High-affinity nanobodies targeting b7h3 (CD276) for treating multiple solid tumors

Abstract

To provide high-affinity nanobodies targeting B7H3 (CD276) for treating multiple solid tumors.SOLUTION: Single-domain monoclonal antibodies that specifically bind B7H3 (also known as CD276) are described. The single-domain antibodies include camel VHH and rabbit VH domain nanobodies selected from phage display libraries. Chimeric antigen receptors (CARs) and other antibody conjugates targeted to B7H3 are also described. The single-domain antibodies and conjugates thereof can be used for the diagnosis and treatment of B7H3-expressing solid tumors.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 924,298, filed October 22, 2019, which is incorporated herein by reference in its entirety.

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

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

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

[0005] Historically, Camelidae V H H-domain antibodies were the mainstay of research until the emergence of human VH-domain antibodies (Feng et al., Proc Natl Acad Sci USA, 110, E1083-1091, 2013; Tang et al., Mol Cancer Ther, 12, 416-426, 2013; Li et al., Proc Natl Acad Sci USA, 114, E6623-E6631, 2017). Camelid sera contain both conventional IgG and significant amounts of heavy chain-only IgG (HCAb), accounting for 45%–75% of total serum immunoglobulins, depending on the specific species (Khodabakhsh et al., Int Rev Immunol, 37, 316-322, 2018). HCAb are VH-domain antibodies.H It consists of an H fragment, a single antigen-binding domain, followed by CH2 and CH3 domains, and a light chain that pairs with the VH-CH1 domain in conventional IgG. H The H domain is the functional entity that is utilized.

[0006] Naturally Evolved Camelidae V H There are several structural features that make H domains highly soluble and stable. First, Val37 (Kabat numbering) in the human VH germline is typically located at VH, which creates a more compact and stable hydrophobic packing of the domain. H The H domain is Phe37 (or Tyr37) (Riechmann and Muyldermans, J Immunol Methods, 231, 25-38, 1999), which is probably the V H This is the driving force that makes VH particularly stable (Shinozaki et al., J Biosci Bioeng, 125, 654-661, 2018). Second, the residues G44, L45, and W47 that contact the light chain in the human VH germline are the same as those in VH. H These residues are E44 (or Q44), R45 (or C45), and G47 (or Ser, Leu, Phe) in H (Holt et al., Trends Biotechnol, 21, 484-490, 2003), which make the accessible surface area more hydrophilic and reduce aggregation. H In some H domains, W103 may be replaced by R103. H H domains usually have longer CDR3s than human / rodent CDR3s, and they typically contain a Cys in the CDR3 that forms an additional disulfide bond with a Cys at the end of CDR1 (camel) or at the beginning of CDR2 (llama) (Wesolowski et al., Med Microbiol Immunol, 198, 157-174, 2009). Furthermore, this additional CDR3 disulfide bond and the canonical C22-C92 disulfide bond form a V H The H domain is more stable (T range 60-78°C)m value), allowing reversible unfolding / refolding (Holt et al. al., Trends Biotechnol, 21, 484-490, 2003).

[0007] In 1989, the first non-V hemocyanin was isolated from a cDNA expression library constructed from the spleen of a mouse immunized with lysozyme and keyhole limpet hemocyanin. H Mammalian VH domain antibodies were screened and two mouse VH domains showed affinities for lysozyme in the 20 nM range (Ward et al., Nature, 341, 544-546, 1989), suggesting the name "single domain antibody (dAb)" for the first time. HWith the discovery of VH, the idea of exploring human VH domain antibodies, which are more attractive for pharmaceutical and other applications, has grown. Both camelid and human domain antibodies have been widely studied, but there are several technical limitations to accessing sources, particularly with regard to the discovery of domain antibodies through immunization. To overcome these limitations, several strategies have been developed to generate human or humanized VH domain antibodies. While phage display of naive human VH domain libraries is a proven method (Feng et al., Proc Natl Acad Sci USA, 110, E1083-1091, 2013; Tang et al., Mol Cancer Ther, 12, 416-426, 2013; Li et al., Proc Natl Acad Sci USA, 114, E6623-E6631, 2017), the affinity is not always as high as that of immunized antibodies. Another method for generating human domain antibodies is to use transgenic animals carrying human VH germline genes (Schusser et al., Eur J Immunol, 46, 2137-2148, 2016; Janssens et al., Proc Natl Acad Sci USA, 103, 15130-15135, 2006). Instead of directly isolating human domain antibodies, generating VH domain antibodies from immunized animals and then humanizing them is also one way to generate human-like antibodies with high affinity. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Khodabakhsh et al., Int Rev Immunol, 37, 316-322, 2018 [Non-patent document 2] Feng et al., Antib Ther, 2, 1-11, 2019 [Non-patent document 3] Prelli and Frangione, J Immunol, 148, 949-952, 1992 Summary of the Invention [Means for solving the problem]

[0009] overview The present disclosure provides ten camelid single domain VH1 antibodies that specifically bind B7H3 (also known as CD276). H A B7H3 monoclonal antibody and two rabbit VH single domain antibodies are described. The B7H3-specific camelid antibodies, designated herein as RWB12 ("B12"), RWG8 ("G8"), RWC4 ("C4"), RWB2, RWH5, RWD5, RWC3, RWG4, RWD9, and RWH1, and the B7H3-specific rabbit antibodies, designated herein as RFA1 and RFB1, bind B7H3 with high affinity. The generation of chimeric antigen receptor (CAR) T cells composed of the disclosed nanobodies is also disclosed.

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

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

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

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

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

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

[0016] The above and other objects and features of the present disclosure will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0017] [Figure 1]FIG. 1A: Schematic diagram showing immunization of rabbits with recombinant B7H3, generation of a VH domain phage display library, and selection of B7H3 binders.

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

[0019] [Figure 2] Figures 2A-2B: Titering of B7H3-hFc-immunized rabbit serum and confirmation of its binding to cells. (Figure 2A) Titering of B7H3-hFc-immunized rabbit serum by protein-binding ELISA. IAB-hFc, derived from the N-terminal fragment of mesothelin, served as the hFc-tagged control. R31M0, R31M1, R31M2, and R31M3 represent the pre-immune, first-, second-, and third-immune sera. (Figure 2B) Cell-binding assay of immune sera. The shaded area represents cells stained with FITC-conjugated secondary antibody alone (goat anti-rabbit); left curve, cells stained with pre-immune serum; right curve, cells stained with immune serum R31M3.

[0020] [Figure 3-1] Figures 3A-3B: Sequence and structural modeling of B7H3 binders. (Figure 3A) Sequence alignment of B7H3 binders (A1, SEQ ID NO: 11; B1, SEQ ID NO: 12) along with a similar VH from a rabbit anti-hypusine monoclonal antibody (PDB#5DUB, SEQ ID NO: 34). The CDR regions were defined by the IMGT drawing system. (Figure 3B) Structural modeling of the A1 and B1 binders using the online software SWISS-MODEL. The crystal structure of 5DUB is also shown for comparison. [Figure 3-2] Same as above.

[0021] [Figure 4-1]Figures 4A-4C: Binding characteristics of B7H3 binders. (Figure 4A) SDS-PAGE analysis of purified A1 and B1 binders (VH-His-FLAG fusions) from E. coli. Two micrograms of purified B7H3-hFc, either unreduced (Non.) or reduced (Red.) with β-mercaptoethanol, was separated on an 8% SDS-PAGE gel. Protein bands were visualized by Coomassie Blue R-250 staining. (Figure 4B) Measurement of protein binding affinity by ELISA. Five micrograms of B7H3-hFc was coated onto an ELISA plate, and various concentrations of domain antibodies were incubated with the plate. Binding was detected with the anti-FLAG mouse monoclonal antibody M2 conjugated to HRP. Binding curves were plotted using GraphPad Prism software, and the EC50 values were calculated using the software's hyperbolic algorithm for single-site binding. (Figure 4C) Cell binding was determined by flow cytometry. Ten micrograms of domain antibodies per mL were co-incubated with one million cells. Antibody binding was visualized with an anti-FLAG monoclonal antibody conjugated to APC. The curve labeled "1" represents cells stained with the secondary antibody alone; the curve labeled "2" represents cells stained with the A1 or B1 domain antibody. [Figure 4-2] Same as above.

[0022] [Figure 5] Figure 5: Production of B7H3-Fc fusion protein in HEK-293 cells. Recombinant B7H3-Fc fusion protein was expressed in HEK-293 cells and purified on a Protein A column (GE Healthcare) using an AKTA Explorer (GE Healthcare). The purified B7H3-Fc fusion protein was >99% pure as shown on an SDA-PAGE gel and had a molecular weight of 154 kDa under non-reducing conditions (left) and 77 kDa under reducing conditions (right). The yield of B7H3-Fc was 2 mg / L. Glypican 2 (GPC2)-Fc was used as a control protein.

