is a cytotoxic drug that is capable of covalently binding between two DNA strands, thereby preventing DNA replication and / or transcription, including but not limited to pyrrolobenzodiazepines (PBDs). PBDs are a class of sequence-selective DNA minor groove binding crosslinking agents originally discovered in Streptomyces species, whose mechanism of action is related to their ability to form adducts in the DNA minor groove, thereby interfering with DNA processing. PBDs include naturally occurring and isolated PBDs, chemically synthesized naturally occurring PBDs, and chemically synthesized non-naturally occurring PBDs. PBDs also include monomeric PBDs, dimeric PBDs, and hybrid PBDs (see, e.g., Gerratana, Med Res Rev, 32(2):254-293, 2012).

[0056] As used herein, the term "small molecule" refers to a molecule that typically has a molecular weight of less than about 1000 Daltons, or in some embodiments, less than about 500 Daltons, wherein the molecule is capable of modulating the activity of a target molecule to a measurable extent.

[0057] As used herein, a "toxin" refers to a molecule that is cytotoxic to a cell, including but not limited to abrin, ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinum toxin, saporin, restrictocin, or gelonin, or a modified toxin thereof. For example, PE and DT are highly toxic compounds that typically cause death through hepatotoxicity. However, PE and DT can be modified into forms useful as immunotoxins by removing the natural targeting component of the toxin (e.g., domain Ia of PE or the B chain of DT) and replacing it with a different targeting moiety (e.g., an antibody).

[0058] As used herein, a "label" refers to a detectable compound or composition, including but not limited to a fluorescent tag, an enzyme, and a radioisotope. Detection of a molecule, such as an antibody or protein, can be facilitated by conjugating a label directly or indirectly to the molecule. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of polypeptide labels include, but are not limited to, the following: fluorescent labels (e.g., fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, b-galactosidase, luciferase, alkaline phosphatase), radiolabels (e.g., 35 S、 11 C、 13 N、 15 O、 18 F、 19 F、99 mTc、 131 I、 3 H、 14 C、 15 N、 90 Y、 99 Tc、 111 In and 125 I), chemiluminescent markers, biotin groups, predetermined polypeptide epitopes recognized by secondary reporter molecules (such as leucine zipper pairs of sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents such as gadolinium chelates. In some embodiments, the markers are attached through spacer arms of various lengths to reduce potential steric hindrance.

[0059] As used herein, a "B7-H3 positive cancer" or "B7-H3 positive tumor" refers to a cancer cell / tumor cell that expresses or overexpresses B7-H3 on the cell surface relative to healthy tissue cells of the same origin or relative to a "B7-H3 negative cancer" or "B7-H3 negative tumor." One of skill in the art can determine whether a cancer is B7-H3 positive by routine methods of detecting tumor associated antigens or tumor typing. In this context, a B7-H3 positive cancer can be a solid tumor or a hematological tumor. In some examples, a solid tumor can be selected from a nervous system tumor, a head and neck tumor, a thoracic tumor, a digestive system tumor, a genitourinary system tumor, a soft tissue and skin tumor, or a bone tumor. In some examples, a hematological tumor can be selected from a leukemia, a lymphoma (HL), a multiple myeloma (MM), or a myelodysplastic syndrome (MDS). By way of non-limiting example, examples of B7-H3 positive cancers include, but are not limited to, a nervous system tumor selected from diffuse glioma, astrocytic tumor (e.g., diffuse astrocytic tumor, anaplastic astrocytic tumor, oligoastrocytic tumor), glioblastoma, oligodendroglioma, childhood diffuse glioma, ependymal tumor, neuronal and mixed neuronal-glial tumor, medulloblastoma, embryonal tumor, schwannoma, meningioma, solitary fibrous tumor, or perivascular cell tumor, a head and neck tumor selected from a nasal cavity and paranasal sinus malignant tumor, nasopharyngeal carcinoma, oral cavity cancer, laryngeal cancer, salivary gland tumor, intracranial tumor (e.g., neuroblastoma or glioblastoma), thyroid cancer, or tongue cancer, a thoracic tumor selected from lung cancer (e.g., non-small cell lung cancer), esophageal cancer, cardiac cancer, breast cancer (e.g., triple negative breast adenocarcinoma cell), mesothelioma, or mediastinal tumor, a digestive system tumor selected from stomach cancer, large intestine cancer, colon cancer (e.g., colon adenoma), colorectal cancer, liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer, periampullary carcinoma, biliary tract cancer, or small intestine cancer, a genitourinary system tumor selected from kidney cancer (e.g., renal cell adenocarcinoma), prostate cancer, bladder cancer, testicular cancer, penile cancer, cervical cancer, endometrial cancer, or ovarian cancer, a soft tissue and skin tumor selected from fibrous histiocytic tumor, rhabdomyosarcoma, synovial sarcoma, or melanoma, a bone tumor selected from osteosarcoma or Ewing's sarcoma, a leukemia selected from B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute myeloid leukemia, monocyte leukemia, or pro-myelocytic leukemia, a lymphoma selected from histiocytic lymphoma or Burkitt's lymphoma.

[0060] A "brain cancer" or "brain tumor" means a cancer or tumor that develops from brain tissue. Brain cancers include, but are not limited to, neuroblastoma, medulloblastoma, glioma, glioblastoma, meningioma, pituitary adenoma, astrocytic tumor, choroid plexus carcinoma, ependymal tumor, and pinealoblastoma.

[0061] “Breast cancer” means a cancer that forms in the tissues of the breast, often in the ducts and lobules. Types of breast cancer include, for example, ductal carcinoma in situ, invasive ductal carcinoma, triple negative breast cancer, inflammatory breast cancer, metastatic breast cancer, medullary carcinoma, tubular carcinoma, and mucinous carcinoma. Triple negative breast cancer refers to a breast cancer in which the cancer cells do not express estrogen receptors, progesterone receptors, or significant levels of the HER2 / neu protein. Triple negative breast cancer is also known as ER-negative PR-negative HER2 / neu-negative breast cancer.

[0062] “Colon cancer” means a cancer that forms in the colon or rectum. The most common type of colon cancer is colorectal adenocarcinoma, which accounts for about 95% of all colon cancers. Adenocarcinomas form in cells inside the colon and / or rectum. Other types of colorectal cancer include gastrointestinal carcinoid, metastatic colorectal cancer, primary colorectal lymphoma (a type of non-Hodgkin lymphoma), gastrointestinal stromal tumor (classified as a sarcoma, originating from Cajal interstitial cells), leiomyosarcoma (originating from smooth muscle cells), and colorectal melanoma.

[0063] As used herein, “liver cancer” refers to any type of cancer that occurs in liver tissue. The most common type of liver cancer is hepatocellular carcinoma (HCC), which forms in liver cells. Other types of liver cancer include cholangiocarcinoma, which forms in the bile ducts; hepatic angiosarcoma, a rare type of liver cancer that begins in the blood vessels of the liver; and hepatoblastoma, a very rare type of liver cancer that most commonly occurs in children.

[0064] As used herein, “lung cancer” refers to any cancer that forms in the lungs. Most cancers that begin in the lungs are malignant. The two main types of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Subtypes of NSCLC include adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.

[0065] As used herein, “ovarian cancer” refers to a cancer that forms in the tissues of the ovary. Most ovarian cancers are ovarian epithelial cancer (a cancer that begins in the surface cells of the ovary) or a malignant germ cell tumor (a cancer that begins in the egg cells). Another type of ovarian cancer is a stromal cell cancer, which originates from cells that release hormones and connect different structures of the ovary.

[0066] As used herein, "pancreatic cancer" refers to a disease in which malignant cells are found in the tissue of the pancreas. Based on the cell of origin of the cancer, pancreatic tumors can be exocrine tumors or neuroendocrine tumors. The vast majority (~94%) of pancreatic cancers are exocrine tumors. 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, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). Pancreatic neuroendocrine tumors, also known as islet cell tumors, are classified according to the type of hormone they produce. Exemplary neuroendocrine tumors include gastrinoma, glucagonoma, insulinoma, somatostatinoma, VIPoma (vasoactive intestinal peptide), and nonfunctioning islet cell tumor.

[0067] As used herein, "pediatric cancer" refers to a cancer that occurs in a child between 0 and 14 years of age. Major types of pediatric cancer include, for example, neuroblastoma, acute lymphoblastic leukemia (ALL), embryonal rhabdomyosarcoma (ERMS), alveolar rhabdomyosarcoma (ARMS), Ewing's sarcoma, desmoplastic small round cell tumor (DRCT), osteosarcoma, brain and other CNS tumors such as neuroblastoma and medulloblastoma, Wilm's tumor, non-Hodgkin's lymphoma, and retinoblastoma.

[0068] The term "diagnosis" refers herein to determining the presence or nature of a pathological 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 diseased individuals that test positive (percentage of true positives). The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the false positive rate is defined as the proportion of disease-free patients that test positive. While a particular diagnostic method can not definitively diagnose a condition, it is sufficient as long as the method provides a positive indication that aids in diagnosis. "Prognosis" is the probability of development (e.g., severity) of a pathological condition such as cancer.

[0069] "Diagnostic tumor imaging" refers to the coupling of antibodies and their derivatives to positron emitting radionuclides for positron emission tomography (PET), a process commonly referred to as immunoPET. While full-length antibodies can be made into good immunoPET reagents, their biological half-life requires waiting several days before imaging, thereby increasing the non-target radiation dose. Smaller single-domain or nanobodies have a biological half-life suitable for same-day imaging.

[0070] As used herein, "preventing" a disease refers to inhibiting the full development of a disease, and "treating" refers to therapeutic intervention that ameliorates signs or symptoms of a disease or pathological condition after it has begun to develop, such as reducing tumor burden or reducing the size of metastatic lesions. "Reducing" refers to decreasing the number or severity of signs or symptoms of a disease, such as cancer.

[0071] As used herein, "therapeutically effective amount" means the amount of a particular substance which is sufficient to effectuate a desired effect in a subject receiving treatment. For example, this can be the amount necessary to arrest or inhibit tumor growth. In one embodiment, a therapeutically effective amount is the amount necessary to eliminate, reduce the size of, or prevent metastasis of a tumor, e.g., an amount that 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 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 100%, as compared to the size / volume / number prior to treatment. When administered to a subject, the dosage used will generally achieve a concentration in the target tissue (e.g., in a tumor) that has been shown to achieve the desired in vitro effect.

[0072] The term "vector" refers herein to a nucleic acid molecule that can be introduced into a host cell thereby producing a transformed host cell. Vectors can include nucleic acid sequences that enable them to replicate in a host cell, such as an origin of replication. Vectors can also include one or more selectable marker genes and other genetic elements known in the art. In some embodiments, the vector is a viral vector, e.g., a lentiviral vector.

[0073] As used herein, the term "individual," "subject," "patient," "host," "needing subject," or similar term refers to any mammal or non-mammal. Mammals include, but are not limited to, cats, other vertebrates such as rodents, humans, non-human primates, e.g., cows, horses, dogs, pigs, sheep, goats, giraffes, deer, camels, llamas, rats, mice, hares, and rabbits.

[0074] As used herein, the term "administering" refers to providing or giving an agent, e.g., a monoclonal antibody, CAR, or CAR-expressing cell of the application, to a subject by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraprostatic, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal, and inhalation.

[0075] As used herein, "operably linked" means that a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked with a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0076] As used herein, a "pharmaceutically acceptable excipient" is an excipient that is conventionally used in the pharmaceutical art. See, e.g., Remington: The Science and practice of Pharmacy, edited by The University of the Sciences in Philadelphia, Lippincott, Williams, & Wilkins, 21st Edition. Philadelphia, PA. (2005). Generally, the nature of the excipient will depend on the particular mode of administration being employed. For example, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions such as powders, pills, tablets, or capsules, conventional non-toxic solid excipients can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral excipients, the pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, e.g., sodium acetate or sorbitan monolaurate.

[0077] As used herein, the term "sequence identity" is used to measure the degree of similarity between amino acid or nucleic acid sequences, typically measured as percent identity. The higher the percentage, the more similar the two sequences. "Identity" is the percentage of identical matches between two or more sequences in an aligned (if any) gap treatment by a particular mathematical model or computer program (e.g., algorithm). Methods of alignment and computer programs are well known in the art, such as the commonly used BLAST suite (Stephen F. Altschul, et al (1997), Nucleic Acids Res. 25:3389-3402), the Smith-Waterman algorithm-based local alignment method (Smith, T. F. & Waterman, M. S. (1981) J. Mol. Biol. 147:195-197), the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D. (1970) J. Mol. Biol. 48:443-453) which is a general global alignment method based on dynamic programming, and the like. It is understood that identity depends on the calculation of percent identity, but can vary in value due to the introduction of gaps and penalties in the calculation.

[0078] The NCBI Basic Local Alignment Search Tool (BLAST) is a commonly used sequence alignment and identity calculation tool, available from multiple sources, such as the National Center for Biotechnology Information (NCBI, Bethesda, Md.). Depending on the type of sequence and the purpose of use, the appropriate analysis program blastp, blastn, blastx, tblastn, and tblastx, etc. can be selected. Instructions for using the program to determine sequence identity are freely available from the NCBI website. For comparison of amino acid sequences greater than about 30 amino acids, the Blast 2 Sequences function can be employed using the default BLOSUM62 matrix (with a gap existence cost of 11 and a gap extension cost of 1) set to default parameters. When aligning short peptides (less than about 30 amino acids), the Blast 2 Sequences function is used with the PAM30 matrix set to default parameters (with a gap penalty of 9 for a single residue gap and 1 for a gap extension). Proteins that are more similar to the reference sequence will display an increasing percentage of sequence identity when evaluated by this method, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When comparing sequence identity over less than the entire sequence, homologues and variants will typically have at least 80% sequence identity over a short window of 10-20 amino acids, and can have at least 85% or at least 90% or 95% sequence identity, depending on their similarity to the reference sequence. Methods to determine sequence identity over a short window are available on the internet at the NCBI website. Those skilled in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible to obtain highly significant homologues outside of the ranges provided.

[0079] Homologues and variants of antibodies that specifically bind to a B7-H3 polypeptide will typically be characterized by having at least about 75%, for example, at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity in a full-length alignment with the amino acid sequence of the antibody, using NCBI Blast 2.0 with the blastp set to default parameters for gaps.

[0080] As used herein, the term "conservative variant" means a protein that contains conservative amino acid substitutions that do not substantially affect or decrease the affinity of the protein, e.g., an anti-B7-H3 antibody. For example, a monoclonal antibody that specifically binds to B7-H3 can include up to about 1, up to about 2, up to about 5, up to about 10, or up to about 15 conservative substitutions, and specifically binds to a B7-H3 polypeptide. The term "conservative variant" also includes the use of substituted amino acids in place of unsubstituted parent amino acids, as long as the antibody specifically binds to B7-H3. Accordingly, a "non-conservative substitution" is a substitution that decreases the activity or binding ability of the protein to B7-H3.

[0081] Conservative amino acid substitutions are well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

[0082] 1) Alanine (A), Serine (S), Threonine (T);

[0083] 2) Aspartic acid (D), Glutamic acid (E);

[0084] 3) Asparagine (N), Glutamine (Q);

[0085] 4) Arginine (R), Lysine (K);

[0086] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0087] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0088] As used herein, "contacting" means directly engaging in physical association, both in solid and liquid form.

[0089] As used herein, "cytotoxicity" refers to the toxicity of a molecule (e.g., an immunotoxin) to the intended target cells, and not to the rest of the organism. In contrast, the term "toxicity" refers to the toxicity of an immunotoxin to cells other than the intended target cells of the targeting moiety of the immunotoxin, and the term "animal toxicity" refers to the toxicity of an immunotoxin to an animal, i.e., the toxicity of the immunotoxin to cells other than the intended target cells of the immunotoxin.

[0090] As used herein, "chemotherapeutic agent" refers to any chemical agent that has a therapeutic effect in the treatment of a disease characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancers, as well as diseases characterized by proliferative growth, such as psoriasis. In one embodiment, the chemotherapeutic agent is an agent used to treat a B7-H3 positive tumor. In one embodiment, the chemotherapeutic agent is a radioactive compound. One of skill in the art can readily determine a chemotherapeutic agent to use (see, e.g., 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 2nd ed., 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, H.J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. One example is the administration of an antibody that binds B7-H3 in combination with a radioactive or chemical compound. In one example, the chemotherapeutic agent is a biologic, such as a therapeutic antibody (e.g., a therapeutic monoclonal antibody), including but not limited to an anti-B7-H3 antibody provided herein, as well as other anti-cancer antibodies such as anti-PD1 or anti-PD-L1 (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).