[0023] [Figure 6] Figure 6: Phage panning of eight camel VHH libraries for B7H3 binders. Eight VHH single-domain antibody libraries generated from eight camels (Camelus dromedaries) were used to perform three rounds of phage panning on recombinant B7H3-Fc protein. The phage titers for each round are shown in the figure. The increase in phage titer in the third round of phage panning indicated enrichment of high-affinity VHH binders to B7H3.

[0024] [Figure 7] Figures 7A-7B: Purification of the RWC4 VHH nanobody. (Figure 7A) SDS-PAGE of the RWC4 VHH camelid nanobody fraction eluted from an AKTA Explorer (GE Healthcare). (Figure 7B) Chromatograph of nanobody elution from a nickel column (GE Healthcare) on an AKTA Explorer (GE Healthcare). The yield of RWC4 was 33.8 mg / L.

[0025] [Figure 8] Figures 8A-8B: Purification of RWG8 VHH nanobody. (Figure 8A) SDS-PAGE of the RWG8 VHH camelid nanobody fraction eluted from an AKTA Explorer (GE Healthcare). (Figure 8B) Chromatograph of RWG8 nanobody elution from a nickel column (GE Healthcare) on an AKTA Explorer (GE Healthcare). The yield of RWG8 was 50 mg / L.

[0026] [Figure 9]Figures 9A-9B: Purification of RWB12 VHH nanobody. (Figure 8A) SDS-PAGE of the RWB12 VHH camelid nanobody fraction eluted from an AKTA Explorer (GE Healthcare). (Figure 9B) Chromatograph of RWB12 nanobody elution from a nickel column (GE Healthcare) on an AKTA Explorer (GE Healthcare). The yield of RWB12 was 132 mg / L.

[0027] [Figure 10] Figure 10: Binding of selected B7H3-targeted VHH nanobodies to NBEB neuroblastoma cells. Binding of B7H3-specific nanobodies was assessed by FACS analysis. Six of the tested antibodies (RWC4, RWB12, RWG8, RWA12, RWG4, and RWD5) were able to bind NBEB cells.

[0028] [Figure 11] Figure 11: Binding of selected B7H3-targeted VHH nanobodies to A431 epidermoid carcinoma cells. Binding of B7H3-specific nanobodies was assessed by FACS analysis. Six of the tested antibodies (RWC4, RWB12, RWG8, RWA12, RWG4 and RWD5) were able to bind A431 cells.

[0029] [Figure 12] Figure 12: Kinetics of RWC4 with human B7H3-Fc. The association / dissociation properties of RWC4 were measured using recombinant human B7H3-Fc protein with the Octet system (Creative Biolabs). KD of RWC4 = 3.8 nM, Kon of RWC4 = 2.65 x 104, and Kdis of RWC4 = 1.01 x 10-4.

[0030] [Figure 13]Figure 13: Kinetics of RWG4 with human B7H3-Fc. The association / dissociation properties of RWG4 were measured using recombinant human B7H3-Fc protein with the Octet system (Creative Biolabs). KD of RWG4 = 6.94 nM, Kon of RWG4 = 9.13 x 103, and Kdis of RWG4 = 6.34 x 10-5.

[0031] [Figure 14] Figure 14: T cell transfection efficiency of lentivirus expressing B7H3-targeted CAR. Transfection efficiency was measured by FACS.

[0032] [Figure 15-1] Figures 15A-15D: Cytotoxicity of B7H3-targeting CAR-T cells in B7H3-positive cells. (Figure 15A) Human neuroblastoma NBEB cells. (Figure 15B) Human neuroblastoma LAN-1 cells. (Figure 15C) Human adenocarcinoma BXPC-3 cells. (Figure 15D) Human pancreatic carcinoma Miacapa2 cells. RWB12, RWG8, and RWC4 CAR-T cells were most effective at inducing specific lysis. [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 15-4] Same as above.

[0033] [Figure 16-1] Figures 16A-16D: Cytotoxicity of B7H3-targeting CAR-T cells in B7H3-positive and B7H3-knockout cells. (Figure 16A) Human neuroblastoma IMR32 cells. (Figure 16B) Mouse colon adenocarcinoma MC38-CD276+ cells. (Figure 16C) Human neuroblastoma IMR32-CD276- / - cells. (Figure 16D) Mouse colon adenocarcinoma MC38-CD276- / - cells. Three of the CAR-T cells (RWB12, RWG8, and RWC4) showed potent cytotoxicity against B7H3-positive cells, but not against B7H3-negative cells. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 16-4] Same as above.

[0034] [Figure 17-1] Figures 17A-17C: Cytotoxicity of B7H3 CAR T cells against pancreatic cancer cells. The cytotoxicity of human B7H3-targeting nanobody-derived CAR T cells (B12, G8, and C4) was evaluated using two luciferase-expressing B7H3-positive pancreatic cancer cell lines, Panc-1 GFP-Luc (GL) and BxPC-3 GL. (Figure 17A) Flow cytometry shows that both Panc-1 and BxPC-1 are B7H3-positive cell lines. (Figure 17B) The T cell transduction efficiencies with the G8, C4, and B12 CARs were 60.4%, 58.6%, and 68.4%, respectively. The T cell transduction efficiency with an unrelated CAR (CD19) was 32%. (Figure 17C) B7H3-targeted CAR (G8 / B12 / C4) T cells and irrelevant control CAR (CD19) T cells were incubated with Panc-1 GL or BxPC-3 GL cells at various effector:target (E:T) ratios for 24 hours. Both Panc-1 GL and BxPC-3 GL cells were effectively lysed in a dose-dependent manner by all three B7H3-targeted CAR T cells, while minimal killing was observed from the control CAR T cells. [Figure 17-2] Same as above.

[0035] [Figure 18-1]Figures 18A-18D: Panc-1 mouse model treated with high doses of human B7H3-specific CAR T cells. Tumor regression was assessed in the Panc-1 xenograft mouse model after injection of 10 million B7H3-targeted CAR T cells. (Figure 18A) Schematic of the Panc-1 xenograft study. One million Panc-1 GFP / Luc tumor cells were implanted intravenously into NSG mice to establish the tumor model. Twenty days later (day 0), the mice were intravenously injected with 10 million C4, G8, or B12 CAR T cells (or control CD19 CAR T cells). Imaging was performed weekly. (Figure 18B) Representative bioluminescence images of Panc-1 tumor growth in mice treated with CAR T cells. Mice treated with 10 million B7H3-targeted CAR T cells (C4, G8, or B12) showed significantly reduced tumor growth compared to injection of control CAR T cells. (Figure 18C) Quantification of tumor bioluminescence as photons per second for the mice shown in Figure 18B. (Figure 18D) Kaplan-Meier survival curves for tumor-bearing mice after treatment with 10 million C4, G8, or B12 CAR T cells. The results demonstrate that C4 CAR T cells, when administered at a high dose (10 million), are more potent in promoting mouse survival than G8 or B12 CAR T cells, and indicate that administration of 10 million CAR T cells is safe for mice. [Figure 18-2] Same as above.

[0036] [Figure 19-1]Figures 19A-19D: Panc-1 mouse model treated with low doses of human B7H3-specific CAR T cells. Tumor regression in the Panc-1 xenograft mouse model after injection of 5 million B7-H3-targeted CAR T cells was measured after tumor rechallenge. (Figure 19A) Schematic of the experiment. Twenty days after inoculation of 1 million Panc-1-Luc cells (day 0), Panc-1 xenografted mice were intravenously injected with 5 million C4 CAR T cells, B12 CAR T cells, untransduced T cells (mock), or PBS. Mice treated with C4 CAR T cells and B12 CAR T cells that did not show detectable tumors were intravenously implanted with 1 million Panc-1 cells on day 35. As a control, naive mice were implanted with Panc-1 cells. Imaging was performed weekly. (Figure 19B) Representative bioluminescence images of Panc-1 tumor growth in mice treated with CAR T cells. Mice treated with 5 million C4 CAR T cells or B12 CAR T cells showed significantly reduced tumor growth compared to mice administered mock T cells or PBS. While tumors grew rapidly in control mice, 100% of mice previously treated with C4 CAR T cells remained tumor-free after rechallenge with Panc-1 tumors, and 60% of mice previously treated with B12 CAR T cells remained tumor-free up to 10 weeks after treatment. (Figure 19C) Quantification of tumor bioluminescence as photons per second in the mice shown in Figure 19B. (Figure 19D) Kaplan-Meier survival curves of tumor-bearing mice after treatment, showing that mice that received 5 million C4 or B12 CAR T cells were still alive at day 70. In contrast, mice treated with PBS or mock T cells did not survive beyond 30 days after injection. [Figure 19-2] Same as above.

[0037] [Figure 20-1]Figures 20A-20D: Testing of B7H3-targeted CAR T cells in an IMR5 neuroblastoma mouse model. (Figure 20A) In vitro killing of the neuroblastoma cell line IMR5 using B7H3-targeted CAR T cells was evaluated. B7H3-targeted G8, B12, or C4 CAR T cells, and commercially available anti-human B7H3 hybridoma antibody 376.96-based CAR T cells were incubated with IMR5 GL cells at various effector:target (E:T) ratios for 24 hours. All CAR T cells effectively lysed IMR5 tumor cells in a dose-dependent manner compared to mock T cells, although B12 CAR T cells were slightly more effective than the other CAR T cells tested. (Figure 20B) Experimental schematic for in vivo studies. IMR5 xenografted mice were intravenously injected with 5 million C4 CAR T cells, B12 CAR T cells, G8 CAR T cells, 376.96 CAR T cells, or untransduced T cells (mock) 35 days after tumor inoculation. (Figure 20C) Representative bioluminescence images of IMR5 tumor growth in the xenograft model. Mice treated with 5 million B12 CAR T cells showed significantly reduced tumor growth compared to 376.96 CAR T cells and mock T cells. C4 CAR T cells also showed moderate antitumor activity. (Figure 20D) Tumor bioluminescence as photons per second in treated mice. [Figure 20-2] Same as above. [Figure 20-3] Same as above.

[0038] [Figure 21-1]Figures 21A-21C: G8 cross-reacts with mouse B7H3 and kills mouse cancer cells. (Figure 21A) Binding activity of anti-B7H3 nanobodies to mouse antigens detected by flow cytometry. G8, but not C4 or B12, showed positive binding to mouse B7H3 expressed in three KPC cell lines (CREP128096, CREP133239, and PDA95775) and the mouse melanoma cell line B16. (Figure 21B) Only G8 CAR T cells showed specific killing of the mouse B7H3-positive B16 cell line. (Figure 21C) Epitope mapping of anti-B7H3 nanobodies and the commercially available antibody 376.96. A total of 48 peptides were designed and synthesized from the human B7H3 protein. Each peptide consisted of 18 amino acids and overlapped with adjacent peptides by 9 amino acids. The antibodies were tested for their ability to bind to each peptide using ELISA technology. The numbers indicate OD450 values. Both G8 and 376.96 bound to peptides 10, 11, and 15 (SEQ ID NOs: 35-37), indicating that they bind similar epitopes. The peptide sequences are shown below the table. C4 and B12 may have conformational epitopes that could not be predicted by the linearized peptide library. [Figure 21-2] Same as above. [Figure 21-3] Same as above.

[0039] [Figure 22] Figure 22: Binding of B7H3-specific antibodies B12, C4, G8 and 376.96 to B7H3 protein by ELISA. DETAILED DESCRIPTION OF THE INVENTION

[0040] Sequence Listing The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids as set forth in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but it is understood that any reference to any of the displayed strands includes the complementary strand. The sequence listing is submitted as a 27.8 KB ASCII text file created on October 19, 2020, which is incorporated herein by reference. In the accompanying sequence listing:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0064] Detailed Description I. Abbreviations ADC antibody-drug conjugate ADCC antibody-dependent cell-mediated cytotoxicity B7H3 B7 homolog 3 BBIR Biotin-binding immunoreceptor CAR Chimeric Antigen Receptor CDR Complementarity Determining Region CTL cytotoxic T lymphocytes ECD extracellular domain EGF epidermal growth factor EGFR epidermal growth factor receptor ELISA enzyme-linked immunosorbent assay EM effector part FACS Fluorescence-Activated Cell Sorting GMCSFRss Granulocyte-macrophage colony-stimulating factor receptor signal sequence hFc human Fc huEGFRt human truncated epidermal growth factor receptor IC50 inhibitory concentration 50 Ig immunoglobulin KO Knockout NK Natural Killer PBD pyrrolobenzodiazepines PE Pseudomonas exotoxin PET Positron Emission Tomography TM transmembrane VH Variable Weight VL Variable Light II. Terminology and Methods

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

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

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

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

[0069] Antibody: A polypeptide ligand comprising at least one variable region that recognizes and binds (e.g., specifically recognizes and binds) an epitope of an antigen. Mammalian immunoglobulin molecules consist of heavy (H) chains and light (L) chains, each of which has a variable heavy (V) domain. H ) area and variable light (V L ) region, which has a variable region called the V H Area and V L The region responsible for binding the antigen recognized by the antibody. There are five major heavy chain classes (or isotypes) of mammalian immunoglobulins, which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is the primary antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0070] Antibody variable regions contain "framework" regions and hypervariable regions known as "complementarity-determining regions" or "CDRs." The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be determined by Kabat et al. (Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991; "Kabat" numbering scheme), Chothia et al. (Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273,927-948, 1997; see "Chothia" numbering scheme), Kunik et al. (Kunik et al., PLoS Comput Biol 8:e1002388, 2012; and Kunik et al., Nucleic Acids Res 40(Web Server issue):W521-524, 2012; see "Paratome CDRs") and the ImMunoGeneTics (IMGT) database (Lefranc, Nucleic Acids Res 29:207-9, 2001; see "IMGT" numbering scheme. The Kabat, Paratome and IMGT databases are maintained online.

[0071] A "single domain antibody" refers to an antibody having a single domain (a variable domain) that is capable of specifically binding an antigen, or an epitope of an antigen, in the absence of additional antibody domains. Single domain antibodies include, for example, V H Domain antibodies, V NAR Antibody, Camelidae V H H antibodies, and V L Domain antibodies are included. NAR Antibodies are produced by cartilaginous fishes such as nurse sharks, wobbe sharks, dogfish, and nurse sharks. H H antibodies are produced by several species, including camels, llamas, alpacas, dromedaries, and guanacos, which produce heavy-chain antibodies that are naturally devoid of light chains.

[0072] A "monoclonal antibody" is an antibody produced by a single clone of lymphocytes or by a cell transfected with a single antibody coding sequence. Monoclonal antibodies are produced by methods known to those skilled in the art. Monoclonal antibodies include humanized monoclonal antibodies.

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

[0074] A "humanized" antibody is an immunoglobulin containing a human framework region and one or more CDRs derived from 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 derived from the donor immunoglobulin of the humanized immunoglobulin. Constant regions need not be present, but if present, they should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or more identical. Thus, all portions of a humanized immunoglobulin, except possibly for the CDRs, are substantially identical to corresponding portions of natural human immunoglobulin sequences. A humanized antibody binds to the same antigen as the donor antibody providing the CDRs. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions.

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

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

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

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

[0079] Binding affinity: The affinity of an antibody for an antigen. In one embodiment, affinity is measured using the Frankel method. The binding affinity is calculated by a modification of the Scatchard method described by Scatchard et al., Mol. Immunol., 16:101-106, 1979. In another embodiment, binding affinity is measured by antigen / antibody dissociation rate. In another embodiment, high binding affinity is measured by competitive radioimmunoassay. In another embodiment, binding affinity is measured by ELISA. In other embodiments, antibody affinity is measured by flow cytometry or surface plasmon resonance. An antibody that "specifically binds" an antigen (such as B7H3) is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens.

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

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

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

[0083] Breast cancer: A type of cancer that forms in the tissues of the breast, usually 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 type of 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 called ER-negative, PR-negative, and HER2 / neu-negative breast cancer.

[0084] Chemotherapeutic agent: Any chemical agent that has therapeutic utility in treating diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancers, as well as diseases characterized by hypertrophic growths, such as psoriasis. In one embodiment, the chemotherapeutic agent is an agent used in treating B7H3-positive tumors. In one embodiment, the chemotherapeutic agent is a radioactive compound. Those skilled in the art can readily identify chemotherapeutic agents for 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 2 nd ed., (Copyright)2000 Churchill Livingstone, Inc;Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. (See, e.g., St. Louis, Mosby-Year Book, 1995; Fischer, DS, Knobf, MF, Durivage, HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one drug to treat cancer. One example is the administration of an antibody that binds B7H3, used in combination with radioactive or chemical compounds. In one example, the chemotherapeutic agent is a biologic, for example, a therapeutic antibody (e.g., a therapeutic monoclonal antibody), such as the anti-B7H3 antibody provided herein, and other anti-cancer antibodies, such as anti-PD1 or anti-PDL1 (e.g., pembrolizumab and nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-EGFR (e.g., cetuximab), anti-VEGF (e.g., bevacizumab), or a combination thereof (e.g., anti-PD-1 and anti-CTLA-4).