[0091] As used herein, "immune response" refers to the reaction of an immune cell (e.g., a B cell, a T cell, or a monocyte) to a stimulus. In one embodiment, the immune response is specific for a particular antigen (i.e., an 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 immune response is a B cell response and produces specific antibodies.

[0092] As used herein, a "linker" is, in some instances, a peptide within an antibody binding fragment (e.g., an Fv fragment) that serves to indirectly bind the variable heavy chain to the variable light chain. A "linker" can also refer to a peptide used to link a targeting moiety (e.g., an antibody) to an effector molecule (e.g., a cytotoxin or a detectable label). The term "conjugated" or "linked" refers to making two polypeptides into one continuous polypeptide molecule, or covalently linking a radionuclide or other molecule to a polypeptide, such as an antibody. Linking can be done chemically or recombinantly. "Chemically" 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.

[0093] As used herein, "photoimmunotherapy" refers to a targeted cancer therapy that utilizes an antigen-specific antibody-light absorber conjugate that can be activated by near infrared light to kill targeted cells. The light absorber is typically based on a phthalocyanine dye, such as a near infrared (NIR) phthalocyanine dye (e.g., 700DX, also known as IR700). The B7-H3 specific antibody binds to an appropriate cell surface antigen (e.g., B7H3), and the photoactivatable dye induces cell membrane lethal damage upon NIR light irradiation. NIR light irradiation (e.g., 690 nm) can induce highly selective necrotic cancer cell death within minutes without damaging neighboring cells (see, e.g., U.S. Application No. 2018 / 0236076).

[0094] As used herein, "purified" or "purification" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified preparation of a peptide is one in which the peptide or protein is enriched compared to the natural environment of the peptide or protein within the cell. In one embodiment, a purified preparation is one in which the protein or peptide comprises at least 50% of the total peptide or protein content of the preparation. Substantially purified refers to 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 90% free of other proteins or cellular components.

[0095] As used herein, "sample" or "biological sample" refers to a biological specimen obtained from a subject that contains genomic DNA, RNA (including mRNA), protein, or a combination thereof. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, a biopsy, a fine needle aspirate, a surgical specimen, and autopsy material. In one example, the sample comprises a tumor biopsy.

[0096] As used herein, "synthetic" means produced in a laboratory by artificial means, e.g., a synthetic nucleic acid or protein (e.g., an antibody) can be chemically synthesized in a laboratory.

[0097] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or adjustments based on the teachings of the present invention, without departing from the spirit and scope of the invention.

[0098] II. B7-H3 specific single-domain monoclonal antibody

[0099] This type of single-domain monoclonal antibody includes V derived from camel antibodies. H H antibody, V antibody derived from cartilage fish NAR Antibodies and V derived from human antibody heavy chains H Antibodies, etc. This disclosure describes the use of recombinant B7-H3 protein to immunize alpacas and generate phage-displayed V. H H-domain antibody library. After three rounds of phage panning and removal of repetitive sequences, a total of 18 conjugates were obtained (named BP2-F12, BP1-C11, BP1-F9, BP1-C10, H-F6, BP2-A9, H-A3, BP1-H8, H-H6, H-A12, BP1-H12, BP2-A12, BP1-F1, BP2-A8, BP1-G2, BP1-C6, BP2-A4, and BP1-F12, respectively). These nanobodies showed the ability to bind to B7-H3 positive tumor cell lines (such as A375 melanoma cells) but not to B7-H3 negative cells (such as Jurkat cells) or B7-H3 knockout cells.

[0100] Table 1 below provides the amino acid sequences of 18 single-domain antibodies, with the amino acid positions of their CDR1, CDR2, and CDR3 indicated by underlines. The CDR positions of the antibodies in this paper were determined using the IMGT method. However, those skilled in the art can readily determine CDR boundaries using alternative numbering schemes (e.g., Kabat, Paratome, or Chothia numbering schemes).

[0101] Table 1

[0102] In some embodiments, the single-domain monoclonal antibodies disclosed herein comprise at least a portion of the amino acid sequence as set forth in any one of SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82, or 87, such as one or more (e.g., one, two, or all three) CDR sequences from any one of the following antibodies: BP2-F12 (SEQ ID NO: 2), BP1-C11 (SEQ ID NO: 7), BP1-F9 (SEQ ID NO: 12), BP1-C10 (SEQ ID NO: 17), H-F6 (SEQ ID NO: 22), BP2-A9 (SEQ ID NO: 27), H-A3 (SEQ ID NO: 32), BP1-H8 (SEQ ID NO: 37), H-H6 (SEQ ID NO: 42), H-A12 (SEQ ID NO: 47), BP1-H12 (SEQ ID NO: 52), BP2-A12 (SEQ ID NO: 57), BP1-F1 (SEQ ID NO: 62), BP2-A8 (SEQ ID NO: 67), BP1-G2 (SEQ ID NO: 72), BP1-C6 (SEQ ID NO: 77), BP2-A4 (SEQ ID NO: 82), BP1-F12 (SEQ ID NO: 87), as determined by any numbering scheme, such as IMGT, Kabat, Paratome, or Chothia, or any combination thereof. In some embodiments, the single-domain antibody comprises the CDR1, CDR2, and CDR3 sequences of any one of SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82, or 87. In particular embodiments, the CDR sequences are determined using the IMGT numbering scheme.

[0103] In some embodiments, the antibody has a combination of CDRs recited in any one of (1) to (18) below: (1) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5; (2) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10; (3) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; (4) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; (5) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25; (6) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30; (7) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35; (8) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40; (9) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45; (10) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 48, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 49, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50; (11) CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 54, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 55;(12) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 60; (13) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 63, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 64, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 65; (14) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 68, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 69, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 70; (15) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75; (16) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80; (17) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 83, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 84, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 85; or (18) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90.

[0104] In some embodiments, the amino acid sequence of the antibody has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82, or 87.

[0105] In some embodiments, the amino acid sequence of the antibody has one or more deletions, substitutions or insertions of one or a few amino acids relative to the amino acid sequence set forth in any one of SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82, or 87, the substitutions preferably being conservative substitutions. In some embodiments, the number of amino acids that are deleted, substituted or inserted is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the deletions, substitutions or insertions occur only in the antibody sequence outside of the CDRs, such as in the framework regions. In particular embodiments, the amino acid sequence of the antibody comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82, or 87. In particular embodiments, the amino acid sequence of the antibody consists of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82, or 87.

[0106] In some embodiments, the amino acid sequence of the antibody is encoded by the nucleotide sequence set forth in any one of SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 76, 81, or 86. In some embodiments, the nucleotide sequence encoding the antibody has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% nucleotide sequence identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 76, 81, or 86. In some embodiments, the nucleotide sequence encoding the antibody has one or more deletions, substitutions or insertions of one or a few nucleotides relative to the nucleotide sequence set forth in any one of SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 76, 81, or 86. In some embodiments, the nucleotide sequence encoding the antibody is codon-optimized.

[0107] In some embodiments, the single-domain monoclonal antibody disclosed herein is selected from a V H H antibody, a VH antibody, a V NAR antibody, or a VL antibody. In some embodiments, the single-domain monoclonal antibody disclosed herein is a humanized antibody or a human antibody. In some embodiments, the single-domain monoclonal antibody disclosed herein is a partially or fully humanized V HH antibodies, human VH antibodies, or camelized human VH antibodies, partially or fully humanized V NAR antibodies, or human VL antibodies. In some embodiments, the single-domain monoclonal antibodies disclosed herein are affinity matured.

[0108] Further provided herein are multivalent antibodies comprising the single-domain antibodies disclosed herein. Multivalent antibodies are further described in Section III herein. Also provided herein are multispecific antibodies comprising the single-domain antibodies 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 NK cell activating receptor. Multispecific antibodies are further described in Section IV herein.

[0109] Also provided herein are chimeric antigen receptors (CARs) comprising an antibody disclosed herein (e.g., a single domain monoclonal antibody, a multivalent antibody, or a multispecific antibody). In some embodiments, the CAR further comprises a hinge region, a transmembrane domain, a costimulatory signaling moiety, an intracellular signaling domain, or any combination thereof. In non-limiting examples, the hinge region comprises a CD8a hinge region, preferably comprising the amino acid sequence set forth in SEQ ID NO: 136; the transmembrane domain comprises a transmembrane domain of any one selected from the group consisting of CD8a, CD4, CD28, CD137, CD80, CD86, CD152, PD1, preferably a transmembrane domain of CD8a or a transmembrane domain of CD28, more preferably comprising the amino acid sequence set forth in SEQ ID NO: 138 or 140; the costimulatory signaling moiety comprises a costimulatory signaling domain of any one selected from the group consisting of a ligand of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and any combination thereof, preferably a cytoplasmic domain of CD28 and / or a cytoplasmic domain of CD137, more preferably comprising the amino acid sequence set forth in SEQ ID NO: 142 or 144, and / or the intracellular signaling domain comprises a CD3 zeta signaling domain, preferably comprising the amino acid sequence set forth in SEQ ID NO: 146. In some preferred embodiments, the chimeric antigen receptor disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 126, 128, 130, 132, or 134. In some embodiments, the chimeric antigen receptor disclosed herein comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 126, 128, 130, 132, or 134. In some embodiments, the amino acid sequence of the chimeric antigen receptor disclosed herein has one or several deletions, substitutions, or insertions of amino acids, preferably conservative substitutions, relative to the amino acid sequence set forth in SEQ ID NO: 126, 128, 130, 132, or 134. In some embodiments, the number of amino acids of the deletions, substitutions, or insertions is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In particular embodiments, the amino acid sequence of the antibody consists of the amino acid sequence set forth in SEQ ID NO: 126, 128, 130, 132, or 134.

[0110] In some embodiments, the amino acid sequence of the chimeric antigen receptor (CAR) is encoded by the nucleotide sequence set forth in any one of SEQ ID NO: 127, 129, 131, 133, or 135. In some embodiments, the nucleotide sequence encoding the CAR has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% nucleotide sequence identity to the nucleotide sequence set forth in any one of SEQ ID NO: 127, 129, 131, 133, or 135. In some embodiments, the nucleotide sequence encoding the CAR has a deletion, substitution, or insertion of one or several nucleotides compared to the nucleotide sequence set forth in any one of SEQ ID NO: 127, 129, 131, 133, or 135. In some embodiments, the nucleotide sequence encoding the CAR is codon-optimized.

[0111] Further provided herein are engineered cells expressing the B7-H3 specific CARs disclosed herein. In some embodiments, the cells are T lymphocytes, such as cytotoxic T cells (CTLs), or natural killer cells (NKs). CARs and CAR-expressing cells are further described in Section III herein.

[0112] Also provided herein are immunoconjugates comprising the antibodies disclosed herein (such as single domain monoclonal antibodies, multivalent antibodies, or multispecific antibodies) and effector molecules. In some embodiments, the effector molecule is a toxin, such as, but not limited to, Pseudomonas exotoxin or variants 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 photon absorbers can be used in photoinmunotherapy. Immunoconjugates are further described in Section VI herein.

[0113] Further provided herein are antibody-drug conjugates (ADCs) comprising a drug conjugated to the antibodies disclosed herein (such as single domain monoclonal antibodies, multivalent antibodies, or multispecific antibodies). In some embodiments, the drug is a small molecule, such as an anti-microtubule agent, an anti-mitotic agent, and / or a cytotoxic agent. ADCs are further described in Section V herein.

[0114] Further provided herein are antibody-nanoparticle conjugates, including a nanoparticle conjugated to an antibody (e.g., a single domain monoclonal antibody, a multivalent antibody, or a multispecific antibody) disclosed herein. In some embodiments, the nanoparticle includes a polymeric nanoparticle, a nanosphere, a nanocapsule, a liposome, a dendrimer, a polymeric micelle, or a vesicle. In some embodiments, the nanoparticle comprises a cytotoxic agent. Antibody-nanoparticle conjugates are further described in Section VII herein.

[0115] Further provided herein are fusion proteins, including an antibody (e.g., a single domain monoclonal antibody, a multivalent antibody, or a multispecific antibody) disclosed herein and a heterologous protein or peptide. In some embodiments, the heterologous protein is selected from a histidine tag, an Fc protein, or a leucine zipper. In some embodiments, the Fc protein or peptide can be an Fc fragment of human IgGl, IgG2, IgG3, or IgG4, or an Fc fragment of other non-human animals, such as, but not limited to, an Fc fragment of mouse IgGl, IgG2a, IgG2b, IgG3, an Fc fragment of rabbit IgG. In some embodiments, the Fc fragment comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 98. In some embodiments, the amino acid sequence of a chimeric antigen receptor disclosed herein has one or more deletions, substitutions, or insertions of amino acids relative to the amino acid sequence set forth in SEQ ID NO: 98, with the substitutions preferably being conservative substitutions. In some embodiments, the number of amino acids that are deleted, substituted, or inserted is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In particular embodiments, the amino acid sequence of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 98.

[0116] Further provided herein are nucleic acid molecules encoding an antibody (e.g., a single domain monoclonal antibody, a multivalent antibody, or a multispecific antibody), a CAR, an immunoconjugate, an antibody-nanoparticle, or a fusion protein disclosed herein. In some embodiments, the nucleic acid molecule is operably linked to a transcriptional regulatory element, such as a promoter, an enhancer, a polyA tail, a 5’ UTR, a 3’ UTR, and the like. Further provided herein are vectors comprising the disclosed nucleic acid molecules.

[0117] Further provided herein are engineered cells expressing an antibody (e.g., a single domain monoclonal antibody, a multivalent antibody, or a multispecific antibody), a CAR, an immunoconjugate, an antibody-nanoparticle, a fusion protein disclosed herein, or comprising a nucleic acid molecule or a vector disclosed herein. In some embodiments, the cell is preferably a cytotoxic T lymphocyte (CTL) or a natural killer (NK) cell.

[0118] Further provided herein are compositions comprising a pharmaceutically acceptable excipient and an antibody (e.g., a single domain monoclonal antibody, a multivalent antibody, or a multispecific antibody), a CAR, an immunoconjugate, an ADC, an antibody-nanoparticle conjugate, a fusion protein, an isolated nucleic acid molecule, an engineered cell (e.g., a CAR-expressing cell, e.g., a CAR T cell or a CAR NK cell) disclosed herein. Compositions and uses thereof are further described in Section IX herein.

[0119] III. Multivalent Antibodies

[0120] Generally, the valency of an antibody is determined by the number of antigen binding sites in one antibody molecule. A single nanobody (e.g., a single domain antibody disclosed herein) can be referred to herein as a "monovalent" antibody or protein or polypeptide or construct. Proteins and polypeptides comprising or consisting essentially of at least two nanobodies (e.g., at least two single domain antibodies disclosed herein, or at least one single domain antibody disclosed herein and at least one other single domain antibody) are referred to herein as "multivalent" proteins or polypeptides or constructs. There is no necessary relationship between the valency of an antibody and its specificity. Two or more antigen binding sites in a multivalent antibody (e.g., two or more single domain antibodies, or at least one single domain antibody and other antigen binding sites) can be directed against the same epitope, substantially equivalent epitopes, or different epitopes.

[0121] Accordingly, also provided herein are multivalent antibodies comprising or consisting essentially of at least two single domain antibodies disclosed herein (e.g., two, three, or four single domain antibodies disclosed herein). Such multivalent antibodies can provide advantages over proteins or polypeptides comprising or consisting essentially of single domain antibodies disclosed herein, e.g., in improving affinity for B7-H3, as further described herein, e.g., a construct having the sequence of a bivalent antibody as set forth in SEQ ID NO: 95.

[0122] Multivalent antibodies disclosed herein can also comprise or consist essentially of at least one single domain antibody disclosed herein and at least one other antigen binding unit (i.e., directed against another epitope or antigen), which can be a conventional four-chain antibody or antigen binding fragment thereof, or another different single domain antibody. Such multivalent antibodies can also be referred to herein as "multispecific" polypeptides or proteins or constructs. Such multivalent antibodies can provide certain additional advantages over single domain antibodies disclosed herein, e.g., the other antigen binding unit can be a single domain antibody capable of binding to a human serum protein (e.g., human serum albumin) to provide increased half-life; or the other antigen binding unit can be a single domain antibody capable of binding to the same or a different epitope on B7-H3 to increase affinity for B7-H3.