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

[0086] Colon cancer: A type of cancer that begins in the colon or rectum. The most common type of colon cancer (also known as "colorectal cancer") is colorectal adenocarcinoma, which accounts for approximately 95% of all colon cancers. Adenocarcinoma develops in the cells that line the colon and / or rectum. Other types of colorectal cancer include gastrointestinal carcinoid tumors, metastatic colorectal cancer, primary colorectal lymphoma (a type of non-Hodgkin's lymphoma), gastrointestinal stromal tumors (classified as sarcomas and arising from interstitial cells of Cajal), leiomyosarcoma (arising from smooth muscle cells), and colorectal melanoma.

[0087] Complementarity determining region (CDR): A region of hypervariable amino acid sequence that determines the binding affinity and specificity of an antibody. Each light and heavy chain of a mammalian immunoglobulin has three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Single domain antibodies contain three CDRs, designated CDR1, CDR2, and CDR3 herein.

[0088] Conjugate: In the context of this disclosure, a "conjugate" is an antibody or antibody fragment (e.g., an antigen-binding fragment) covalently linked to an effector molecule or a second protein (such as a second antibody). The effector molecule can be, for example, a drug, 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 (such as a cytotoxic agent), the conjugate is often referred to as an "antibody-drug conjugate" or "ADC." Other antibody conjugates include, for example, multispecific (such as bispecific or trispecific) antibodies and chimeric antigen receptors (CARs).

[0089] Conservative variant: A protein containing conservative amino acid substitutions that do not substantially affect or reduce the affinity of a protein, such as an antibody to B7H3. For example, a monoclonal antibody that specifically binds B7H3 may contain at most about 1, at most about 2, at most about 5, at most about 10, or at most about 15 conservative substitutions and be capable of specifically binding a B7H3 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 B7H3. Non-conservative substitutions are those that reduce activity or binding to B7H3.

[0090] Conservative amino acid substitution tables providing functionally similar amino acids are well known to those skilled in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0091] Contacting: To bring into direct physical association; includes both solid and liquid forms.

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

[0093] Cytotoxicity: The toxicity of a molecule, such as an immunotoxin, to cells that are intended to be targeted, as opposed to cells in the rest of the organism. In contrast, the term "toxicity" refers to the toxicity of an immunotoxin to cells other than those intended to be targeted by the targeting moiety of the immunotoxin, and the term "animal toxicity" refers to the toxicity of an immunotoxin to animals due to the toxicity of the immunotoxin to cells other than those intended to be targeted by the immunotoxin.

[0094] Degenerate variant: A polynucleotide encoding a polypeptide that contains a sequence that is degenerate as a result of the genetic code. There are 20 naturally occurring amino acids, most of which are specified by more than one codon. Thus, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide is unchanged.

[0095] Diagnosis: Identifying the presence or nature of a condition, such as a B7H3-positive cancer. Diagnostic methods vary in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent 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 those without the disease who test positive. While a particular diagnostic method may not be able to provide a definitive diagnosis of a condition, it is sufficient if the method provides a positive indication that aids in diagnosis. "Prognosis" is the probability of development (e.g., severity) of a condition, such as cancer.

[0096] Diagnostic tumor imaging: Coupling antibodies and their derivatives with positron-emitting radionuclides for positron emission tomography (PET) is a process often referred to as immunoPET. While full-length antibodies can make good immunoPET agents, their biological half-lives necessitate waiting several days before imaging, resulting in increased non-targeted radiation doses. Smaller single-domain antibodies, or nanobodies, have biological half-lives that are suitable for same-day imaging.

[0097] Drug: Any compound used to treat, alleviate, or prevent a disease or condition in a subject. In some embodiments herein, the drug is an anti-cancer drug, e.g., a cytotoxic agent, e.g., an antimitotic agent or a microtubule inhibitor.

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

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

[0100] Framework region: Amino acid sequences interposed between the CDRs. The framework region of an immunoglobulin molecule comprises the variable light and variable heavy framework regions.

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

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

[0103] Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, the immune response is a response of a CD4 + Response or CD8 + In another embodiment, the response is a B cell response, such as a T cell response, which results in the production of specific antibodies.

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

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

[0106] Interstrand cross-linking agent: a class of cytotoxic drugs that can covalently bond between two strands of DNA, thereby preventing DNA replication and / or transcription.

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

[0108] Label: A detectable compound or composition directly or indirectly conjugated to another molecule, such as an antibody or a protein, to facilitate its detection. Specific, non-limiting examples of labels include fluorescent tags, enzyme conjugation, and radioisotopes. In one example, a "labeled antibody" refers to the incorporation of another molecule in an antibody. For example, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid or the attachment of a biotinyl moiety to a polypeptide that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (e.g., 35 S, 11 C. 13 N, 15 O. 18 F,19 F, 99m Tc, 131 I, 3 H, 14 C. 15 N, 90 Y, 99 Tc, 111 In and 125 I), fluorescent labels (e.g., fluorescein isothiocyanate (FITC), rhodamine, lanthanide fluorophores), enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0109] Linker: In some cases, a linker is a peptide within an antibody-binding fragment (such as an Fv fragment) that serves to indirectly link the variable heavy chain to the variable light chain. "Linker" can also refer to a peptide that serves to link a targeting moiety, such as an antibody, to an effector molecule, such as a cytotoxin or a detectable label. The terms "conjugating," "joining," "bonding," or "linking" refer to the making of two polypeptides into one contiguous polypeptide molecule, or the covalent attachment of a radionuclide or other molecule to a polypeptide, such as an antibody. Linking can be by chemical or recombinant means. "Chemical means" refers to a reaction between an antibody moiety and an effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.

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

[0111] Lung cancer: Any cancer that forms in the lung. Most cancers that start in the lung are carcinomas. The two main types of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Subclasses of NSCLC include adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.

[0112] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to 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, are in the same reading frame.

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

[0114] Pancreatic cancer: A disease in which malignant cells are found within the tissues of the pancreas. Pancreatic tumors can be either exocrine or neuroendocrine tumors based on the cellular origin of the cancer. The majority of pancreatic cancers (approximately 94%) 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 cases, pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). Pancreatic neuroendocrine tumors, also called islet cell tumors, are classified by the hormones they produce. Exemplary neuroendocrine tumors include gastrinomas, glucagonomas, insulinomas, somatostatinomas, VIP (vasoactive intestinal peptide) tumors, and non-functioning islet cell tumors.

[0115] Childhood cancer: Cancer occurring in children aged 0 to 14 years. Major types of childhood 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), Wilms' tumor, non-Hodgkin's lymphoma, and retinoblastoma.

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

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

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

[0119] Purified: The term purified does not require absolute purity, but rather is intended as a relative term. Thus, for example, a purified peptide preparation is one in which a peptide or protein is enriched more than the peptide or protein is in its natural environment within a cell. In one embodiment, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation. Substantial purification 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 from other proteins or cellular components.

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

[0121] Recombinant: A recombinant nucleic acid or protein is one having a sequence that is not found in nature or that is made by the artificial combination of two otherwise isolated segments of sequence, which is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid, e.g., by genetic engineering techniques.

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

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

[0124] Methods for aligning sequences for comparison are well known in the art.Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed discussion of sequence alignment methods and homology calculations.

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

[0126] Homologs and variants of antibodies that specifically bind B7H3 polypeptides are typically characterized by sharing at least about 75%, e.g., at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, as calculated over a full-length alignment with the amino acid sequence of the antibody, using NCBI Blast 2.0, gapped blastp, set to default parameters. Comparisons of amino acid sequences of more than about 30 amino acids use the Blast 2 alignment function with the default BLOSUM62 matrix set to default parameters (gap existence cost of 11, and per-residue gap cost of 1). When aligning short peptides (fewer than approximately 30 amino acids), alignments should be performed using the Blast 2 alignment function with the PAM30 matrix set to default parameters (open gap penalty of 9, extension gap penalty of 1). Proteins with even greater similarity to the reference sequence, when assessed by this method, exhibit increasing percentages of identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When comparing less than the entire sequence for sequence identity, homologs and variants typically have at least 80% sequence identity over a short window of 10-20 amino acids, and may have at least 85%, or at least 90%, or 95% sequence identity, depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available on the Internet at the NCBI website. Those skilled in the art will understand that these sequence identity ranges are provided for guidance only, and that it is quite possible to obtain highly significant homologs that fall outside the provided ranges.

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

[0128] Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals.

[0129] Synthetic: Produced by artificial means in a laboratory, for example, a synthetic nucleic acid or protein (e.g., an antibody) may be chemically synthesized in a laboratory.

[0130] Therapeutically effective amount: A quantity of a particular substance sufficient to achieve a desired effect in a treated subject. For example, this may be the amount necessary to inhibit or suppress tumor growth. In one embodiment, a therapeutically effective amount is the amount necessary to eliminate a tumor, reduce tumor size, or prevent tumor metastasis, e.g., reduce 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 reduce 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%, e.g., compared to the size / volume / number before treatment. When administered to a subject, a dosage is generally used that achieves a target tissue concentration (e.g., in a tumor) shown to achieve the desired in vitro effect.

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

[0132] Vector: A nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector may contain a nucleic acid sequence that allows it to replicate in the host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements known in the art. In some embodiments, the vector is a viral vector, such as a lentiviral vector. III. Single-Domain Monoclonal Antibodies ("Nanobodies") Specific for B7H3

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

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

[0135] RWB12 (SEQ ID NO: 1) [ka] [Table A-1]

[0136] RWG8 (SEQ ID NO: 2) [ka] [Table A-2]

[0137] RWC4 (SEQ ID NO: 3) [ka] [Table A-3]

[0138] RWB2 (SEQ ID NO: 4) [ka] [Table A-4]

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

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

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

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

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

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

[0145] RFA1 (SEQ ID NO: 11) [ka] [Table A-11]

[0146] RFB1 (SEQ ID NO: 12) [ka] [Table A-12]

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

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

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

[0150] In some embodiments, the CDR1, CDR2, and CD3 sequences of the antibody comprise residues 31-35, 50-66, and 97-105 of SEQ ID NO:2, residues 26-33, 51-58, and 97-106 of SEQ ID NO:2, or residues 27-35, 47-61, and 97-106 of SEQ ID NO:2, respectively. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:2.

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

[0152] In some embodiments, the CDR1, CDR2, and CD3 sequences of the antibody comprise residues 31-35, 50-65, and 96-110 of SEQ ID NO:4, residues 26-33, 51-57, and 96-110 of SEQ ID NO:4, or residues 27-35, 47-60, and 96-109 of SEQ ID NO:4, respectively. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:4. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:4.

[0153] In some embodiments, the CDR1, CDR2, and CD3 sequences of the antibody comprise residues 27-30, 45-61, and 90-109 of SEQ ID NO:5, residues 26-28, 46-53, and 90-110 of SEQ ID NO:5, respectively; or residues 27-30, 43-55, and 90-110 of SEQ ID NO:5. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:5. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:5.

[0154] In some embodiments, the CDR1, CDR2, and CD3 sequences of the antibody comprise residues 31-35, 50-65, and 96-111 of SEQ ID NO:6, residues 26-33, 51-57, and 96-112 of SEQ ID NO:6, or residues 27-35, 47-60, and 96-111 of SEQ ID NO:6, respectively. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:6. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:6.

[0155] In some embodiments, the CDR1, CDR2, and CD3 sequences of the antibody comprise residues 31-35, 50-65, and 96-111 of SEQ ID NO:7, residues 26-33, 51-57, and 96-112 of SEQ ID NO:7, or residues 27-35, 47-60, and 96-111 of SEQ ID NO:7, respectively. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:7.

[0156] In some embodiments, the CDR1, CDR2, and CD3 sequences of the antibody comprise residues 31-35, 50-65, and 96-111 of SEQ ID NO:8, residues 26-33, 51-57, and 96-112 of SEQ ID NO:8, or residues 27-35, 47-60, and 96-111 of SEQ ID NO:8, respectively. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:8.

[0157] In some embodiments, the CDR1, CDR2, and CD3 sequences of the antibody comprise residues 31-35, 50-65, and 96-111 of SEQ ID NO:9, residues 26-33, 51-57, and 96-112 of SEQ ID NO:9, or residues 27-35, 47-60, and 96-111 of SEQ ID NO:9, respectively. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:9.

[0158] In some embodiments, the CDR1, CDR2, and CD3 sequences of the antibody comprise residues 31-35, 50-65, and 96-113 of SEQ ID NO: 10, residues 26-33, 51-57, and 96-114 of SEQ ID NO: 10, or residues 27-35, 47-60, and 97-114 of SEQ ID NO: 10, respectively. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO: 10.

[0159] In some embodiments, the CDR1, CDR2, and CD3 sequences of the antibody comprise residues 30-34, 50-64, and 93-105 of SEQ ID NO:11, residues 25-32, 50-56, and 93-104 of SEQ ID NO:11, or residues 26-34, 46-59, and 93-105 of SEQ ID NO:11, respectively. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11 or to residues 1-113 of SEQ ID NO:11. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:11. In other specific examples, the amino acid sequence of the antibody comprises or consists of residues 1-113 of SEQ ID NO:11.

[0160] In some embodiments, the CDR1, CDR2, and CD3 sequences of the antibody comprise residues 32-35, 51-65, and 94-107 of SEQ ID NO:12; residues 26-33, 51-57, and 94-107 of SEQ ID NO:12; or residues 27-35, 47-60, and 94-108 of SEQ ID NO:12, respectively. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:12 or to residues 1-116 of SEQ ID NO:12. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:12. In other specific examples, the amino acid sequence of the antibody comprises or consists of residues 1-116 of SEQ ID NO:12.

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

[0162] Also provided herein is a chimeric antigen receptor (CAR) comprising the single-domain monoclonal antibody disclosed herein. In some embodiments, the CAR further comprises a hinge region, a transmembrane domain, a costimulatory signaling moiety, a signaling domain, or any combination thereof. In a specific, non-limiting example, the hinge region comprises a CD8α hinge region, the transmembrane domain comprises a CD8α transmembrane domain, the costimulatory signaling moiety comprises a 4-1BB signaling moiety, and / or the signaling domain comprises a CD3ζ signaling domain.

[0163] Also provided herein is the B7H3 specific antibody that is modified so that it can be used in universal CAR system.In some embodiments, the B7H3 specific antibody is fused to one component of specific binding pair.In some examples, the antibody is fused to leucine zipper or biotin.

[0164] Further provided is the cell that expresses B7H3-specific CAR.In some examples, the cell is a T lymphocyte, such as a CTL, or a natural killer cell.CAR and CAR-expressing cells are further described in section IV.

[0165] Also provided herein is an immunoconjugate comprising the single domain antibody disclosed herein and an effector molecule.In some embodiments, the effector molecule is a toxin, such as, but not limited to, Pseudomonas exotoxin or its variant, for example, 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, for example, IR700.The immunoconjugate comprising the photon absorber can be used for photoimmunotherapy.Immunoconjugates are further described in Section V.

[0166] Further provided herein is antibody-drug conjugate (ADC), which comprises a drug conjugated to the single domain antibody disclosed herein.In some embodiments, the drug is a small molecule, such as a microtubule inhibitor, an antimitotic agent and / or a cytotoxic agent.ADC is further described in section VI.

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

[0168] Further provided herein is an antibody-nanoparticle conjugate comprising a nanoparticle conjugated to the single domain antibody disclosed herein.In some embodiments, the nanoparticle comprises a polymer nanoparticle, a nanosphere, a nanocapsule, a liposome, a dendrimer, a polymer micelle, or a niosome.In some embodiments, the nanoparticle comprises a cytotoxic agent.Antibody-nanoparticle conjugates are further described in Section VIII.

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

[0170] Further provided herein is a nucleic acid molecule that encodes the antibody, CAR, immunoconjugate, multispecific antibody or fusion protein disclosed herein.In some embodiments, the nucleic acid molecule is operably linked to a promoter.Also provided is a vector that comprises the disclosed nucleic acid molecule.Further provided is an isolated cell that comprises the nucleic acid molecule or vector disclosed herein.

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

[0172] Further provided is an isolated cell that co-expresses the B7H3-specific CAR and huEGFRt disclosed herein. In some examples, the cell is a CTL or NK cell.

[0173] The present disclosure further provides compositions comprising a pharmaceutically acceptable carrier and the single domain monoclonal antibody, CAR, isolated cells (such as CAR-expressing cells, e.g., CAR T cells or CAR NK cells), immunoconjugates, ADCs, multispecific antibodies, antibody-nanoparticle conjugates, or fusion proteins disclosed herein. Compositions and their uses are further described in Section IX. IV. Chimeric Antigen Receptors (CARs)

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

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

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

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

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

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

[0180] In some embodiments disclosed herein, the CAR construct encodes the following amino acid sequence from N-terminal to C-terminal: [ka]

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

[0182] The truncated version of human EGFR, referred to herein as "huEGFRt", only contains domain III, domain IV and TM domain.Thus, huEGFRt lacks domain I, domain II and all three intracellular domains.huEGFRt cannot bind EGF and lacks signal transduction activity.However, this molecule retains the ability to bind certain EGFR-specific monoclonal antibodies, such as FDA-approved cetuximab (PCT Publication No. WO2011 / 056894, incorporated herein by reference).