[0123] In the above constructs, the one or more single-domain antibodies and / or other structural units can be directly linked and / or linked via one or more linker sequences. Linkers or spacer sequences that can be used in multivalent and multispecific polypeptides will be known to the skilled person and can generally be any linker or spacer sequence used in the art to link amino acid sequences, preferably a linker or spacer sequence suitable for use in a pharmaceutical protein or polypeptide, more preferably a linker or spacer sequence used to link antibody fragments or antibody domains. For example, linkers used in the construction of diabodies or ScFv fragments. And since, unlike diabodies or ScFv fragments, single-domain antibodies form a complete antigen binding site by themselves, without the need to consider restrictions to bring the VH and VL domains in close spatial proximity to form a complete antigen binding site, the length or flexibility of the linkers used herein can be as long as it does not hinder the binding of the single-domain antibodies in the multivalent construct to the respective target epitope or target antigen. As a non-limiting example, the linkers herein can be, for example, an amino acid sequence of 1 to 50, preferably 1 to 30, such as 1 to 15 amino acid residues. The skilled person will be able to determine a linker suitable for use in the antibodies of the application based on the disclosure herein, optionally after a limited amount of routine experimentation. In some examples, the linker comprises (Gly x Ser y XAA z ) n(Gly4Ser)3or (Gly3Ser2)3. In some embodiments, the linker sequence is GGSGGSGGGGSGGG (SEQ ID NO: 101) or GGGGSGGGGSGGGS (SEQ ID NO: 102). Other illustrative linkers include, but are not limited to, those having the sequence LE, (GGGGS)n (n is 1, 2, 3, or 4) (SEQ ID NOs: 103-106), (Gly)n (n is 6, 7, or 8) (SEQ ID NOs: 107-109), (EAAAK)n (n is 1, 2, or 3) (SEQ ID NOs: 110-112), A(EAAAK)nA (n is 2, 3, 4, or 5) (SEQ ID NOs: 113-116), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 117), PAPAP (SEQ ID NO: 118), KESGSVSSEQLAQFRSLD (SEQ ID NO: 119), EGKSSGSGSESKST (SEQ ID NO: 120), GSAGSAAGSGEF (SEQ ID NO: 121). In some embodiments, the linker is one or more of, e.g., GGGSE (SEQ ID NO: 122), GSESG (SEQ ID NO: 123), GSEGS (SEQ ID NO: 124), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 125), or a linker in which G, S, and E are randomly placed every 4 amino acids. The multivalent antibodies disclosed herein can also use other naturally occurring heavy chain antibody hinge regions or analogous hinge-like region sequences as linkers (see, e.g., WO 99 / 42077, WO 06 / 040153, WO 06 / 122825, WO 94 / 04678). Other suitable linkers can also include organic compounds or polymers, such as polyethylene glycol moieties for linking antibody domains (see, e.g., WO 04 / 081026). When two or more linkers are used in the polypeptides disclosed herein, the linkers can be the same or different.

[0124] IV. Multispecific Antibodies

[0125] Multispecific antibodies are recombinant proteins composed of two or more different monoclonal antibodies (such as the single domain antibodies disclosed herein) or antigen binding fragments thereof. For example, bispecific antibodies can be composed of antigen binding fragments of two different monoclonal antibodies. Thus, bispecific antibodies bind to two different epitopes or antigens, while trispecific antibodies bind to three different epitopes or antigens. Multispecific antibodies can be used for cancer immunotherapy by, for example, targeting immune effector cells (such as cytotoxic T cells or natural killer cells, by targeting T cell surface receptors such as CD3 or NK cell activating receptors such as CD16) and at least one tumor antigen. The B7-H3 specific single domain monoclonal antibodies disclosed herein can be used to generate multispecific (e.g., bispecific or trispecific) antibodies that target both B7-H3 and CTLs or that target both B7H3 and NK cells, thereby providing methods of treating B7-H3 positive cancers.

[0126] Thus, as examples of bispecific antibodies disclosed herein, one can cite bispecific T cell engagers that fuse a first single domain monoclonal antibody specific for a tumor antigen B7-H3 with a second antibody (such as a scFV or single domain antibody) specific for a T cell surface receptor CD3, or bispecific NK cell engagers that fuse a first single domain monoclonal antibody specific for a tumor antigen B7-H3 with a second antibody (such as a scFV or single domain antibody) specific for an NK cell surface receptor CD16, Ly49 or CD94.

[0127] V. Chimeric Antigen Receptors (CARs)

[0128] The antibodies disclosed herein (e.g., nanobodies, multivalent antibodies, multispecific antibodies) can also be used to produce CARs and / or engineered cytotoxic T lymphocytes or natural killer cells. Generally, CARs comprise an extracellular antigen binding moiety, an extracellular hinge region and spacer element, a transmembrane domain, and an intracellular domain that performs a signaling function (see, e.g., Cartellieri et al., J Biomed Biotechnol, 2010:956304, 2010; Dai et al., J Natl Cancer Inst, 108(7):djv439, 2016). In many cases, the extracellular antigen binding moiety is an antigen binding fragment of a monoclonal antibody, such as a single domain antibody per se or a scFv. The spacer element / hinge region often comprises sequences from an IgG subclass, e.g., IgGl, IgG4, IgD, and CD8 domains. The transmembrane domain can be derived from a variety of different T cell proteins, e.g., CD3 zeta, CD8 alpha, CD4, CD28, CD137, CD80, CD86, CD152, PD1. Several different intracellular domains have been used to generate CARs. For example, the intracellular domain can consist of a signaling chain with ITAMs, including, e.g., at least one of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, DAP10, CD3 zeta, or Fc epsilon RI gamma. In some cases, the intracellular domain also includes an intracellular portion of at least one additional costimulatory domain, e.g., CD28, 4-1BB (CD137, TNFRSF9), OX-40 (CD134), ICOS, CD27, and / or DAP10.

[0129] CTLs, NK cells, or other immune cells expressing CARs can target specific cell types, e.g., B7-H3 positive tumor cells. Thus, the nanobodies disclosed herein can be used to engineer CTLs or NK cells expressing CARs, thereby enabling the engineered CTLs or NK cells to target B7-H3 positive tumor cells. Engineered T cells have previously been used to adoptively treat certain types of cancer. The use of T cells expressing CARs is more general than standard CTL-based immunotherapy, as CAR-expressing CTLs are not HLA restricted, and thus can be used for any patient with a tumor expressing the target antigen.

[0130] The present disclosure also encompasses multispecific CARs. In some embodiments, the multispecific CAR comprises a nanobody specific for B7-H3 (e.g., any of BP2-F12, BP1-C11, BP1-F9, BP1-C10, H-F6, BP2-A9, H-A3, BP1-H8, H-H6, H-A12, BP1-H12, BP2-A12, BP1-F1, BP2-A8, BP1-G2, BP1-C6, BP2-A4, BP1-F12) and a monoclonal antibody specific for a different antigen (e.g., a T cell antigen).

[0131] Accordingly, provided herein are chimeric antigen receptors (CARs) prepared from the B7-H3 specific antibodies disclosed herein, which can be any of the single domain antibodies disclosed herein, or a multivalent or multispecific antibody comprising at least one such single domain antibody. The extracellular antigen binding portion of the prepared CAR comprises at least one single domain antibody disclosed herein, or an antigen binding fragment (e.g., scFv) of a multivalent or multispecific antibody disclosed herein, thereby enabling targeting of B7-H3 positive tumor cells. In some embodiments, the extracellular antigen binding portion of the CAR disclosed herein comprises two or three single domain antibodies disclosed herein, which are optionally the same or different from each other. In some embodiments, the CAR disclosed herein is a bispecific CAR. In other embodiments, the CAR is a bi-cistronic CAR.

[0132] In some embodiments, the CAR comprises a signal peptide sequence, e.g., at the N-terminus of the antigen binding domain. The signal peptide sequence can be any suitable signal peptide sequence, e.g., a signal peptide sequence from granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), immunoglobulin light chain kappa, or IL-2. While the signal peptide sequence can facilitate expression of the CAR on the surface of a cell, it is not required for the CAR to function. The signal peptide sequence can be cleaved from the CAR when the CAR is expressed on the surface of a cell. Accordingly, in some embodiments, the CAR lacks the signal peptide sequence.

[0133] In some embodiments, the CAR construct disclosed herein encodes the following amino acid sequence in the N-terminal to C-terminal direction:

[0134] Signal peptide sequence: MLLLVT SLLLCELPHPAFLLIP (SEQ ID NO: 148)

[0135] Antigen binding portion: an antibody (including, e.g., an antigen binding fragment of a single domain antibody, bispecific antibody, or bivalent antibody disclosed herein) that binds to and / or is specific for B7-H3

[0136] Hinge region (CD8a hinge): FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 136)

[0137] Transmembrane domain (CD28-TM): FWVLVVVGGVLACYSLLVTVAFIIFW (SEQ ID NO: 140)

[0138] Intracellular signaling portion:

[0139] 4-1BB costimulatory signaling domain: VKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 144)

[0140] CD3 zeta costimulatory signaling domain: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 146)

[0141] VI. Immunoconjugates

[0142] The antibodies (e.g., nanobodies, multivalent antibodies, multispecific antibodies) disclosed herein can be conjugated to a therapeutic agent or effector molecule. Immunoconjugates include, but are not limited to, molecules in which a therapeutic agent is covalently attached to an antibody. Therapeutic agents are agents that are capable of affecting a particular biological activity of a target molecule or a cell bearing a target molecule. Therapeutic agents can include various drugs (e.g., vinblastine, daunorubicin, etc.), cytotoxins (e.g., natural or modified Pseudomonas exotoxin or diphtheria toxin), encapsulants containing active sites internally (e.g., liposomes, LNPs), radioactive agents (e.g., 125 I, 32 P, 14 C, 3 H and 35 S), photon absorbers (e.g., IR700), as well as other markers, target moieties, and ligands.

[0143] One skilled in the art can select an appropriate therapeutic agent depending on the application scenario, e.g., depending on the target molecule or cell, the desired biological effect. Thus, the therapeutic agent can be a cytotoxin for causing death of a particular target cell (e.g., a tumor cell).

[0144] Using the therapeutic agents and antibodies described herein, one of skill in the art can readily construct a variety of clones containing functionally equivalent nucleic acids (e.g., nucleic acids that differ in sequence but encode the same effector moiety or antibody sequence). Accordingly, the present disclosure provides nucleic acids encoding antibodies and conjugates and fusion proteins thereof.

[0145] An effector molecule can be attached to the antibodies disclosed herein in any number that a person of skill in the art deems appropriate. The mode of attachment can use covalent and non-covalent means. The procedure for attaching an effector molecule to an antibody can vary depending on the chemical structure of the effector molecule. For example, polypeptide-based effector molecules often contain functional groups such as carboxylic acid (COOH), amino (-NH2), or thiol (-SH) that can be used to react with a suitable functional group on the antibody to attach the effector molecule. Alternatively, the antibody can be derivatized to expose or attach additional reactive functional groups, such as by attaching any of a variety of known linker molecules. A linker can be any molecule used to attach an effector molecule to an antibody. The linker is capable of forming a covalent bond to both the antibody and the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight chain or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. In the case where both the antibody and the effector molecule are polypeptides, the linker can be attached to a side group of either constituent amino acid or to the alpha carbon amino and carboxyl groups of terminal amino acids (e.g., by a disulfide bond to a cysteine).

[0146] In certain cases, it is desirable to release the effector molecule from the conjugate when the immunoconjugate reaches its target site. In these cases, the immunoconjugate will contain a linkage that can be cleaved in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the conjugate can be promoted by enzymatic activity or by conditions to which the immunoconjugate is subjected inside the target cell or in the vicinity of the target site.

[0147] One of skill in the art can determine the appropriate method for attaching a given agent to an antibody or other polypeptide, without adversely affecting the target activity of the antibody and agent (e.g., the binding of the antibody to the target antigen), according to the various methods of attaching a wide variety of radio-diagnostic compounds, radiotherapeutic compounds, markers (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies that have been extensively reported.

[0148] The immunoconjugates disclosed herein can be produced by cross-linking two or more antibodies (of the same or different types, e.g., to produce a bispecific antibody). Suitable cross-linking agents include heterobifunctional cross-linking agents, having two apparently reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., succinimidyl succinate). Such linkers are commercially available.

[0149] The immunoconjugates disclosed herein can be conjugated to a detectable marker, such as a detectable marker that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber-optic inspection, and laparoscopy). Non-limiting examples of detectable markers include magnetic beads, fluorophores, chemiluminescers, enzyme linkages, radioisotopes, 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-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like; bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP); and enzymes which can be used for detection such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When the antibodies or antigen-binding fragments disclosed herein are conjugated to a detectable enzyme, detection can be accomplished by addition of other reagents that cause the enzyme to produce a recognizable reaction product. For example, when horseradish peroxidase is conjugated, detection can be accomplished visually by addition of hydrogen peroxide and diaminobenzidine, which results in a colored reaction product. When the antibodies or antigen-binding fragments disclosed herein are conjugated to biotin, detection can be accomplished by indirect measurement of binding to avidin or streptavidin. Additionally, the detection reagent itself (e.g., avidin) can itself be conjugated to an enzyme or fluorescent marker.

[0150] Antibodies can be labeled with magnetic reagents, such as gadolinium. Antibodies can also be labeled with lanthanides (e.g., europium and dysprosium) and manganese. Paramagnetic particles such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be labeled with predetermined polypeptide epitopes that are recognized by secondary reporters (e.g., leucine zipper pairs, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labels are attached through various length spacers to reduce potential steric hindrance.

[0151] Antibodies can also be labeled with radiolabeled amino acids. Radiolabeled antibodies can be used for both diagnostic and therapeutic purposes. For example, radiolabeled antibodies can be used to detect expression of a target antigen by x-ray, emission spectroscopy, or other diagnostic techniques. Examples of radiolabels include, but are not limited to, the use of the following radioisotopes: 3 H、 14 C、 15 N、 35 S、 90 Y、 99 Tc、 111 In、 125 I、 131 I.

[0152] The antibodies disclosed herein can also be conjugated to a photon absorber. In some embodiments, the photon absorber is a phthalocyanine dye, such as, but not limited to, 700DX (also known as “IR700”). Antibody-photon absorber conjugates can be used in photodynamic therapy.

[0153] Antibodies can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl or carbohydrate groups. These groups can be used to improve the biological properties of the antibody, such as to extend serum half-life or to enhance tissue binding.

[0154] The antibodies disclosed herein can also be used with toxins to produce an immunotoxin. Exemplary toxins include ricin, abrin, diphtheria toxin and subunits thereof, and botulinum toxins A through F. Contemplated toxins also include variants of the toxins described herein (see, e.g., U.S. Pat. Nos. 5,079,163 and 4,689,401). In one embodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Pat. No. 5,602,095). As used herein, “Pseudomonas exotoxin” can include full-length native PE or cytotoxic fragments thereof, or modified PE. Cytotoxic fragments of PE include cytotoxic fragments with or without subsequent proteolytic processing or other processing in a target cell. Cytotoxic fragments of PE include PE40, PE38, and PE35. See, e.g., U.S. Pat. Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; 5,854,044; U.S. Pat. App. Pub. No. 2015 / 0099707; PCT Pub. Nos. WO 99 / 51643 and WO 2014 / 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, for additional description of PE and variants thereof. Modified PE can include, but is not limited to, ablation of domain la, multiple amino acid deletions in domains lb, II, and III, single amino acid substitutions, and addition of one or more sequences at the carboxy terminus (see, e.g., Siegall et al., J. Biol. Chem. 264:14256-14261, 1989).

[0155] Also contemplated herein are protease-resistant PE variants and immunogenicity-reduced PE variants, such as, but not limited to, PE-LR, PE-6X, PE-8X, PE-LR / 6X, and PE-LR / 8X (see, e.g., Weldon et al., Blood 113(16):3792-3800, 2009; Onda et al., Proc Natl Acad Sci USA 105(32):11311-11316, 2008; and PCT Publication Nos. WO 2007 / 016150, WO 2009 / 032954, and WO 2011 / 032022, which are incorporated herein by reference).

[0156] 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. WO 2009 / 032954). In other examples, the PE is a variant designated PE-LR / 6X (PCT Publication No. WO 2011 / 032022). In other examples, the PE variant is an immunogenicity-reduced PE. In yet other examples, the PE is a variant designated PE-LR / 8M (PCT Publication No. WO 2011 / 032022).