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

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

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

[0186] The disclosed single domain monoclonal antibodies 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 linked to an antibody. A therapeutic agent is an agent with a specific biological activity directed at a specific target molecule or a cell that has a target molecule. Those skilled in the art will recognize that therapeutic agents can be various drugs such as vinblastine, daunomycin, cytotoxins such as natural or modified Pseudomonas exotoxin or diphtheria toxin, encapsulating media (such as liposomes) containing pharmacological compositions, or the like. 125 I, 32 P, 14 C. 3 H and 35 It will be appreciated that the antibodies may include radioactive agents such as S, photon absorbers such as IR700, as well as other labels, targeting moieties and ligands.

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

[0188] The therapeutic agents and antibodies described herein enable one of skill in the art to readily construct various clones that differ in sequence but contain functionally equivalent nucleic acids, such as nucleic acids encoding the same effector moiety or antibody sequence. Thus, the present disclosure provides nucleic acids encoding antibodies and conjugates and fusion proteins thereof.

[0189] Effector molecules can be linked to the antibody of interest using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. The procedure for attaching an effector molecule to an antibody varies depending on the chemical structure of the effector. Polypeptides typically contain various functional groups, such as carboxylic acid (COOH), free amine (-NH), or sulfhydryl (-SH) groups, which are available for reaction with suitable functional groups on the antibody, resulting in the attachment of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization can involve the attachment of any of several known linker molecules. The linker can be any molecule used to attach an antibody to an effector molecule. The linker is capable of forming covalent bonds to both the antibody and the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and effector molecule are polypeptides, the linkers may be attached to the constituent amino acids through their side chain groups (e.g., to cysteine via a disulfide linkage) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

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

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

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

[0193] One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, with two different reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are commercially available.

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

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

[0196] Antibodies may also be labeled with radiolabeled amino acids. Radiolabels can be used for both diagnostic and therapeutic purposes. For example, radiolabels can be used to detect the expression of target antigens by x-rays, emission spectroscopy, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I can be mentioned.

[0197] 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, IRDye® 700DX (also known as "IR700"). The antibody-photon absorber conjugate can be used for photoimmunotherapy.

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

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

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

[0201] Protease-resistant PE variants and PE variants with reduced immunogenicity, such as, but not limited to, PE-LR, PE-6X, PE-8X, PE-LR / 6X, and PE-LR / 8X, are also contemplated herein (see, 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. WO2007 / 016150, WO2009 / 032954, and WO2011 / 032022, which are incorporated herein by reference).

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

[0203] Modifications of PE can occur in any of the previously described variants, including cytotoxic fragments of PE (e.g., PE38, PE-LR, and PE-LR / 8M). Modified PE can include, for example, any substitution(s) of one or more amino acid residues within one or more T cell 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).

[0204] Contemplated forms of PE also include deimmunized forms of PE, such as versions in which domain II is deleted (e.g., PE24). Deimmunized forms of PE are described, for example, in PCT Publication Nos. WO2005 / 052006, WO2007 / 016150, WO2007 / 014743, WO2007 / 031741, WO2009 / 32954, WO2011 / 32022, WO2012 / 154530, and WO2012 / 170617.

[0205] The antibodies described herein can also be used to target any number of different diagnostic or therapeutic compounds to cells expressing B7H3 on their surface. Thus, the antibodies of the present disclosure can be conjugated, either directly or via a linker, to drugs that are delivered directly to cells expressing B7H3 on their surface. This can be for therapeutic, diagnostic, or research purposes. Therapeutic agents include compounds such as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, photon absorbers, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent crosslinking to single- or double-stranded DNA, and triplex-forming oligonucleotides.

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

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

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

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

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

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

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

[0213] Provided herein is an ADC comprising a drug (such as a cytotoxic agent) conjugated to a monoclonal antibody that binds (for example, specifically binds) B7H3.In some embodiments, the drug is a small molecule.In some examples, the drug is a crosslinking agent, a microtubule inhibitor and / or an antimitotic agent, or any cytotoxic agent suitable for mediating tumor cell killing. Exemplary cytotoxic agents include, but are not limited to, PBDs, auristatins, maytansinoids, dolastatins, calicheamicins, nemorubicin and its derivatives, PNU-159682, anthracyclines, vinca alkaloids, taxanes, trichothecenes, CC1065, camptothecins, elinafides, combretastatins, dolastatins, duocarmycins, enediynes, geldanamycins, indolino-benzodiazepine dimers, puromycins, tubulysins, hemiasterins, spliceostatins, or pladienolides, and stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.

[0214] In some embodiments, the ADC comprises a pyrrolobenzodiazepine (PBD). The natural product anthramycin (PBD) was first reported in 1965 (Leimgruber et al., J Am Chem Soc, 87:5793-5795, 1965; Leimgruber et al., J Am Chem Soc, 87:5791-5793, 1965). Since then, several PBDs, both naturally occurring and synthetic analogs, have been described (Gerratana, Med Res Rev 32(2):254-293, 2012; and U.S. Patent Nos. 6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; and 7,557,099). As an example, PBD dimers have been shown to recognize and bind to specific DNA sequences and to be useful as cytotoxic agents. PBD dimers have been conjugated to antibodies, and the resulting ADCs have been shown to have anti-cancer properties (see, for example, US2010 / 0203007). Exemplary linking sites in PBD dimers include five-membered pyrrolo rings, tethers between PBD units, and N10-C11 imine groups (see WO2009 / 016516; US2009 / 304710; US2010 / 047257; US2009 / 036431; US2011 / 0256157; and WO2011 / 130598).

[0215] In some embodiments, the ADC comprises an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine, which are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinoids are described, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.

[0216] In some embodiments, the ADC comprises an antibody conjugated to a dolastatin or auristatin, or an analog or derivative thereof (see U.S. Patent Nos. 5,635,483; 5,780,588; 5,767,237; and 6,124,431). 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 anticancer (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob Agents Chemother 42:2961-2965, 1998). Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin F, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (monomethyl auristatin D or monomethyl dolastatin 10), MMAF (monomethyl auristatin F or N-methylvaline-valine-dolaisoloiin-dolaproine-phenylalanine), MMAE (monomethyl auristatin E or N-methylvaline-valine-dolaisoloiin-dolaproine-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other auristatins (see, e.g., U.S. Publication No. 2013 / 0129753).

[0217] In some embodiments, the ADC comprises an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics 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 containing calicheamicin drug moieties are described in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.

[0218] In some embodiments, the ADC comprises an anthracycline. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. It is believed that anthracyclines can act to kill cells through several different mechanisms, including intercalation of the drug molecule into cellular DNA and thereby inhibiting DNA-dependent nucleic acid synthesis; induction of free radical production, which then reacts with cellular macromolecules and causes cell damage; and / or interaction of the drug molecule with the cell membrane. Non-limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, daunorubicin, doxorubicin, epirubicin, nemorubicin, valrubicin, and mitoxantrone, and their derivatives. For example, PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al., Clin Cancer Res 11(4):1608-1617, 2005). Nemorubicin is a semisynthetic analog of doxorubicin that has a 2-methoxymorpholino group at the glycoside amino of doxorubicin (Grandi et al., Cancer Treat Rev 17:133, 1990; Ripamonti et al., Br J Cancer 65:703-707, 1992).

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

[0220] In some cases, the linker has a functional group that can react with a free cysteine present on the antibody to form a covalent bond. Exemplary linkers with such reactive functional groups include maleimides, haloacetamides, α-haloacetyls, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

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

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

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

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

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

[0226] Bispecific killer cell engagers (BiKEs) are a type of bispecific monoclonal antibody that is a fusion of a first monoclonal antibody (e.g., scFv or single domain antibody) that targets a tumor antigen (e.g., B7H3) and a second scFv that binds an NK cell activating receptor, e.g., CD16.

[0227] Provided herein are multispecific, e.g., trispecific or bispecific, monoclonal antibodies, including B7H3-specific monoclonal antibodies. In some embodiments, the multispecific monoclonal antibody further comprises a monoclonal antibody that specifically binds a component of a T cell receptor, e.g., CD3. In other embodiments, the multispecific monoclonal antibody further comprises a monoclonal antibody that specifically binds an NK cell activating receptor, e.g., CD16, Ly49, or CD94. Also provided are isolated nucleic acid molecules and vectors encoding the multispecific antibodies, as well as host cells containing the nucleic acid molecules or vectors. Multispecific antibodies, including B7H3-specific antibodies, can be used to treat cancers that express B7H3. Thus, provided herein are methods for treating a subject with cancer by selecting a subject with a B7H3-expressing cancer and administering to the subject a therapeutically effective amount of a multispecific antibody that targets B7H3. VIII. Antibody-Nanoparticle Conjugates

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

[0229] Liposomes are a common type of nanoparticle 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 phospholipid is composed of a hydrophilic head group and two hydrophobic chains, allowing for the encapsulation of both hydrophobic and hydrophilic drugs. Conventional liposomes are rapidly cleared from the circulation by macrophages of the reticuloendothelial system (RES). The composition, size, and charge of the liposome can be modulated to generate long-circulating liposomes. The surface of the liposome can also be modified, for example, with glycolipids or sialic acid. For example, the inclusion of polyethylene glycol (PEG) significantly increases circulation half-life. Liposomes for use as drug delivery agents, including for the preparation of immunoliposomes, have been described in the art (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; and 2010 / 00329981).

[0230] Niosomes are nonionic surfactant-based vesicles with a structure similar to that of liposomes. Their membranes consist solely of nonionic surfactants, such as polyglyceryl alkyl ethers or N-palmitoyl glucosamine. Niosomes range from small unilamellar particles to large multilamellar particles. These nanoparticles are monodisperse, water-soluble, chemically stable, low in toxicity, biodegradable, and non-immunogenic, increasing the bioavailability of encapsulated drugs.

[0231] Dendrimers include a variety of branched polymer conjugates. These nanoparticles are water-soluble, biocompatible, and sufficiently non-immunogenic for human use. Dendrimers generally consist of an initiator core surrounded by layers of selected polymers grafted onto the core, forming a branched macromolecular complex. Dendrimers are typically produced using polymers such as poly(amidoamine) or poly(L-lysine). Dendrimers have been used for a variety of therapeutic and diagnostic applications, including delivery of DNA, RNA, bioimaging contrast agents, and chemotherapeutic agents.

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

[0233] Polymeric nanoparticles include both nanospheres and nanocapsules. Nanospheres consist of a solid matrix of polymer, while nanocapsules contain an aqueous core. The formulation chosen typically 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, 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(ester), poly(alkyl cyanoacrylate), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(D,L-lactic-co-glycolic acid) (PLGA).

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

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

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

[0237] A typical pharmaceutical composition for intravenous administration contains about 0.1 to 10 mg of antibody (or ADC, CAR, multispecific antibody, antibody-nanoparticle conjugate, or immunoconjugate) per subject per day. Dosages of 0.1 up to about 100 mg per subject per day may be used, particularly if the agent is administered to an isolated site and not into the circulation or lymphatic system, such as into a body cavity or lumen of an organ. Actual methods for preparing administrable compositions will be known or apparent to those of skill in the art and may be found in, for example, Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st This is described in more detail in publications such as Edition (2005).

[0238] The monoclonal antibodies disclosed herein may also be administered by other routes, including via inhalation, orally, topically, or intraocularly. In some examples, the monoclonal antibody (or conjugate thereof) is administered by microneedle.

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

[0240] Controlled-release parenteral formulations may be made as implants, oily injections, or microparticle systems. For a review of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Microparticle systems include, for example, microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Because capillaries have a diameter of approximately 5 μm, only nanoparticles are administered intravenously. Microparticles are typically approximately 100 μm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0241] Polymers can be used for ion-controlled release of the antibody-based compositions disclosed herein. A variety of degradable and non-degradable polymer matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer polaxamer 407 exists as a viscous but mobile liquid at low temperatures, but forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm.112:215-224, 1994). In yet another embodiment, liposomes are used for the controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496). A. Treatment method

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

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

[0244] A therapeutically effective amount of a B7H3-specific monoclonal antibody, ADC, CAR (e.g., a CTL expressing a CAR), multispecific (such as bispecific or trispecific) antibody, immunoconjugate, immunoliposome, or composition disclosed herein will vary depending on the severity of the disease, the type of disease, and the general state of the patient's health. A therapeutically effective amount of an antibody-based composition is an amount that provides a subjective relief of symptom(s) or an objectively identifiable improvement as noted by a physician or other qualified observer.

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

[0246] Administration of the B7H3-specific antibodies, ADCs, CARs (or cells expressing CARs), immunoconjugates, multispecific antibodies, antibody-nanoparticle conjugates, immunoliposomes, and compositions disclosed herein may also be accompanied by administration of other anti-cancer agents or therapeutic treatments (e.g., surgical resection of tumors). Any suitable anti-cancer agent may be administered in combination with the antibodies, compositions, and immunoconjugates disclosed herein. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormonal agents (e.g., anti-androgens), and anti-angiogenic agents. Other anti-cancer treatments include radiation therapy and other antibodies (e.g., biologics) that specifically target cancer cells.

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

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

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

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

[0251] Non-limiting examples of hormones and antagonists include corticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include adriamycin, alkeran, Ara-C, BiCNU, busulfan, CCNU, carboplatinum, cisplatinum, cytoxan, daunorubicin, DTIC, 5-FU, fludarabine, hydrair, idarubicin, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, taxol (or other taxanes, e.g., docetaxel), velban, vincristine, VP-16, although some newer drugs include gemcitabine (Gemzar), Herceptin, irinotecan (Camptosar, CPT-11), leustatin, navelbine, Rituxan STI-571, taxotere, topotecan (Hycamtin), Xeloda (capecitabine), Zevelin, and calcitriol.

[0252] Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte-macrophage colony-stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), IMREG (from Imreg, New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0253] Non-limiting examples of biologics that can be used in combination with the disclosed B7H3-specific antibodies, ADCs, CARs (or cells expressing CARs), immunoconjugates, multispecific antibodies, antibody-nanoparticle conjugates, immunoliposomes include therapeutic monoclonal antibodies, such as 3F8, abagovomab, adecatumumab, afutuzumab, alacizumab, alemtuzumab, altumomab pentetate, anatumomab mafenatox, apolizumab, arcitumomab, bavituximab, bectumomab, belimumab, bezilesomab, and bevacizumab. , bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromab pendetide, catumaxomab, CC49, cetuximab, sitatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan, conatumumab, dacetuzumab, detumomab, ecromeximab, eculizumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab, galiximab, gemtuzumab ozogamicin, girentuximab, glembatumumab vedotin, eve Ritumomab tiuxetan, igovomab, imciromab, intetumumab, inotuzumab ozogamicin, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab mertansine, lucatumumab, rumiliximab, mapatumumab, matuzumab, mepolizumab, metelimumab, milatuzumab, mitumomab, morolimumab, nacolomab butafenatox, naptumomab estafenatox, necitumumab, nimotuzumab, nofetumomab merpentan, ofatumumab, olaratumumab, oportuzumab monatox, oleic and one or more of govomab, panitumumab, pemtumomab, pertuzumab, pintumomab, pritumumab, ramucirumab, rilotumumab, rituximab, lobatumumab, satumomab pendetide, sibrotuzumab, sonepcizumab, tacatuzumab tetraxetan, taplitumomab paptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, trastuzumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab, volociximab, votumumab, and zalutumumab.In some examples, the therapeutic antibody specifically binds and antagonizes PD-1 or PD-L1, such as one or more of atezolizumab, MPDL3280A, BNS-936558 (nivolumab), pembrolizumab, pidilizumab, CT011, AMP-224, AMP-514, MEDI-0680, BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, MSB-0010718C, MGA-271, Indoximod, Epacadostat, BMS-986016, MEDI-4736, MEDI-4737, MK-4166, BMS-663513, PF-05082566 (PF-2566), lirilumab, and durvalumab.

[0254] In some examples, the additional therapeutic agent 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, such as 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, such as a mAb. Specific agonist mAbs that can be used with the disclosed methods include PF-05082566 (utomilumab) and BMS-663513 (urelumab). In one example, the 4-1BB agonist is a 4-1BB ligand (4-1BBL), such as a natural 4-1BBL (such as human 4-1IBBL) or a streptavidin-conjugated 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, such as a mAb. Specific 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 natural GITRL or a multivalent GITR ligand fusion protein. In one example, the GITR agonist is MEDI1873, a hexameric GITRL molecule with a human IgG1 Fc domain. In some examples, the additional therapeutic agent administered is an immunotherapeutic agent.Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte-macrophage colony-stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), IMREG (from Imreg, New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

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

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

[0257] In other cases, B7H3 expression is detected in biological samples. Samples can be any samples, including but not limited to tissues from biopsies, autopsies, and pathology specimens. Biological samples also include tissue sections, such as frozen sections taken for histological purposes. Biological samples can also include bodily fluids, such as blood, serum, plasma, sputum, spinal fluid, or urine. In some cases, the sample is a serum sample containing exosomes. Biological samples are typically obtained from mammals, such as humans or non-human primates.