[0157] The modifications to the PE can occur in any of the previously described variants, including cytotoxic fragments of PE (e.g., PE38, PE-LR, and PE-LR / 8M). The modified PE can include any substitution, such as substitution of one or more amino acid residues within one or more T cell epitopes and / or B cell epitopes of PE, or deletion of one or more T cell and / or B cell epitopes (see, e.g., U.S. Patent Application Publication No. 2015 / 0099707).

[0158] Also contemplated forms of PE include de-immunized forms of PE, such as versions that delete domain II (e.g., PE24). De-immunized forms of PE are described in, e.g., PCT Publication Nos. WO 2005 / 052006, WO 2007 / 016150, WO 2007 / 014743, WO 2007 / 031741, WO 2009 / 32954, WO 2011 / 32022, WO 2012 / 154530, and WO 2012 / 170617.

[0159] The antibodies disclosed herein can also be used to target any number of different diagnostic or therapeutic compounds to cells expressing B7-H3 on their surface. Thus, the antibodies of the present disclosure can be attached, directly or through a linker, to a drug that will be delivered directly to cells expressing B7-H3 on their surface for therapeutic, diagnostic, or research purposes. Drugs or effector molecules that can be delivered by the antibodies of the present disclosure include nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, photon absorbers, lipids, carbohydrates, or recombinant viruses, among other compounds. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking to single- or double-stranded DNA, and oligonucleotides that form triplexes.

[0160] The molecule attached to the antibody can also be an encapsulating system, such as a nanoparticle, liposome, or micelle, containing a therapeutic composition (such as a drug), nucleic acid (such as an antisense nucleic acid, mRNA), or another therapeutic moiety, preferably shielded from direct exposure to the circulatory system. Methods of making antibody-attached liposomes are known to those of skill in the art (see, e.g., U.S. Patent No. 4,957,735; Connor et al., Pharm. Ther. 28:341-365, 1985).

[0161] VII. Antibody-drug conjugates (ADCs)

[0162] ADCs are compounds composed of a tumor antigen-specific antibody (such as a single-domain antibody, multivalent antibody, multispecific antibody, or antigen-binding fragment thereof disclosed herein) and a drug, usually a cytotoxic agent, such as an anti-microtubule agent or a cross-linking agent. ADCs take advantage of the specific targeting of antibodies to cancer cells, which can be more effective than standard chemotherapy drugs. The IC50 of the most common cytotoxic drugs used as ADCs is 100 to 1000 times stronger than conventional chemotherapy drugs. Cytotoxic drugs commonly used in ADCs include anti-microtubule agents, such as maytansinoids, auristatins such as auristatin E and auristatin F. Other cytotoxins include pyrrolobenzodiazepines (PBDs), which covalently bind to the minor groove of DNA to form interstrand crosslinks. In many cases, the antibody to drug ratio of ADCs is 1:2 to 1:4 (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).

[0163] Antibodies and drugs can be linked via cleavable or noncleavable linkers. In some cases, ADCs require linkers that are stable in circulation, thereby preventing significant off-target toxicity of the cytotoxic drug due to systemic release. Noncleavable linkers prevent release of the cytotoxic agent until the ADC is internalized by the target cell. Upon endocytosis into the lysosome, enzymatic cleavage by lysosomal proteases enables release of the cytotoxic agent (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).

[0164] One method of site-specific and stable conjugation of drugs to monoclonal antibodies is through glycan engineering. Monoclonal antibodies have a conserved N-linked oligosaccharide chain at the Asn297 residue of each heavy chain CH2 domain (Qasba et al., Biotechnol Prog 24:520-526, 2008). Using a mutant β1,4-galactosyltransferase (Y289L-Gal-T1; U.S. Patent Application Publication Nos. 2007 / 0258986 and 2006 / 0084162, incorporated herein by reference), 2-keto-galactose is transferred to the free GlcNAc residue on the antibody heavy chain, enabling conjugation chemistry.

[0165] Oligosaccharide chains attached to monoclonal antibodies can be grouped into three groups based on the terminal galactose residue— fully galactosylated (two galactose residues; IgG-G2) oligosaccharide chains, one galactose residue (IgG-G1) oligosaccharide chains, or fully agalactosylated (IgG-G0) oligosaccharide chains. Treatment of monoclonal antibodies with β1,4-galactosidase can convert the antibody to the IgG-G0 glycoform. Mutant β1,4-galactosyltransferase can transfer 2-keto-galactose or 2-azido-galactose from their respective UDP derivatives to the GlcNAc residue on the IgG-G1 and IgG-G0 glycoforms. Chemical manipulation of the transferred sugar enables a variety of molecules to be conjugated to the monoclonal antibody through the glycan residue (Qasba et al., Biotechnol Prog 24:520-526, 2008).

[0166] Provided herein are ADCs comprising a drug conjugated to a monoclonal antibody directed to and / or specifically binding to B7-H3. In some embodiments, the drug is a small molecule. In some examples, the drug is a cross-linking agent, an anti-microtubule agent, and / or an anti-mitotic agent, or any cytotoxic agent suitable to mediate killing of tumor cells. Exemplary cytotoxic agents include, but are not limited to, PBDs, auristatins, maytansinoids, dolastatins, calicheamicins, nemorubicin and its derivatives, PNU-159682, anthracyclines, vinca alkaloids, taxoids, a ciliatins, CC1065, camptothecins, elliptinium, combretastatins, dolastatins, duocarmycins, enediynes, geldanamycin, indolinobenzodiazepine dimers, puromycins, tubulysins, semustine, splicing inhibitors, or pladiellins, as well as stereoisomers, isosteres, analogs, and derivatives having cytotoxic activity.

[0167] In some embodiments, the ADC comprises an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine and are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was originally isolated from the East African shrub, Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinoids are disclosed, for example, in U.S. Pat. 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.

[0168] In some embodiments, the ADC comprises an antibody conjugated to a dolastatin or auristatin or analog or derivative thereof (see U.S. Patent Nos. 5,635,483; 5,780,588; 5,767,237; and 6,124,431). Auristatins are derivatives of the marine mollusk compound dolastatin-10. Dolastatins and auristatins have been shown to 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 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- sea horse isoleucine-sea horse proline-phenylalanine), MMAE (monomethyl auristatin E or N-methylvaline-valine-sea horse isoleucine-sea horse proline-nor epinephrine), 5-benzoylvaleric acid-AE ester (AEVB), and other auristatins (see, e.g., U.S. Pub. No. 2013 / 0129753).

[0169] In some embodiments, the ADC comprises an antibody conjugated to one or more calicheamicin molecules. Antibiotics of the calicheamicin family and their analogs are capable of producing double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al., Cancer Res 53:3336-3342, 1993; Lode et al., Cancer Res 58:2925-2928, 1998). Exemplary methods for preparing ADCs with calicheamicin drug moieties are described in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.

[0170] In some embodiments, the ADC comprises an anthracycline. Anthracyclines are antibiotic compounds with cytotoxic activity. It is believed that anthracyclines can kill cells through a variety of different mechanisms, including intercalation of the drug molecule into the DNA of the cell, thereby inhibiting DNA-dependent nucleic acid synthesis; induction of free radicals that then react with cellular macromolecules, causing damage to the cell; and / or interaction of the drug molecule with the cell membrane. Non-limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunorubicin, daunomycin, doxorubicin, epirubicin, nemorubicin, valrubicin, and mitoxantrone, and derivatives thereof. For example, PNU-159682 is an effective metabolite or derivative of nemorubicin (Quintieri et al., Clin Cancer Res 11(4): 1608-1617, 2005). Nemorubicin is a semisynthetic analogue of doxorubicin with a 2-methoxymorpholinyl group on the glycoside amino group of doxorubicin (Grandi et al., Cancer Treat Rev 17: 133, 1990; Ripamonti et al., Br J Cancer 65: 703-707, 1992).

[0171] In some embodiments, the ADC can further include a linker. In some examples, the linker is a bifunctional or multifunctional moiety that can be used to attach one or more drug moieties to an antibody to form an ADC. In some embodiments, the ADC is prepared using a linker having a reactive functional group for covalent attachment of a drug and an antibody. For example, a cysteine thiol of an antibody can form a bond with a reactive functional group of a linker or drug-linker intermediate to prepare an ADC.

[0172] In some examples, the linker has a functional group that is capable of reacting with a free cysteine present on the antibody to form a covalent bond. Exemplary linkers having such a reactive functional group include maleimides, haloacetamides, a-haloacetyl, activated esters (such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters), acid anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0173] In some examples, the linker has a functional group that is 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 linker reactive functional group can react with an electrophilic group on the antibody and form a covalent bond with the antibody unit. Non-limiting examples include hydrazides, oximes, amines, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazides.

[0174] In some examples, the linker is a cleavable linker that facilitates drug release. Examples of cleavable linkers include acid-labile linkers (e.g., containing a hydrazone), protease-sensitive linkers (e.g., peptidase-sensitive linkers), photolabile linkers, and disulfide-containing linkers (Chari et al., Cancer Res 52: 127-131, 1992; U.S. Pat. No. 5,208,020).

[0175] The ADCs disclosed herein can be used alone to treat a B7-H3 positive cancer, or in combination with another therapeutic agent and / or in combination with any standard therapy for treating a cancer (e.g., surgical resection of a tumor, chemotherapy, or radiation therapy).

[0176] VIII. Antibody-nanoparticle conjugates

[0177] The monoclonal antibodies disclosed herein can be conjugated to a variety of different types of nanoparticles, delivering the nanoparticle or its encapsulated contents directly to a tumor cell via the binding of the antibody to B7-H3 expressed on the surface of the tumor cell. Nanoparticles can reduce off-target side effects, improve bioavailability of a drug, and reduce the dose of a drug needed to achieve a therapeutic effect. Nanoparticles can be formulated depending on the drug carried or encapsulated within the particle. For example, hydrophobic molecules can be incorporated within the core of the nanoparticle, while hydrophilic drugs can be carried within an aqueous core protected by a polymeric or lipid shell. Examples of nanoparticles include, but are not limited to, nanospheres, nanocapsules, liposomes, dendrimers, polymeric micelles, vesicles, and polymeric nanoparticles (Fay and Scott, Immunotherapy 3(3):381-394, 2011).

[0178] Liposomes are a common nanoparticle type used for drug delivery. Antibodies conjugated to liposomes are often referred to as “immunoliposomes.” The liposome component of immunoliposomes is typically a lipid vesicle of one or more concentric phospholipid bilayers. In some cases, the phospholipids consist of a hydrophilic head group and two hydrophobic chains, enabling 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 liposome can be adjusted. The surface of the liposome can also be modified, for example, with glycolipids or sialic acids. For example, the addition of polyethylene glycol significantly extends the circulation half-life. Liposomes used as drug delivery agents, including liposomes used to make immunoliposomes, have been described in the art (see, e.g., Paszko and Senge, Curr Med Chem 19(31) 5239-5277, 2012; Immordino et al., Int J Nanomedicine 1(3):297-315, 2006; U.S. Patent Application Publication Nos. 2011 / 0268655; 2010 / 00329981).

[0179] Niosomes are vesicles based on non-ionic surfactants, with a structure similar to liposomes. The niosome membrane consists only of non-ionic surfactants, such as polyglyceryl alkyl ethers or N-palmitoylglucosamine. Niosomes range from small unilamellar particles to large multilamellar particles. These nanoparticles are monodisperse, water-soluble, chemically stable, have low toxicity, are biodegradable and non-immunogenic, and enhance the bioavailability of encapsulated drugs.

[0180] Dendrimers include a series of branched polymer complexes. These nanoparticles are water-soluble, biocompatible, and sufficiently non-immunogenic for human use. Typically, dendrimers consist of an initiator core, around which is a layer of selected polymers (grafted onto the core), forming a branched macromolecular complex. Dendrimers are often produced using polymers such as poly(amidoamine) or poly(L-lysine). Dendrimers have been used for a variety of therapeutic and diagnostic applications, including for the delivery of DNA, RNA, bioimaging contrast agents, and chemotherapeutic agents.

[0181] The composition of the polymer micelles is that amphiphilic copolymers (composed of hydrophilic monomer units and hydrophobic monomer units) assemble into a hydrophobic core surrounded by a corona of hydrophilic polymer chains exposed to the aqueous environment. In many cases, the polymer used to make the polymer micelles is a heterobifunctional copolymer composed of hydrophilic blocks of PEG, poly(vinylpyrrolidone), and a hydrophobic poly(L-lactide) or poly(L-lysine) that forms the core of the particle. Polymer micelles can be used to carry poorly soluble drugs. These nanoparticles have been used to encapsulate a number of anticancer drugs, including doxorubicin and camptothecin. Cationic micelles have also been developed to carry DNA or RNA molecules.

[0182] Polymeric nanoparticles include nanospheres and nanocapsules. Nanospheres are composed of a solid matrix of polymer, while nanocapsules contain an aqueous core. The selected formulation generally depends on the solubility of the therapeutic agent to be carried / encapsulated; poorly water-soluble drugs are more easily encapsulated in nanospheres, while water-soluble and unstable drugs, such as DNA and proteins, are more easily encapsulated in nanocapsules. Polymers used to produce these nanoparticles include, for example, poly(acrylamide), poly(esters), poly(cyanoalkyl acrylate), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(D,L-lactic-co-glycolic acid) (PLGA).

[0183] Antibodies can be conjugated to suitable nanoparticles according to standard methods known in the art. For example, the conjugation can be covalent or non-covalent. In some embodiments, where the nanoparticle is a liposome, the antibody is attached to the sterically stabilized, long-circulating liposome via a PEG chain. Coupling of the antibody or antibody fragment to the liposome can also involve a thioester bond, for example, through the reaction of a thiol and a maleimide group. Cross-linking agents can be used to generate thiols to attach the antibody to the nanoparticle (Paszko and Senge, Curr Med Chem 19(31) 5239-5277, 2012).

[0184] IX. Compositions and Methods of Use

[0185] The compositions provided herein comprise one or more of the monoclonal antibodies disclosed herein that bind to and / or are specific for B7-H3. Also provided are compositions comprising the CARs disclosed herein, engineered cells expressing the CARs, immunoconjugates, ADCs, antibody-nanoparticle conjugates, immunoliposomes. The combinations disclosed herein can be prepared in unit dosage form for administration to a subject, the amount and timing of administration being determined by a clinician based on the intended effect. The various antibodies, CARs, engineered cells expressing the CARs, immunoconjugates, ADCs, antibody-nanoparticle conjugates, immunoliposomes disclosed herein can be formulated for systemic or local (e.g., intra- or peri-tumoral) administration. In one example, the combinations herein are formulated for parenteral administration, e.g., intravenous administration.

[0186] The compositions disclosed herein can include solutions of various antibodies, CARs, engineered cells expressing CARs, immunoconjugates, ADCs, antibody-nanoparticle conjugates, immunoliposomes in pharmaceutically acceptable carriers (e.g., aqueous vehicles). A variety of aqueous vehicles can be used, e.g., buffered saline and the like. These solutions are sterile, and generally free of unacceptably large amounts of particulate matter. The compositions disclosed herein can be sterilized by conventional, well-known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliaries as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antibodies in the composition can vary widely, depending upon such factors as the volume of the body space or fluid to be treated, the viscosity of the composition, the specific mode of administration selected and the subject's body weight, among other things.

[0187] Typical pharmaceutical compositions for intravenous administration include about 0.1 mg to 10 mg of antibody (or CAR, immunoconjugate, ADC, antibody-nanoparticle conjugate, immunoliposome) per subject per day. Doses of 0.1 mg to about 100 mg per subject per day can also be employed, particularly where the agent is administered to a secluded site (e.g., a body cavity or lumen of an organ), rather than the circulatory or lymphatic system. Actual methods for preparing administrable compositions are known or apparent to those skilled in the art, see, e.g., Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, Pa., 21st Edition (2005).

[0188] The monoclonal antibodies (or conjugates thereof) disclosed herein can also be administered by other routes, including by inhalation, orally, topically, or intraocularly. In some examples, the monoclonal antibodies (or conjugates thereof) are administered by fine needle.

[0189] In addition to being provided in the form of sterile solutions, the antibodies disclosed herein can also be provided in lyophilized form and reconstituted with sterile water prior to administration. The antibody solution can then be added to an infusion bag containing 0.9% sodium chloride (USP) and, in some cases, administered at a dose of 0.5 to 15 mg / kg body weight. Since 1997, Rituxan TMSince approval, the field has accumulated a great deal of experience in administering marketed antibody drugs. Antibodies, CARs, immunoconjugates, ADCs, antibody-nanoparticle conjugates, immunoliposomes can be administered by slow infusion, rather than intravenous bolus or large bolus administration. In one example, a higher loading dose is administered first, followed by lower level maintenance doses. For example, an initial loading dose of 4 mg / kg can be infused over a period of about 90 minutes, followed by a maintenance dose of 2 mg / kg infused over 30 minutes every week for 4-8 weeks, if the previous dose was well tolerated.

[0190] Controlled release parenteral formulations can be made into implants, oily injections, or microparticulate systems. A comprehensive overview of protein delivery systems can be found in, for example, Banga, A. J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, Pa., (1995). Microparticulate systems include, for example, microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, for example, a cytotoxin or a drug, as a core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules that are less than about 1 μm are commonly referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries are about 5 μm in diameter, so only nanoparticles can be administered intravenously. Microparticles are typically about 100 μm in diameter and can be administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339, (1992).

[0191] Polymers can be used to ionically control the release of the compositions disclosed herein. Various degradable and non-degradable polymeric matrices for 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, yet flowable liquid at low temperatures, but forms a semisolid gel at body temperature, and has proven to be an effective vehicle for formulating 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 controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In another aspect, liposomes are used for controlled release of lipid-encapsulated drugs as well as drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, Pa. (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are also 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).

[0192] A. Methods of Treatment

[0193] The antibodies (e.g., single domain antibodies, multivalent antibodies, multispecific antibodies), CARs, engineered cells (e.g., CTLs or NK cells) expressing CARs, immunoconjugates, ADCs, antibody-nanoparticle conjugates, immunoliposomes, compositions disclosed herein can be administered to slow or inhibit the growth of B7-H3 positive tumor cells, or to inhibit metastasis of B7-H3 positive tumor cells. In these applications, a therapeutically effective amount of the composition is an amount that is administered to a subject sufficient to inhibit the growth, replication, or metastasis of cancer cells or to inhibit a sign or symptom of cancer. Suitable subjects include subjects diagnosed with a B7-H3 positive solid tumor, such as, but not limited to, liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer, renal cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer (e.g., neuroblastoma or glioblastoma), pediatric cancer (e.g., osteosarcoma, neuroblastoma, rhabdomyosarcoma, or Ewing's sarcoma), melanoma, or mesothelioma.

[0194] Provided herein are methods of treating a B7-H3 positive cancer in a subject by administering to the subject a therapeutically effective amount of a B7-H3 specific antibody disclosed herein, a CAR, an immunoconjugate, an ADC, an antibody-nanoparticle conjugate, or an immunoliposome prepared based on the antibody, an engineered cell (e.g., CTL or NK cell) expressing the CAR, a composition. Also provided herein are methods of inhibiting tumor growth or metastasis of a B7-H3 positive cancer in a subject by administering to the subject a therapeutically effective amount of a B7-H3 specific antibody disclosed herein, a CAR, an immunoconjugate, an ADC, an antibody-nanoparticle conjugate, or an immunoliposome prepared based on the antibody, an engineered cell (e.g., CTL or NK cell) expressing the CAR, a composition. In some embodiments, the B7H3 positive cancer is liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer, renal cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer (e.g., neuroblastoma or glioblastoma), pediatric cancer (e.g., osteosarcoma, neuroblastoma, rhabdomyosarcoma, or Ewing's sarcoma), melanoma, or mesothelioma.

[0195] A therapeutically effective amount of a B7-H3 specific monoclonal antibody disclosed herein, a CAR, an immunoconjugate, an ADC, an antibody-nanoparticle conjugate, or an immunoliposome prepared based on the antibody, an engineered cell (e.g., CTL or NK cell) expressing the CAR, a composition will depend on the severity of the disease, the type of disease, and the overall condition of the patient. A therapeutically effective amount of an antibody-based composition is an amount that is capable of producing a subjective improvement in symptoms or an objectively identifiable improvement as noted by a clinician or other qualified observer.

[0196] The B7-H3 specific monoclonal antibodies disclosed herein, CARs, immunoconjugates, ADCs, antibody-nanoparticle conjugates or immunoliposomes prepared based on the antibodies, engineered cells expressing the CARs (such as CTLs or NK cells), compositions can also be administered in conjunction with the administration of other anti-cancer agents or therapies (e.g., surgical removal of a tumor). Examples of suitable anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-angiogenesis agents, biological response modifiers, hormones and anti-hormones (e.g., anti-androgens), and anti-vascular agents. Other anti-cancer therapies include radiation therapy and other antibodies (e.g., biologies) that specifically target cancer cells.

[0197] Non-limiting examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkyl sulfonates (e.g., busulfan), and nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin, or dacarbazine).

[0198] Non-limiting examples of anti-metabolites include folic acid analogues (e.g., methotrexate), pyrimidine analogues (e.g., 5-FU or cytarabine), and purine analogues (e.g., mercaptopurine or thioguanine).

[0199] Non-limiting examples of natural products include vinca alkaloids (e.g., vinblastine, vincristine, or vindesine), epipodophyllotoxins (e.g., etoposide or teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (e.g., L-asparaginase).

[0200] Non-limiting examples of miscellaneous agents include platinum coordination complexes (e.g., cis-diamminedichloroplatinum II, also known as cisplatin), substituted ureas (e.g., hydroxyurea), methyl hydrazine derivatives (e.g., procarbazine), and adrenocortical suppressors (e.g., mitotane and aminoglutethimide).

[0201] Non-limiting examples of hormones and antagonists include adrenocorticosteroids (e.g., prednisone), progestogens (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (e.g., stilbestrol and ethynyl estradiol), antiestrogens (e.g., tamoxifen), and androgens (e.g., testosterone propionate and fluoxymesterone).

[0202] Examples of the most commonly used chemotherapeutic agents include adriamycin, mafosfamide, Ara-C, BiCNU, busulfan, CCNU, carboplatin, cisplatin, cyclophosphamide, daunorubicin, DTIC, 5-FU, fludarabine, hydroxyurea, idarubicin, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel (or other taxanes, such as docetaxel), vinblastine, vincristine, VP- 16, while some newer agents include gemcitabine (GEMZAR®), herceptin, irinotecan (CAMPTOSAR®, CPT-11), LEUKOVORIN®, LURTOTON® injection, NELARABINE®, NOVANTRON®, OFERON® STI-571, TAXOTERE®, topotecan (HYCAMTIN®), XELODA® (capecitabine), Zevelin, and calcitriol.

[0203] Non-limiting examples of immunomodulatory agents include AS-10 (Wyeth-Ayerst Labs), bropirimine, gamma interferon, GM-CSF (granulocyte macrophage colony stimulating factor), IL-2, human immunoglobulin, Imreg, SK&F 106528, and TNF.

[0204] Non-limiting examples of biologics that can be used in combination with the B7-H3-specific monoclonal antibodies disclosed herein, CARs prepared based on said antibodies, immunoconjugates, ADCs, antibody-nanoparticle conjugates or immunoliposomes, engineered cells expressing said CARs (such as CTLs or NK cells), and compositions thereof include various therapeutic monoclonal antibodies, such as 3F8, abagovomab, adecatumumab, afutuzumab, alacizumab, alemtuzumab, atumomab pentetate, and anatumomab. mafenatox, apolizumab, arcitumomab, bavituximab, beectumomab, belimumab, besilesomab, bevacizumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromab pendetide, catuxomab, CC49, cetuximab, citatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan, conatumumab, dacetuzumab, detumomab, ecromeximab, eculizumab, edrecolomab, eprecolomab, epratuzumab, ertumaxomab, edaracizumab, farletuzumab, figitumumab, galiximab, gemtuzumab ozogamicin, girenteximab, glembatumumab vedotin, iprimumumabtiuxetan, Igovomab, Imciromab, Intetumumab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab tafenatox, Naptumomabestafenatox, Necitumumab, Nimotuzumab, Nofetumomab merpentan, Ofatumumab, Olaratumab, Oportuzumab Monatox, Oregovomab, Panitumumab, Pemtumomab, Pertuzumab, Pintumomab, Pritumumab, Ramucirumab, Rilotumumab, Rituximab, Robatumumab, Satumomab pendetide, Sibrotuzumab, Sonepizumab, Tacatuzumab tetraxetan, Taplitumomabpaptox), Tenatumomab, TGN1412, Ticilimumab (tremelimumab), Tigatuzumab, TNX-650, Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Veltuzumab, Volociximab, Votumumab, and Zalutumumab. In some examples, the therapeutic antibody specifically binds to and antagonizes PD-1 or PD-L1, for example, 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.

[0205] In some examples, the additional therapeutic agent 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 administered is an OX40 agonist, such as an antibody, e.g., 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 administered is a 4-1BB agonist, such as a 4-1BB agonist antibody, e.g., a mAb. Particular agonist mAbs that can be used in the methods disclosed herein include PF-05082566 (Utolumab) and BMS-663513 (Urelumab). In one example, the 4-1BB agonist is a 4-1BB ligand (4-1BBL), such as a native 4-1BBL (e.g., human 4-1IBBL) or a streptavidinized 4-1BBL (SA-4-1BBL) complex. In some examples, the additional therapeutic agent administered is a GITR (glucocorticoid-induced tumor necrosis factor (TNF) receptor, or TNFRSF18) agonist, such as a GITR agonist antibody, e.g., a mAb. Particular GITR agonist mAbs that can be used with 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 a GITR ligand (GITRL), such as a native GITRL or a multivalent GITR ligand fusion protein. In one example, the GITR agonist is MEDI1873, a hexameric GITRL molecule with a human IgGl Fc domain. In some examples, the additional therapeutic agent administered is an immunotherapy. Non-limiting examples of immunomodulatory agents that can be used include AS-101, bromopindol, gamma interferon, GM-CSF, IL-2, human immunoglobulin, Imreg, SK&F 106528, and TNF.

[0206] In one example, the additional therapy used is surgical treatment, such as surgical resection of a cancer or a portion thereof. Another example of an additional therapy is radiation therapy, such as administration of a radioactive substance or energy (e.g., external beam therapy) to a tumor site prior to surgical resection to help eradicate or shrink the tumor.

[0207] B. Diagnostic and Detection Methods

[0208] Also provided herein are methods for detecting B7-H3 protein in vitro or in vivo. For example, the monoclonal antibodies (e.g., single-domain antibodies, multivalent antibodies, multispecific antibodies), immunoconjugates, antibody-nanoparticle conjugates, fusion proteins, or compositions disclosed herein can be used for in vivo tumor imaging. To use the disclosed antibodies as in vivo diagnostic reagents, the antibodies are labeled with a detectable moiety, such as a radioisotope, a fluorescent marker, or a positron-emitting radionuclide. As one example, the monoclonal antibodies disclosed herein can be conjugated to a positron-emitting radionuclide for use in positron emission tomography (PET); this diagnostic procedure is commonly referred to as immunoPET. In comparison to full-length antibodies, single-domain antibodies / nanobodies are smaller in size, have shorter biological half-lives, do not require waiting for several days before imaging, and can be imaged on the same day, thus improving the convenience of diagnosis and detection and reducing the associated non-target radiation dose.

[0209] The monoclonal antibodies (e.g., single-domain antibodies, multivalent antibodies, multispecific antibodies), immunoconjugates, antibody-nanoparticle conjugates, fusion proteins, or compositions disclosed herein can also be used to detect B7-H3 expression in a biological sample. The sample can be any sample, including but not limited to tissue from a biopsy, autopsy sample, and pathological sample. Biological samples also include tissue sections, e.g., frozen sections for histological purposes. Biological samples also include body fluids, such as blood, serum, plasma, sputum, spinal fluid, or urine. In some examples, the sample is an exosome-containing serum sample. The biological sample is typically obtained from a mammal, such as a human or non-human primate.

[0210] Provided herein is a method of determining / diagnosing whether a subject has a B7-H3 positive cancer, comprising detecting binding of the monoclonal antibodies (e.g., single-domain antibodies, multivalent antibodies, multispecific antibodies), immunoconjugates, antibody-nanoparticle conjugates, fusion proteins, or compositions disclosed herein to a sample from the subject after contacting the sample with the antibodies. An increase in binding of the antibodies to the sample compared to binding in a control will confirm the subject as diagnosed with a B7-H3 positive cancer.

[0211] In some examples, the monoclonal antibodies (e.g., single-domain antibodies, multivalent antibodies, multispecific antibodies), immunoconjugates, antibody-nanoparticle conjugates, fusion proteins, or compositions disclosed herein are directly labeled.

[0212] In other examples, the methods disclosed herein further comprise contacting a second antibody (detection antibody) that specifically binds to a monoclonal antibody (e.g., a single domain antibody, a multivalent antibody, a multispecific antibody) of the disclosure, an immunoconjugate, an antibody-nanoparticle conjugate, a fusion protein, or a composition of the disclosure with the sample; and detecting binding of the second antibody. An increase in binding of the second antibody to the sample compared to binding in a control sample will confirm a diagnosis that the subject has a B7-H3 positive cancer.

[0213] In some cases, the cancer diagnosed by the methods of the disclosure can be selected from a solid tumor or a hematological tumor. In some examples, the solid tumor can be selected from a nervous system tumor, a head and neck tumor, a thoracic tumor, a digestive system tumor, a genitourinary system tumor, a soft tissue and skin tumor, or a bone tumor. In some examples, the nervous system tumor can be selected from a diffuse glioma, a astrocytoma (e.g., diffuse astrocytoma, anaplastic astrocytoma, oligoastrocytoma), a glioblastoma, an oligodendroglioma, a childhood diffuse glioma, an ependymoma, a neuronal and mixed neuronal-glial tumor, a medulloblastoma, an embryonal tumor, a schwannoma, a meningioma, an solitary fibrous tumor, or a perivascular cell tumor. In some examples, the head and neck tumor can be selected from a nasal cavity and paranasal sinus malignant neoplasm, a nasopharyngeal carcinoma, an oral cavity cancer, a laryngeal cancer, a salivary gland tumor, an intracranial tumor (e.g., a neuroblastoma or a glioblastoma), a thyroid cancer, or a tongue cancer. In some examples, the thoracic tumor can be selected from a lung cancer (e.g., a non-small cell lung cancer), an esophageal cancer, a cardiac cancer, a breast cancer (e.g., a triple negative breast adenocarcinoma cell), a mesothelioma, or a mediastinal tumor. In some examples, the digestive system tumor can be selected from a gastric cancer, a large intestinal cancer, a colon cancer (e.g., a colon adenoma), a colorectal cancer, a liver cancer (e.g., a hepatocellular carcinoma), a pancreatic cancer, a periampullar carcinoma, a biliary tract cancer, or a small intestinal cancer. In some examples, the genitourinary system tumor can be selected from a renal cancer (e.g., a renal cell adenocarcinoma), a prostate cancer, a bladder cancer, a testicular cancer, a penile cancer, a cervical cancer, an endometrial cancer, or an ovarian cancer. In some examples, the soft tissue and skin tumor can be selected from a fibrous histiocytoma, a rhabdomyosarcoma, a synovial sarcoma, or a melanoma. In some examples, the bone tumor can be selected from a osteosarcoma or an Ewing's sarcoma. In some examples, the hematological tumor is selected from a leukemia, a lymphoma (HL), a multiple myeloma (MM), or a myelodysplastic syndrome (MDS). In some examples, the leukemia is selected from a B-cell acute lymphoblastic leukemia, a T-cell acute lymphoblastic leukemia, an acute myeloid leukemia, a monocyte leukemia, or a myeloblast leukemia. In some examples, the lymphoma is selected from a histiocytic lymphoma or a Burkitt's lymphoma.

[0214] In some examples, the control sample is a sample from a subject that does not have cancer. In particular examples, the sample is an ex vivo blood sample or a tissue sample.

[0215] In some embodiments of the diagnostic and detection methods, the B7-H3 monoclonal antibodies disclosed herein can be directly labeled with a detectable label. In another embodiment, the B7-H3 monoclonal antibodies disclosed herein (primary antibodies) are unlabeled, while a secondary antibody or other molecule directed against the antibody is labeled. The selection of a secondary antibody, depending on the species of the primary antibody, is well known to those skilled in the art. For example, if the primary antibody is a human IgG, then the secondary antibody can be anti-human IgG. Other molecules that can be conjugated to antibodies include, but are not limited to, protein A and protein G, both of which are commercially available.

[0216] Suitable labels for the antibody or secondary antibody include various enzymes, prosthetic groups, fluorescent, luminescent, magnetic and radioactive agents. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic groups include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent agents include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium; and non-limiting exemplary radioactive labels include 125 I, 131 I, 35 S or 3 H.

[0217] In an alternative embodiment, B7-H3 in a biological sample can be determined by a competitive immunoassay utilizing a B7-H3 protein standard labeled with a detectable substance and an unlabeled antibody that specifically binds to B7-H3. In this assay, the biological sample, the labeled B7-H3 protein standard, and the antibody that specifically binds to B7-H3 are combined, and the amount of labeled B7-H3 protein standard that binds to the unlabeled antibody is determined. The amount of B7-H3 in the biological sample is inversely proportional to the amount of labeled B7-H3 protein standard that binds to the antibody that specifically binds to B7-H3.

[0218] The immunoassays and methods disclosed herein can be used for a variety of purposes. In one embodiment, the detection methods disclosed herein can be used to detect production of B7-H3 in a cell culture. In another embodiment, the detection methods disclosed herein can be used to detect the amount of B7-H3 in a biological sample, such as a tissue sample or a blood sample or a serum sample. In some examples, the B7-H3 is cell surface B7-H3. In other examples, the B7-H3 protein is soluble (e.g., in a cell culture supernatant, or in a bodily fluid sample such as blood or serum). In some examples, the detection methods disclosed herein can detect or assess B7H3 antibody targeted therapy, such as by an immunohistochemical assay of a tumor for B7H3 expression. In some examples, the detection methods disclosed herein can detect a phenotypic change in an immune cell. In some examples, the detection methods disclosed herein can detect or assess a change in a B7H3 positive tumor before and after treatment.

[0219] In one embodiment, a kit for detecting B7-H3 in a biological sample is provided, comprising any of the monoclonal antibodies disclosed herein that bind to and / or are specific for B7-H3. For example, to confirm a diagnosis of cancer in a subject, a biopsy can be taken to obtain a tissue sample for histological examination, and a suitable bodily fluid sample, such as a blood sample, a serum sample, a synovial fluid sample, and the like, can also be obtained. In another embodiment, the antibodies comprised by the kit are labeled, such as with a fluorescent label, a radioactive label, or an enzymatic label.

[0220] In one embodiment, the kit includes instructional materials setting forth methods of use, particularly methods of use of the B7-H3 specific antibodies disclosed herein. The instructional materials can be written in electronic form (e.g., computer floppy disk or compact disk), or can be visual (e.g., video file). The kit can also include additional components to facilitate the particular application for which the kit is designed. Thus, the kit can additionally contain components to aid in detection of the label, such as enzyme substrates for enzymatic labels, filter sets for detection of fluorescent labels, appropriate secondary labels such as secondary antibodies, and the like. The kit can also include buffers and other reagents that are routinely used in the practice of the particular method. Such reagents and appropriate contents are well known to those skilled in the art.

[0221] In one embodiment, the kit includes an immunoassay for diagnosis. While the details of the immunoassay can vary depending on the particular format employed, generally it includes the step of contacting a biological sample to be tested with a B7-H3 specific antibody under immunoreactive conditions. The presence of B7-H3 protein in the sample to be tested is determined by specific binding of the antibody to form an immunocomplex under immunoreactive conditions, and direct or indirect detection of the presence of the immunocomplex.

[0222] The monoclonal antibodies disclosed herein that are directed against and / or specifically bind to B7-H3 can also be used in immunoassays, including but not limited to radioimmunoassays (RIA), ELISA, immunohistochemical assays, Western blots, or immunoprecipitations. The antibodies can also be used in fluorescence-activated cell sorting (FACS). FACS employs multiple color channels, low angle and obtuse light scatter detection channels, impedance channels, and other more sophisticated levels of detection to separate or sort cells.

[0223] The application is further described in the following examples, which do not limit the scope of the application. Various modifications and adjustments can be made by those skilled in the art based on the teachings of the present application without departing from the spirit and scope of the application.

[0224] Examples

[0225] The application is further described in the following examples, which do not limit the scope of the application. Various modifications and adjustments can be made by those skilled in the art based on the teachings of the present application without departing from the spirit and scope of the application.

[0226] The experimental methods in the following examples are routine methods, and are performed according to the techniques or conditions described in the literature in the art or according to the instructions of the products, unless otherwise specified. The materials, reagents, instruments, etc. used in the following examples can be obtained commercially, unless otherwise specified. The relevant nucleic acid strands, genes, enzymes can also be obtained based on the information of public databases through routine molecular biology experimental techniques.

[0227] Experimental materials, reagents, instruments and experimental methods

[0228] Table 1: Experimental materials and reagents

[0229] Table 2: Cell lines

[0230] Table 3: Equipment

[0231] Example 1:

[0232] This example describes the preparation of recombinant B7-H3 protein for immunizing animals.

[0233] Zhejiang Deenovo Biotech Co., Ltd. synthesized plasmids pCDNA3.4-B7H3-FC and pCDNA3.4-B7H3-HIS for expressing B7-H3 protein. Sequencing verification showed that the plasmids contained nucleotide sequences encoding B7H3-FC protein (SEQ ID NO: 92) and B7H3-His protein (SEQ ID NO: 94), respectively. The B7H3-FC protein was fused with the extracellular region of B7-H3 (amino acid residues 29 (Leu29) to 461 (Thr461) of Uniprot accession number Q5ZPR3) and the Fc of human IgG1, and had an amino acid sequence as shown in SEQ ID NO: 91; the B7H3-His protein was fused with the extracellular region of B7-H3 and a HIS tag, and had an amino acid sequence as shown in SEQ ID NO: 93.

[0234] The above plasmids were amplified in TOP10 bacteria and recovered by purification using a plasmid maxi extraction kit according to the manufacturer's instructions to obtain plasmids for transient cells. 100 μg of the purified sterile plasmid, 500 μl of TA-293 transfection reagent and 10 mL of KPM medium were gently mixed and allowed to stand at room temperature for 10 minutes to obtain a plasmid-carrier complex. 293F cells in the logarithmic growth phase were resuspended in KOP293-EX medium at a density of 4 x 10 6 The plasmid-carrier complex was added to the above 293F cells, which were cultured in a CO2 incubator at 37°C, 8% CO2, 125 rpm for 16-22 hours. Then, a cell protein expression enhancer (such as KE-293) was added to increase the expression of the product. 5% (v / v) 293-ProFeed feed was added. The cells and supernatant were collected on the 6th day after transfection for subsequent purification.

[0235] According to the manufacturer's instructions, the B7H3-FC fusion protein was purified using a Protein A column, and the B7H3-HIS fusion protein was purified using a nickel column. The purity of the fusion protein was detected by SDS-PAGE, and the results are shown in Figure 1.

[0236] Example 2:

[0237] This example describes the preparation of a phage display library for screening B7-H3 specific llama nanobodies.

[0238] Using the B7H3-FC protein prepared in Example 1, a llama was immunized according to a standard immunization procedure. Specifically, 0.5 mg of the purified protein was mixed with an adjuvant and injected into the back of a healthy adult llama in multiple points intradermally and subcutaneously. The immunization was performed 5 times, with an interval of 2 weeks between each time. The adjuvant used was complete Freund's adjuvant for the first time and incomplete Freund's adjuvant for the next four times.

[0239] To test the immunization effect, the serum of the vaccinated llama after the fifth immunization was collected, and the antibody titer against human B7-H3 was determined by ELISA. Briefly, a 96-well enzyme-labeled plate was coated with a solution of 5 μg / ml B7-H3 protein, and after washing and blocking, 100 μL / well of gradient-diluted llama serum (starting from 1:4000) was added, and after incubation at 37°C for a sufficient time, a horseradish peroxidase (HRP)-labeled rabbit anti-llama (Lama) antibody was used as a secondary antibody, a color development reaction was performed in the presence of substrate 3,3',5,5'-tetramethylbenzidine (TMB) (Biopanda, item TMB-S-003), and the absorbance value was measured at 450 nm. The OD 450nm The OD values of the serum of the three llamas at a dilution of 1:16000 were 0.889, 0.251, and 0.641, confirming the presence of anti-B7-H3 antibodies in the serum of the immunized llama.

[0240] For the immunized llama that was successful in immunization, 50 ml of fresh peripheral blood was collected at the 35th week (third immunization), the 49th week (fourth immunization), and the 63rd week (fifth immunization), respectively, to prepare peripheral blood mononuclear cells (PBMCs). Specifically, the fresh peripheral blood was diluted with an equal volume of PBS, then slowly added to an equal volume of lymphocyte separation medium, and centrifuged at 800g to separate the cells. The middle layer (corresponding to the suspended white blood cells) was transferred to a new centrifuge tube, washed with PBS and centrifuged, and the precipitated white blood cells were resuspended in 0.3 mL of PBS, 5 μL of which was diluted and counted using a hemocytometer, and the rest was added to 0.6 mL of Trizol solution, mixed, and stored at -80°C. Then, total RNA samples were obtained from the cells stored with Trizol solution using the phenol / chloroform method, and dissolved in 50 μl of H2O as a template for RT-PCR to obtain cDNA.

[0241] In the RT-PCR, the primer pairs AlpVh-LD and CH2-R, and the primer combinations Alp-F1 and AlpVHH-R1 / R2 were used in sequence to amplify the V HH gene fragment. The reaction system used in the nested PCR was as follows: 25 μl of 2x Phanta MIX buffer, 4 μl of dNTP mixed solution (2.5 mM of each dNTP), 1 μl of cDNA, 2 μl of each primer constituting a primer pair, 1 μl of high-fidelity polymerase, and water added to 50 μl; the first step of the nested reaction conditions were as follows: 50 °C annealing for 15 s, 72 °C extension for 1 min for one cycle, and 25 cycles; the second step of the nested reaction conditions were as follows: 55 °C annealing for 15 s, 72 °C extension for 30 s for one cycle, and 30 cycles. Then, the RT-PCR product was purified using a DNA product purification kit, and the product was quantified using an ultraviolet spectrophotometer (Thermo Fisher) that can detect protein concentration.

[0242] Table 4:

[0243] According to the manufacturer's instructions, the purified nested reaction product fragment was digested with Sfil enzyme at 50 °C overnight, and the digested product of the fragment was ligated to the vector pSGPHA01 that had been pretreated with the same enzyme, to obtain a vector pSGPHA01-V H Recombinant vector pSGPHA01-V of H single-domain antibody coding sequence H H.

[0244] 1 μg of the above ligation product was electroporated (0.2 cm shock cup, 2.5 kV voltage) to transform 50 μL of E. coli TG1 competent cells, and a phage library with a size of 9.61 x 1011cfu library capacity was prepared under the rescue of helper phage M13K07. 8 V H H sequence phage library.

[0245] Example 3:

[0246] This example describes B7-H3 specific llama nanobodies obtained by panning from a phage library.

[0247] The purified B7H3-HIS protein prepared according to Example 1 was diluted with PBS to a final concentration of 100 μg / mL and added to the wells of an ELISA plate at 100 μL / well, and incubated at 4°C for 12 h. After discarding the coating solution, the wells were washed 3 times with PBS, and 300 μL / well of a blocking solution containing 3% BSA in PBS was added and incubated at 37°C for 2 h. After discarding the blocking solution, the wells were washed 6 times with PBS, and the library panning was initiated. In the first round of panning, 10 μL of the phage library prepared according to Example 2 was mixed with 90 μL of PBS and added to each well, and incubated at 37°C for 2 h. The mixture was then discarded, and the wells were washed 5 times with PBST and 10 times with PBS to remove unbound phage. Then, 100 μL / well of a BSA-containing Gly-HCl elution solution was added, and incubated at 37°C for 8 min to elute the phage specifically bound to B7-H3. The eluate was transferred to a sterile centrifuge tube and neutralized immediately with 50 μL of Tris-HCl buffer (pH 9.5). The eluted output phage was used to re-infect fresh TG1 competent cells and re-amplified, and the re-amplified phage was used as the input for the next round of panning. Three rounds of panning were performed, and the second and third rounds of panning were essentially the same, except that the phage / PBS mixture added to the blocked wells also contained 1% BSA or OVA.

[0248] Single colonies of TG1 cells infected with the output phage from the third round of panning were randomly selected and subjected to monoclonal phage ELISA to identify B7-H3-specific binders. Specifically, 50 μL / well of the cultured monoclonal phage solution was added to the ELISA plate coated with 100 μL of the target molecule (1 μg / mL B7H3-HIS) and blocked with 3% skim milk, and incubated at 37°C for 1 h. After washing the ELISA plate 5 times with PBST (PBS + 0.1% Tween 20), the binding of the phage was detected by HRP-labeled anti-M13 antibody. The OD of each well at 450 nm was measured using antigen-coated wells with added M13K07 as a negative control and antigen-coated wells with added PBS as a blank control. Monoclonal phage with an OD value (S) ≥ 0.5 for the test sample were identified as positive clones, and the results are shown in Table 5 below. TM 20) washes, the binding of the phage was detected by HRP-labeled anti-M13 antibody. The OD of each well at 450 nm was measured using antigen-coated wells with added M13K07 as a negative control and antigen-coated wells with added PBS as a blank control. Monoclonal phage with an OD value (S) ≥ 0.5 for the test sample were identified as positive clones, and the results are shown in Table 5 below.

[0249] Table 5: Results of phage ELISA screening against B7-H3 target

[0250] Among the 288 randomly selected monoclonal antibodies, 108 specifically bind to B7-H3. Sequencing of these positive clones, after removing repeated sequences from the sequencing results, obtained 18 B7-H3 specific VHH single domain antibodies, and their antibody sequences are shown in SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82, 87, respectively, and the CDR sequences are shown in Table 6.

[0251] Table 6: Anti-B7-H3 single domain antibodies

[0252] Example 4:

[0253] This example describes the preparation and binding activity characterization of B7-H3 specific nanobodies.

[0254] The nucleotide sequences (SEQ ID NO: 31, 1, 6, 11, 41, 61, 66, 16, 86, 71, 76, 36) encoding 12 nanobodies (H-A3, BP2-F12, BP1-C11, BP1-F9, H-H6, BP1-F1, BP2-A8, BP1-C10, BP1-F12, BP1-G2, BP1-C6, BP1-H8) were cloned into the restriction enzyme cleavage sites AgeI and AscI between the pCDNA3.4-FC vector, respectively, to obtain V H The expression plasmids of H-FC nanobodies were prepared, and the corresponding purified nanobodies were prepared. Figure 2 shows the SDS-PAGE detection results of each antibody.

[0255] Further, the binding activity of the above prepared antibodies to B7-H3 protein was determined by ELISA experiment. Specifically, 100 μl of B7H3-HIS protein antigen containing 1 μg / ml B7H3-HIS protein antigen in carbonate / bicarbonate coating buffer (pH 9.8) was used to coat the enzyme-labeled plate, and incubated at 4°C overnight. Discard the coating solution, and wash the enzyme-labeled plate once. Add 250 μL of blocking buffer (add 3 g of BSA to 100 ml of PBS) to each well, and incubate at 37°C for at least 1 hour. Wash the enzyme-labeled plate twice with 250 μL of washing buffer (add 0.05 ml of Tween 20 to 100 ml of PBS). Add 100 μL of diluted B7H3-V HH-FC antibody samples (highest antibody concentration 10 pg / mL, 2-fold gradient dilution), incubated at 37°C for 1 hour. Discard the sample, wash the enzyme-labeled plate twice with 250 pL of washing buffer. Add 100 pL of secondary antibody (goat anti-human IgG-HRP) diluted 1:10000 with blocking buffer to each well, mix gently, and incubate at 37°C for 20 minutes. Wash the enzyme-labeled plate three times with washing buffer. Add TMB developing solution, incubate at 37°C for 10 minutes in the dark. Add 50 pL of stop solution, and then immediately measure the OD value of each well at a wavelength of 450 nm. The results are shown in Figure 3 and Table 7. The results show that 12 antibodies can effectively bind to B7-H3 protein.

[0256] Table 7: EC50 values of B7-H3 nanobodies

[0257] The binding specificity of the above antibodies to a positive cell line (A375 melanoma cell line) expressing B7-H3 and a negative cell line (acute T cell leukemia Jurkat cell line) not expressing B7-H3 was determined by flow cytometry. Specifically, the two cell lines in culture were subjected to enzymatic digestion, and the cells after digestion were collected by centrifugation. After counting, the cells were resuspended in PBS containing 2% BSA to a cell concentration of 1x10 7 / mL. Different B7H3-V H H-FC antibodies were used as primary antibodies for staining. For A375 cells, 12 concentrations were set for each antibody (highest concentration 10 pg / ml, 2-fold gradient dilution), and for Jurkat cells, 1 concentration was set for each antibody (10 pg / mL); an isotype antibody was used as a control. The primary antibody was mixed well with the cell sample, and the cells and antibodies were incubated at 4°C for 30 min to allow sufficient binding. After washing the cells with 1 ml of PBS containing 2% BSA, centrifugation was performed at 1000 rpm for 5 min, and the supernatant was discarded. The washing was repeated twice, and then the cells were resuspended in PBS containing 2% BSA. 2 pL of goat anti-human IgG-APC was added as a fluorescently labeled secondary antibody, and after mixing well, the cells were incubated at 4°C in the dark for 30 min. After washing the cells with 1 ml of PBS containing 2% BSA, centrifugation was performed at 1000 rpm for 5 min, and the supernatant was discarded. The washing was repeated twice, and then the cells were resuspended in PBS containing 2% BSA. The fluorescence signal of each sample was detected by flow cytometry. Among them, for all the test antibodies, almost no fluorescence signal produced by the bound antibodies was observed in Jurkat cells, and the results of the determination in A375 cells are shown in Table 8.

[0258] The results show that, compared with the isotype negative control, the other 11 antibodies, except for BP1-C11 antibody, all achieved binding with B7-H3 positive A375 cells, but not with B7-H3 negative Jurkat cells, indicating that these antibodies can specifically target B7-H3 positive cells.

[0259] Example 5:

[0260] This example describes the preparation of a bivalent antibody against B7-H3.

[0261] According to the sequences of the B7-H3 specific nanobodies prepared according to Example 3, a bivalent nanobody can be further prepared. In this example, according to the sequences of BP2-F12 and H-H6, a bivalent antibody BP2F12-HH6 was prepared, which comprises two structural units corresponding to B7-H3 nanobodies BP2-F12 and H-H6, the two structural units are fused by a (G4S)3 linker, as shown in Table 9 below.

[0262] Table 9: Anti-B7-H3 bivalent nanobodies

[0263] The bivalent antibody can also be constructed by the same method as described in Example 4, and an expression plasmid capable of expressing the bivalent antibody Fc fusion protein BP2F12-HH6-FC (SEQ ID NO: 100) was constructed, and the corresponding purified antibody was prepared. Figure 4 shows the SDS-PAGE detection results of the bivalent antibody FC fusion protein.

[0264] Example 6:

[0265] This example determines the affinity test experiment of the exemplary antibody of the application against B7-H3 protein.

[0266] The B7H3-HIS antigen protein was gradient diluted with a buffer at pH 7.4 to 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.13nM, respectively. The diluted B7H3-V H The H-FC antibody was loaded onto the Protein A sensor, with a loading time of 180s, a baseline time of 180s, an antigen binding time of 180s, and a dissociation time of 600s. 50mM pH1.7 Gly-HCl was used as the regeneration buffer. The binding constant Ka, dissociation constant Kd and affinity constant KD (KD = Kd / Ka) of the antibody were obtained based on the analysis software (HT12) of the Octet RED384 protein interaction instrument, which respectively reflect the binding speed, dissociation speed and firmness of the antibody and the antigen. The results are shown in Table 10.

[0267] Table 10: Binding activity parameters of B7-H3 bivalent nanobody

[0268] The results show that the bivalent antibody BP2F12-HH6-FC and other exemplary monovalent antibodies of the application all exhibit excellent pharmacokinetics and can effectively bind to B7H3 protein. Moreover, compared with the parent antibodies BP2-F12-FC and H-H6-FC, the bivalent antibody has further improved affinity to the target protein.

[0269] The antigen protein B7H3-HIS molecules were coated on an enzyme-labeled plate, and the binding of the three antibodies BP2F12-HH6-FC, H-H6-FC and BP2-F12-FC to B7H3 protein was determined by ELISA. The results show that the three antibodies can effectively bind to B7H3-HIS antigen, and the binding activity of the bivalent antibody BP2F12-HH6-FC is the best (Figure 5). The detection results of Fortebio molecular interaction instrument also show that the affinities of the three antibodies BP2F12-HH6-FC, H-H6-FC and BP2-F12-FC to B7H3 protein are 2.84 nM, 9.8 nM and 7.55 nM, respectively.

[0270] Example 7:

[0271] In this example, the flow cytometry technique was used to determine that the exemplary antibodies of the application have binding specificity to B7-H3 protein and can specifically target cells expressing B7-H3 protein on the cell surface.

[0272] Using the same method as in Example 4, three kinds of nanobodies (BP2-F12-Fc, H-H6-Fc, BP2F12-HH6-Fc) were tested for specific binding with positive cell lines expressing B7-H3 and negative cell lines not expressing B7-H3, wherein the B7-H3 positive cell lines were human neuroblastoma cells (SH-SY5Y and SK-N-AS), human malignant melanoma cells (A375) and human non-small cell lung cancer cells (A549), respectively; the negative cell line was human T lymphocyte leukemia cells (Jurkat). The above positive cells (SH-SY5Y, SK-N-AS, A375, A549) were each divided into 11 portions, each containing 2E5 cells in 100 μL, of which 9 portions were added with 0.0625 μg, 0.25 μg and 1 μg of the corresponding nanobody protein (BP2-F12-Fc, H-H6-Fc, BP2F12-HH6-Fc), respectively; the negative cells (Jurkat) were divided into 5 portions, of which 3 portions were added with 0.0625 μg, 0.25 μg and 1 μg of the nanobody protein BP2F12-HH6-Fc, respectively. To the wells without inoculated cells but incubated with the antibody protein, 100 μL of IgG antibody working solution (containing 1 μL of anti-human IgG antibody per 100 μL) was added as a blank negative control. To the wells inoculated with various target tumor cells and incubated with the above nanobodies, one was also added with 100 μL of the above IgG antibody working solution as a negative antibody control, and the other was added with 100 μL of B7-H3 antibody working solution (containing 0.5 μL of B7H3-PE / Cy7 antibody per 100 μL) as a positive antibody control, and gently mixed and incubated at room temperature for 15 min in the dark. The antibodies were dissolved in a PBS solution containing 2% FBS. After incubation, 100 μL of FBS buffer was added to each well, resuspended and mixed, centrifuged at 300 rcf for 3 min, the supernatant was aspirated and discarded, and the washing was repeated once before being detected by a BD FACSC canto II flow cytometer. According to the instructions of the equipment supplier, the flow rate was set and 50000 evts cells were collected. The results are shown in Figures 6-10.

[0273] The experimental results show that the monovalent and bivalent B7H3 nanobodies can specifically bind to the cell lines expressing B7H3, such as human neuroblastoma cells (SH-SY5Y and SK-N-AS), human malignant melanoma cells (A375) and human non-small cell lung cancer cells (A549), and the fluorescence binding intensity measured also increases with the increasing concentration of the nanobodies. Among them, the binding force of the bivalent antibody BP2F12-HH6-FC is significantly higher than that of either of the two single antibodies, and the average fluorescence intensity MFI (at an addition amount of 1 μg, corresponding to an antibody concentration of 10 μg / mL) reaches 5901, 20590, 10673, 19401, respectively, and the positive sample B7H3-PE / Cy7 has a binding force stronger than the two monovalent antibodies but weaker than the recombinant bivalent antibody BP2F12-HH6-FC. On the other hand, in the negative cell line not expressing B7H3, such as human T lymphocyte leukemia cells (Jurkat), no specific binding of any monovalent or bivalent B7H3 nanobody is observed. This suggests that the nanobodies prepared in the present application can bind to the target B7H3 antigen with stronger specificity and higher affinity than the known B7H3 antibodies. Moreover, the nanobodies of the present application, as single heavy chain antibodies, have the advantages of shorter sequence and higher similarity to human antibodies, and exhibit weak immunogenicity when applied to the human body, and have a wide market in the fields of tumor research and immunology research.

[0274] Example 8

[0275] This example describes the preparation of T cell chimeric antigen receptor (CAR) targeting B7-H3 and the construction of Car-T cells using the same, and the verification of the activity of the CAR and Car-T.

[0276] Preparation of CAR lentiviral plasmid targeting B7-H3

[0277] According to the coding sequences of five B7-H3 nanobodies (H-H6, BP2-F12, BP1-H8, BP1-F9, BP1-C10) described in Example 3, the corresponding chimeric antigen receptors (CARs) were full-gene synthesized. The nucleotide sequences of the five different CARs obtained are shown in SEQ ID NO. 127, 129, 131, 133, 135, respectively, and the encoded CAR proteins have the amino acid sequences shown in SEQ ID NO. 126, 128, 130, 132, 134, respectively, which are referred to as CAR 001 to CAR 005, respectively. The purified nucleotide sequences were ligated into the multiple cloning sites of pLVX-EF1a-ZY11 (OBI O) respectively, to construct lentiviral target vectors capable of expressing the corresponding B7-H3-targeting CARs. The plasmids that passed sequencing were purified, transformed into DH5a competent cells (Thermo Fisher), and single colonies were picked. After large-scale culture, the plasmid extraction kit PureLink TM HiPure Plasmid Maxiprep Kit (Thermo Fisher) was used for purification, and the corresponding B7-H3-targeting CAR lentiviral plasmids were obtained.

[0278] Preparation of B7-H3-targeting Car-T cells

[0279] The above-prepared B7-H3-targeting CAR lentiviral plasmids were co-transfected with packaging plasmids psPAX2 and pMD2.G into 293T cells at a ratio of 1.64 pmol: 1.3 pmol: 0.72 pmol. The preparation method of the packaging plasmids was performed according to the instructions of the PureLink TM HiPure Plasmid Maxiprep Kit. The transfection was performed according to the instructions, using polyethyleneimine transfection reagent at a ratio of DNA: PEI = 1:3. After 16 hours of transfection, the complete culture medium was replaced. After 24 hours, 48 hours and 72 hours of culture, the supernatant containing lentivirus was collected. The supernatants were combined and centrifuged at 3000 rpm for 10-15 minutes at -80°C. After filtration through a 0.45 μm filter membrane, the lentivirus concentrate was collected by ultracentrifugation at 25000 rpm for 2-3 hours at 4°C, and stored at -80°C before use.

[0280] Fresh peripheral blood mononuclear cells were prepared by isolating fresh peripheral blood from healthy donors, and CD3 TM T cells were sorted using CD3 + T cell sorting magnetic beads, MS separation column and MiniMACS + T cells were sorted using CD3 6at a concentration of 1 cell / mL were added to 24-well plates, and magnetic beads coupled with anti-CD3 antibody and anti-CD28 antibody were added at a ratio of 1:1 and mixed well. The magnetic beads were separated and resuspended in OpTmizer TM T-Cell Expansion SFM medium, and cultured in a 37°C, 5% CO2 incubator for 1 day. Then, the above-described lentiviral concentrate loaded with CAR elements targeting B7-H3 (MOI = 2) was added to the cultured cells, and Polybrene was added to a concentration of 10 pg / mL. The CAR-T cells targeting B7-H3 001 to 005 (hereinafter also collectively referred to as CAR-T cells) were obtained by low-speed (500-1000 g / min) centrifugation in a flat-angle centrifuge for 30-60 minutes, and then cultured in a 37°C incubator for 48 hours, and respectively expressed CAR elements 001 to 005.

[0281] Phenotype detection of CAR-T cells

[0282] Flow cytometry (FACS) was used to determine the expression of CD8, B7H3-Fc, and EGFR in untransfected blank control T cells (CON-T) and the above-described five CAR-T cells (CAR-T 001 to CAR-T 005). The above-described cells were stained with antibodies CD8-Pacific Blue, B7H3-FC+APC-anti-human IgG-FC, EGFR-PE, 7-AAD-Percp5.5 (all antibodies were purchased from Biolegend) in OpTmizer TM After 12 days of culture in T-Cell Expansion SFM medium, 2 x 10 5 T cells were taken for antibody CD8-Pacific Blue, B7H3-FC+APC-anti-human IgG-FC, EGFR-PE, 7-AAD-Percp5.5 (all antibodies were purchased from Biolegend) staining. Then, cell phenotyping was performed using a flow cytometer (BD FACSCanto II), and the results are shown in FIG. 11. It can be seen that CD8 and CD4 T cells in the control T cells that were not transfected did not express B7-H3 and EGFR, while CAR-T 001 to 005, CD8 and CD4 T cells that were transfected expressed B7-H3 and EGFR.

[0283] Functional experiment of CAR-T cells

[0284] This example further tests the cell-killing toxicity of the above five CAR-T cells (CAR-T 001 to CAR T-005) in different B7-H3 positive and B7-H3 negative tumor cell lines. The B7-H3 positive tumor cell lines used include human neuroblastoma cell lines SH-SY5Y and SK-N-AS, hepatocellular carcinoma (HCC) Huh7, melanoma cell A375 and human myeloblast leukemia cell line HL60-MDSC; the B7-H3 negative tumor cell line (Jurkat) used is human T lymphocyte leukemia cell line Jurkat.

[0285] Each tumor cell was plated in a 96-well plate at a density of 5 x 10 4 After the tumor cells were fully adherent or the growth state was restored, the untransfected blank control T cells and the above CAR-T cells (CAR-T 001 to CAR T-005) were added for co-culture at an effector-to-target ratio (E:T) of 1:1 or 1:2, respectively. After 48 hours, the tumor-killing activity of the above five CAR-T cells was quantitatively evaluated by flow cytometry (FACS). The tumor cell lines co-cultured with the CAR-T cells for 48 hours were enzymatically digested. For cell lines Huh7, A375, SK-N-AS and SH-SY5Y, CD8-Pacific Blue, 7-AAD-Percp5.5, CD45-APC-CY7 staining was used; for cell line HL60-MDSC, CD33-APC-CY7 staining was used, and then flow cytometry was used for detection. Among them, the purpose of CD45 staining is to distinguish adherent tumor cells (CD45 negative) and T cells (CD45 positive), and the purpose of CD33 staining is to distinguish suspended tumor cells (CD33 negative) and T cells (CD33 positive). The results are shown in FIG. 12.

[0286] FIG. 12 shows that the unmodified control T cells (CON-T) have little effect on the growth and proliferation of tumor cells. In contrast, the five CAR-T cells disclosed herein all exhibit good cell-killing toxicity under low effector-to-target ratio (E:T = 1:1) and high effector-to-target ratio (E:T = 1:2) conditions. Among them, CAR-T 001, 003 and 004 show excellent killing effect on the four B3-H7 positive tumors tested (human neuroblastoma, melanoma, hepatocellular carcinoma, chronic myeloid leukemia), and can eliminate most of the tumor cells within 48 hours.

[0287] Example 9:

[0288] B7-H3 is a tumor associated antigen (TAA) known to have limited expression in normal tissues and specific high expression in some malignancies. Immunohistochemistry (IHC) analysis results of normal tissues also showed that B7-H3 was only lowly expressed in a few tissues, or even not detected. This example verified the binding activity of the Car-T cells disclosed herein against various tumor cells. Human neuroblastoma cells (SH-SY5Y and SK-N-AS) expressing B3-H7 protein identified correctly by STR, human renal cell adenocarcinoma cells (ACHN and 786-O), human non-small cell lung cancer cells (A549), human breast cancer cells (MDA-MB-231 and MCF-7), human hepatocarcinoma cells (Hep G2 and PLC), human monocytic leukemia cells (MV-4-11 and THP-1), human histiocytic lymphoma cells (U-937), human pancreatic cancer cells (PANC-1), human gastric cancer cells (SNU-1 and NCI-N87), human ovarian cancer cells (NIH: OVCAR-3), human colon cancer cells (HT-29 and HCT116), human Burkitt's lymphoma cells (Raji), human myeloblast leukemia cells (HL-60 and K562), human osteosarcoma cells (MG-63), and human multiple myeloma cells (MM.1S) were included.

[0289] Specifically, 1 μg of B7-H3 Nanobody protein BP2F12-HH6-Fc was added to 100 μL of a reaction system containing 2E5 tumor cells (PBS containing 2% FBS), and incubated at room temperature in the dark for 20 min. FBS buffer was added to a total volume of 200 μL, centrifuged at 300 rcf for 3 min, the supernatant was aspirated and discarded, and the washing was repeated once. Then, 100 μL of IgG antibody working solution (containing 1 μL of anti-human IgG antibody purchased from Biolend per 100 μL) was added to each of the tumor cell samples incubated with the Nanobody, mixed well, and incubated at room temperature in the dark for 15 min. Then, 100 μL of FBS buffer was added to each sample, mixed well, centrifuged at 300 rcf for 3 min, the supernatant was aspirated and discarded, and the washing was repeated once, as the test sample. The tumor cells without incubation with the Nanobody were treated in exactly the same way as described above, as the control sample. All samples were detected by a BD FACSC canto II flow cytometer according to the instrument instructions. The total number of cells collected was set to 50000 ects, and the results are shown in FIG. 13.

[0290] As shown in FIG. 13, the exemplary B7-H3 antibodies herein are capable of specifically recognizing and binding to cells derived from various tumors expressing B7-H3 protein on the cell surface, including but not limited to human neuroblastoma, human renal cell adenocarcinoma, human non-small cell lung cancer, human breast cancer, human liver cancer, human monocyte leukemia, human histiocytic lymphoma, human pancreatic cancer, human gastric cancer, human ovarian cancer, human colon cancer, human Burkitt's lymphoma, human myeloblast leukemia, human osteosarcoma, and human multiple myeloma. These tumor cells all show high binding force with the B7-H3 nanobody protein (BP2F12-HH6-Fc) disclosed herein. Therefore, it is indicated that B7-H3 is a broad-spectrum target for primary malignancies, and the binding of the B7-H3 antibodies of the present application to tumors can not be affected by the specific tumor type and source, and can successfully target all B7-H3 specifically expressing tumors (B7-H3 positive tumors). The bivalent B7-H3 antibody as an example of the B7-H3 nanobody of the present application, those skilled in the art can reasonably expect that other B7-H3 nanobodies of the present application also have the same targeting activity. On this basis, each B7-H3 antibody of the present application has a good application prospect as a tumor therapeutic agent, diagnostic agent, detection agent, etc. In addition, the B7-H3 antibodies of the present application can also be used to design various molecular entities that utilize their binding activity to tumors, such as multi-specific binding molecules, chimeric antigen molecules, CAR immune cells (CAR-T cells or CAR-NK cells, etc.), antibody drug conjugates (ADC), etc., or other technical means, such as targeted protein degradation technology (PROTAC, AbTAC, PROTAB, KineTAC), etc.

Claims

1. A single-domain monoclonal antibody targeting and / or specifically binding to B7-H3, wherein the antibody comprises the complementarity-determining region 1 (CDR1), CDR2, and CDR3 sequences contained in any one of SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82, or 87; Optionally, the CDR sequence is determined using the IMGT scheme.

2. The antibody according to claim 1, wherein, (1) The CDR1 contains the amino acid sequence shown in SEQ ID NO:3, the CDR2 contains the amino acid sequence shown in SEQ ID NO:4, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:5; (2) The CDR1 contains the amino acid sequence shown in SEQ ID NO:8, the CDR2 contains the amino acid sequence shown in SEQ ID NO:9, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:

10. (3) The CDR1 contains the amino acid sequence shown in SEQ ID NO:13, the CDR2 contains the amino acid sequence shown in SEQ ID NO:14, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:15; (4) The CDR1 contains the amino acid sequence shown in SEQ ID NO:18, the CDR2 contains the amino acid sequence shown in SEQ ID NO:19, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:

20. (5) The CDR1 contains the amino acid sequence shown in SEQ ID NO:23, the CDR2 contains the amino acid sequence shown in SEQ ID NO:24, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:25; (6) The CDR1 contains the amino acid sequence shown in SEQ ID NO:28, the CDR2 contains the amino acid sequence shown in SEQ ID NO:29, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:

30. (7) The CDR1 contains the amino acid sequence shown in SEQ ID NO:33, the CDR2 contains the amino acid sequence shown in SEQ ID NO:34, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:35; (8) The CDR1 contains the amino acid sequence shown in SEQ ID NO:38, the CDR2 contains the amino acid sequence shown in SEQ ID NO:39, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:

40. (9) The CDR1 contains an amino acid sequence as shown in SEQ ID NO:43, the CDR2 contains an amino acid sequence as shown in SEQ ID NO:44, and the CDR3 contains an amino acid sequence as shown in SEQ ID NO:45; (10) The CDR1 contains an amino acid sequence as shown in SEQ ID NO:48, the CDR2 contains an amino acid sequence as shown in SEQ ID NO:49, and the CDR3 contains an amino acid sequence as shown in SEQ ID NO:50; (11) The CDR1 contains the amino acid sequence shown in SEQ ID NO:53, the CDR2 contains the amino acid sequence shown in SEQ ID NO:54, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:55; (12) The CDR1 contains the amino acid sequence shown in SEQ ID NO:58, the CDR2 contains the amino acid sequence shown in SEQ ID NO:59, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:

60. (13) The CDR1 contains the amino acid sequence shown in SEQ ID NO:63, the CDR2 contains the amino acid sequence shown in SEQ ID NO:64, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:

65. (14) The CDR1 contains the amino acid sequence shown in SEQ ID NO:68, the CDR2 contains the amino acid sequence shown in SEQ ID NO:69, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:

70. (15) The CDR1 contains the amino acid sequence shown in SEQ ID NO:73, the CDR2 contains the amino acid sequence shown in SEQ ID NO:74, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:75; (16) The CDR1 contains the amino acid sequence shown in SEQ ID NO:78, the CDR2 contains the amino acid sequence shown in SEQ ID NO:79, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:

80. (17) The CDR1 contains the amino acid sequence shown in SEQ ID NO:83, the CDR2 contains the amino acid sequence shown in SEQ ID NO:84, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:85; or (18) The CDR1 contains the amino acid sequence shown in SEQ ID NO:88, the CDR2 contains the amino acid sequence shown in SEQ ID NO:89, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:

90.

3. The antibody according to claim 1 or 2, wherein it is selected from V H H antibody, VH antibody, V NAR Antibody or V L Antibody; Optionally, the antibody is a humanized antibody or a human antibody; Optionally, the antibody is a partially or fully humanized V antibody. H H antibody, human VH antibody or camelified human VH antibody, partially or fully humanized VH antibody NAR Antibody, or human VL antibody; Optionally, the antibody has undergone affinity maturation.

4. The antibody according to any one of claims 1 to 3, wherein, The amino acid sequence of the antibody is selected from any of the following: (i) It has at least about 90% amino acid sequence identity with the amino acid sequence shown in any one of SEQ ID NO:2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82 or 87; (ii) The amino acid sequence having one or more deletions, substitutions, or insertions of amino acids compared to the amino acid sequence represented by any one of SEQ ID NO:2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82, or 87, wherein the substitutions are preferably conservative substitutions; or (iii) Contains an amino acid sequence represented by any one of SEQ ID NO:2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82 or 87, or is composed of an amino acid sequence represented by any one of SEQ ID NO:2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, 72, 77, 82 or 87.

5. A multivalent antibody comprising at least two single-domain antibodies targeting and / or specifically binding to B7-H3, or at least one single-domain antibody targeting and / or specifically binding to B7-H3 and at least one additional monoclonal antibody or antigen-binding fragment thereof, wherein the single-domain antibody targeting and / or specifically binding to B7-H3 is selected from the antibodies of any one of claims 1 to 4. Optionally, the single-domain antibodies targeting and / or specifically binding to B7-H3 may be the same or different. Optionally, the multivalent antibody is a bivalent, trivalent, or quadrivalent antibody.

6. The multivalent antibody according to claim 5, wherein, The single-domain antibodies are linked to each other, or to the other monoclonal antibodies or their antigen-binding fragments, via adapters or spacer sequences. Optionally, the joint or spacer sequence is rigid or flexible, preferably flexible. Optionally, the linker or spacer sequence is an amino acid sequence having 1 to about 50, preferably 1 to about 30, more preferably 1 to about 15 amino acid residues; Optionally, the connector or spacer sequence is a GS type connector, preferably a (Gly4Ser)3 connector or a (Gly3Ser2)3 connector; Optionally, the linker or spacer sequence comprises an amino acid sequence selected from any of SEQ ID NO:101 to 125.

7. The multivalent antibody according to claim 5 or 6, wherein the antibody (i) It has at least about 90% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:95; (ii) Compared to the amino acid sequence shown in SEQ ID NO:95, it has one or more amino acid deletions, substitutions, or insertions, wherein the substitutions are preferably conservative substitutions; or (iii) Contains or consists of the amino acid sequence shown in SEQ ID NO:

95.

8. A multispecific antibody comprising at least two single-domain antibodies targeting and / or specifically binding to B7-H3, or one single-domain antibody targeting and / or specifically binding to B7-H3 and at least one additional monoclonal antibody or antigen-binding fragment thereof, wherein the single-domain antibody targeting and / or specifically binding to B7-H3 is selected from the antibodies of any one of claims 1 to 4. Optionally, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

9. The multispecific antibody according to claim 8, wherein, The at least one additional monoclonal antibody or its antigen-binding fragment specifically binds to the components of the T-cell receptor or NK-cell activation receptor.

10. A chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof as an extracellular antigen-binding domain, as described in any of the preceding claims; Optionally, it further includes a hinge region, a transmembrane domain, a co-stimulatory signal transduction portion, an intracellular signal transduction domain, or any combination thereof. Optionally, the hinge region includes a CD8α hinge region, preferably including an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:136; Optionally, the transmembrane domain comprises a transmembrane domain selected from any one of CD3ζ, CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1, preferably comprising a transmembrane domain of CD8α or CD28, and more preferably comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 138 or 140; Optionally, the co-stimulatory signal transduction portion comprises a co-stimulatory signal transduction domain selected from at least one of the ligands CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, DAP10, CD3ζ, or FcεRIγ, and any combination thereof, preferably comprising a cytoplasmic domain of CD28 and / or a cytoplasmic domain of CD137, more preferably comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 142 or 144; and / or Optionally, the intracellular signal transduction domain includes a CD3ζ signal transduction domain, preferably including an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:146; Optionally, the CAR(i) has at least about 90% amino acid sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 126, 128, 130, 132 or 134; (ii) The amino acid sequence having one or more amino acid deletions, substitutions, or insertions compared to the amino acid sequence shown in any of SEQ ID NO: 126, 128, 130, 132, or 134, wherein the substitutions are preferably conservative substitutions; or (iii) Contains or consists of the amino acid sequence shown in any of SEQ ID NO: 126, 128, 130, 132 or 134.

11. An immunoconjugate comprising the antibody of any one of claims 1 to 9, and an effector molecule covalently linked thereto; Optionally, the effector molecule is selected from any one of toxins, photon absorbers, detectable markers, or small molecule drugs; Optionally, the detectable marker is selected from fluorophores, enzymes, or radioisotopes; Optionally, the small molecule drug is selected from antimicrotubule agents, antimitotic agents, and / or cytotoxic agents; Optionally, the antibody and the effector molecule are covalently linked via a cleavable or incleavable linker.

12. An antibody-nanoparticle conjugate comprising the antibody according to any one of claims 1 to 9, and nanoparticles covalently linked thereto; Optionally, the nanoparticles are selected from polymer nanoparticles, nanospheres, nanocapsules, liposomes, lipid nanoparticles, dendritic polymers, polymer micelles, or vesicles. Optionally, the nanoparticles contain a cytotoxic agent.

13. A fusion protein comprising the antibody of any one of claims 1 to 9, and a heterologous protein or peptide covalently linked thereto; Optionally, the heterologous protein is selected from polyhistidine tags, Fc proteins, or leucine zippers.

14. A nucleic acid molecule encoding an antibody according to any one of claims 1 to 9, a CAR according to claim 10, an immunoconjugate according to claim 11, an antibody-nanoparticle according to claim 12, or a fusion protein according to claim 13.

15. A vector comprising the nucleic acid molecule of claim 14; Optionally, the nucleic acid molecule is operatively connected to a regulatory element.

16. Engineered cells expressing any one of claims 1 to 9, the CAR of claim 10, the immunoconjugate of claim 11, the antibody-nanoparticle of claim 12, or the fusion protein of claim 13, or comprising the nucleic acid molecule of claim 14 or the vector of claim 15, wherein the cells are preferably cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells.

17. A composition comprising the antibody of any one of claims 1 to 9, the CAR of claim 10, the immunoconjugate of claim 11, the antibody-nanoparticle of claim 12, or the fusion protein of claim 13, the nucleic acid molecule of claim 14, the vector of claim 15, or the engineered cell of claim 16, and Pharmaceutically acceptable excipients.

18. A method for detecting B7-H3 expression in a sample, comprising the following steps: The sample is brought into contact with the antibody of any one of claims 1 to 9, the immunoconjugate of claim 11, the antibody-nanoparticle of claim 12, or the fusion protein of claim 13; The binding of the antibody, immunoconjugate, antibody-nanoparticle, or fusion protein to the sample was determined; and Based on the measured binding, determine whether B7-H3 is expressed in the sample.

19. A method for diagnosing a subject with B7-H3-positive cancer, comprising the following steps: Contact the biological sample obtained from the subject with the antibody of any one of claims 1 to 9, the immunoconjugate of claim 11, the antibody-nanoparticle of claim 12, or the fusion protein of claim 13; The binding of the antibody, immunoconjugate, antibody-nanoparticle, or fusion protein to the biological sample was determined; and Based on the measured binding, the subject is diagnosed as having B7-H3 positive cancer. Optionally, the biological sample is selected from the subject's body fluid sample, cell sample, tissue sample, tissue section, primary and / or passaged cell culture derived from the patient's tissue.

20. A method for treating B7-H3 positive cancer, the method comprising: Administering a therapeutically effective amount of any one of claims 1 to 9, the CAR of claim 10, the immunoconjugate of claim 11, the antibody-nanoparticle of claim 12, the fusion protein of claim 13, the nucleic acid molecule of claim 14, the carrier of claim 15, the engineered cell of claim 16, or the composition of claim 17 to a patient in need; Optionally, the B7-H3 positive cancer is selected from solid tumors or hematologic malignancies; Optionally, the solid tumor is selected from tumors of the nervous system, head and neck, chest, digestive system, genitourinary system, soft tissue and skin, or bone. Optionally, the nervous system tumor is selected from diffuse glioma, astrocytoma, glioblastoma, oligodendroglioma, pediatric diffuse glioma, ependymoma, neuronal and mixed neuronal-glial tumors, medulloblastoma, embryonal tumor, schwannoma, meningioma, solitary fibrous tumor or hemangiopericytoma. Optionally, the astrocytoma is selected from diffuse astrocytoma, anaplastic astrocytoma, or oligodendroastrocytoma; Optionally, the head and neck tumor is selected from malignant tumors of the nasal cavity and sinuses, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, salivary gland tumors, intracranial tumors, thyroid cancer, or tongue cancer; Optionally, the intracranial tumor is a neuroblastoma or a glioblastoma; Optionally, the thoracic tumor is selected from lung cancer, esophageal cancer, gastric cardia cancer, breast cancer, mesothelioma, or mediastinal tumor; Optionally, the lung cancer is non-small cell lung cancer; Optionally, the breast cancer is triple-negative breast cancer cells; Optionally, the digestive system tumor is selected from gastric cancer, colorectal cancer, colon cancer, colorectal cancer, liver cancer, pancreatic cancer, periampullary cancer, biliary tract cancer, or small bowel cancer; Optionally, the colon cancer is a colonic adenoma; Optionally, the liver cancer is hepatocellular carcinoma; Optionally, the urogenital tumor is selected from kidney cancer, prostate cancer, bladder cancer, testicular cancer, penile cancer, cervical cancer, endometrial cancer, or ovarian cancer; Optionally, the renal cell carcinoma is a renal cell adenocarcinoma; Optionally, the soft tissue and skin tumors are selected from fibrous histiocytoma, rhabdomyosarcoma, synovial sarcoma, or melanoma; Optionally, the bone tumor is selected from osteosarcoma or Ewing's sarcoma; Optionally, the hematologic malignancy is selected from leukemia, lymphoma (HL), multiple myeloma (MM), or myelodysplastic syndrome (MDS); Optionally, the leukemia is selected from B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute myeloid leukemia, monocytic leukemia, or promyelocytic leukemia; Optionally, the lymphoma is selected from histiocytic lymphoma or Burkitt's lymphoma; Optionally, the method further includes administering additional therapeutic agents to the patient.

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