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

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

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

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

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

[0263] In some examples, the control sample is a sample from a subject who does not have cancer. In particular examples, the sample is a blood or tissue sample.

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

[0265] Suitable labels for antibodies or secondary antibodies include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials 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 a non-limiting exemplary radioactive label is125 I, 131 I, 35 S or 3 Contains H.

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

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

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

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

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

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

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

[0273] Example 1 material and method This example describes the materials and experimental procedures used in the study described in Example 2. cell line

[0274] Eight cancer cell lines (Hep3B, HepG2, IMR32, MC38-B7H3+, A431, IMR32-B7H3 KO, and MC38-B7H3 KO) were cultured in DMEM medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 1% L-glutamine, and 1% penicillin-streptomycin (Invitrogen) and incubated at 37°C in 5% CO2 with the balance air. The neuroblastoma cell line NBEB was cultured in RPMI-1640 medium containing the same supplements as DMEM. The medium was refreshed twice a week. Protein expression and purification

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

[0276] To amplify the rabbit VH cDNA fragment, forward and reverse primers annealing to the 5' and 3' ends of the VH cDNA were used as described in Peng et al. The primers were synthesized according to the method described in

[1999] . The primers are listed in Table 1, and the underlined nucleotides correspond to the SfiI restriction enzyme site. Table 1: Primers for construction of rabbit VH phage library [Table 1-1] [Table 1-2]

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

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

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

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

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

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

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

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

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

[0286] After confirming the success of immunization, the spleens of the immunized rabbits were collected, and the VH gene fragments were cloned using degenerate primers (Peng et al., J Mol Biol, 429, 2954-2973, 2017), which was then ligated with the phage display vector pComb3x. Ten micrograms of the ligation was used to transform TG1 competent cells by electroporation, and 7 × 10 9 A VH library was constructed with individual clones of this size. Both library phage generation and subsequent antigen panning were performed at 37°C.

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

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

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

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

[0291] Although whole rabbit monoclonal antibodies have been widely used as excellent research reagents for many years, the potential advantages of rabbit VH domain antibodies have not been fully exploited. Recently, one group demonstrated that high-affinity rabbit VH domain antibodies can be generated by low-temperature (i.e., 16°C) phage display (Shinozaki et al., Sci Rep, 7, 5794, 2017), which will greatly accelerate research into rabbit VH domain antibodies. However, low-temperature phage display methods tend to enrich a significant portion of unstable and difficult-to-express binders, which may require considerable efforts to improve their physicochemical properties, particularly expression and thermal stability (Shinozaki et al., J Biosci Bioeng, 125, 654-661, 2018). The current study explored the possibility of using high-temperature phage display methods to screen for thermostable and well-expressed binders.

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

[0293] This example shows 10 camel V from a phage display library. H We describe the selection of H nanobodies and the characterization of their binding properties. H Chimeric antigen receptors composed of H nanobodies are also described. 8 Camel V on B7H3 binders H Phage panning with the H library

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

[0295] Eight Vs made from eight camel (Camelus dromedaries) specimens H Using the H single domain antibody library, phage panning was performed three times on the recombinant B7H3-Fc protein. The phage titers for each round are shown in Figure 6. The increase in phage titer in the third round of phage panning indicates that the phage have high affinity for B7H3. H Phage binding to B7H3-Fc was also assessed by ELISA; the results are shown in Table 2. All of the selected phages were able to bind to B7H3-Fc but not to the IgG control. Table 2: Selected phage binding on B7H3-Fc by ELISA [Table 2-1] [Table 2-2]

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

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

[0298] Selected Vs in hB7H3-Fc, hB7H3-His, mouse B7H3-His, monkey B7H3-His, and rat B7H3-His fusion proteins H Binding of the H nanobodies was measured by ELISA. The results are shown in Table 4 and Figure 22. With the exception of RWB2, V H H was able to bind to B7H3-Fc and B7H3-His fusion proteins. RWG8 showed cross-reactivity to mouse B7H3 (Figure 22). Furthermore, RWA12 showed cross-reactivity with PBS and PD-L1, and the remaining V H H did not cross-react with PDL1 (Table 5). Table 4: Selected Vs in B7H3 fusion proteins by ELISA H H bond [Table 4] Table 5: Cross-reactivity of selected B7H3 binders with PD-L1 by ELISA [Table 5] Selected B7H3-targeted V to B7H3-expressing cells H H bond

[0299] Selected B7H3-targeted V H Binding of H nanobodies to NBEB neuroblastoma cells was assessed by FACS analysis (Figure 10). Six of the tested antibodies (RWC4, RWB12, RWG8, RWA12, RWG4 and RWD5) were able to bind NBEB cells.

[0300] Selected B7H3-targeted V H Binding of H nanobodies to A431 epidermoid carcinoma cells was assessed by FACS analysis (Figure 11). Six of the tested antibodies (RWC4, RWB12, RWG8, RWA12, RWG4 and RWD5) were able to bind A431 cells.

[0301] Five of the B7H3-targeting antibodies (RWC4, RWB12, RWG8, RWG4, and RWD5) were also evaluated for their ability to bind MC38-CD276+ and MC38-CD276KO cells. All tested antibodies bound to B7H3-positive cells but not to B7H3-negative cells (see Table 6). Table 6: The Five Vs H Overview of cell binding ability of H [Table 6] Binding kinetics

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

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

[0304] The kinetics of RWG4 on human B7H3-Fc are shown in Figure 13 and summarized in Table 8. K D is 6.94 x 10 -9 It was. Table 8: Kinetics of RWG4 with human B7H3-Fc [Table 8] B7H3 expression in various cancer cell lines

[0305] Several cancer cell lines were evaluated for B7H3 expression by FACS, and expression was detected in 14 cell lines but not in two cell lines in which B7H3 (CD276) was knocked out (see Table 9). Table 9: Summary of B7H3-expressing cells [Table 9-1] [Table 9-2] Generation of B7H3-targeting CAR

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

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

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

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

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

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

[0312] The second in vivo study evaluated the treatment of Panc-1 tumor-bearing mice with lower doses of B7H3-specific CAR T cells injected after tumor rechallenge. A schematic diagram of the experimental design for this study is shown in Figure 19A. Panc-1 xenografted mice were intravenously injected with 5 million C4 CAR T cells, 5 million B12 CAR T cells, 5 million untransduced T cells (mock), or PBS 20 days after inoculation of 1 million Panc-1-Luc cells (day 0). Mice treated with C4 CAR T cells and B12 CAR T cells that did not exhibit detectable tumors were intravenously transplanted with 1 million Panc-1 cells on day 35. As a control, naive mice were transplanted with Panc-1 cells. Imaging was performed weekly. Representative bioluminescence images of Panc-1 tumor growth in mice treated with CAR T cells are shown in Figure 19B. Mice treated with 5 million C4 CAR T cells or 5 million B12 CAR T cells showed significantly reduced tumor growth compared with mice administered mock T cells or PBS. While tumors grew rapidly in control mice, 100% of mice previously treated with C4 CAR T cells remained tumor-free after rechallenge with Panc-1 tumors, and 60% of mice previously treated with B12 CAR T cells remained tumor-free up to 10 weeks after treatment. Quantitation of tumor bioluminescence is shown in Figure 19C. The survival time of mice treated with B7H3-targeting CAR T cells was also determined. Figure 19D shows Kaplan-Meier survival curves of tumor-bearing mice after treatment. Mice receiving 5 million C4 or B12 CAR T cells were still alive at day 70. In contrast, mice treated with PBS or mock T cells did not survive more than 30 days after injection. Example 5 B7H3-targeted CAR T cells kill neuroblastoma tumor cells in vitro and in vivo

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

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

[0315] The binding activity of anti-B7H3 nanobodies G8, C4, and B12 to mouse B7H3 was measured by flow cytometry. G8, but not C4 or B12, showed positive binding to mouse B7H3 expressed on three KPC cell lines (CREP128096, CREP133239, and PDA95775; pancreatic ductal adenocarcinoma cells) and the mouse melanoma cell line B16. The in vitro cytotoxicity of B7H3-targeted CARs was evaluated using B16 melanoma cells and B7H3 (CD276) knockout cells. Only G8 CAR T cells showed specific killing of mouse B7H3-positive B16 cells (Figure 21B). Example 7 Epitope mapping of B7H3 nanobodies

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

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

Claims

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

2. the CDR1, CDR2 and CDR3 sequences are each residues 31-35, 50-66 and 97-118 of SEQ ID NO:1; Residues 26-33, 51-58, and 97-119 of SEQ ID NO:1; or Residues 27-35, 47-62 and 98-118 of SEQ ID NO:1 The antibody of claim 1, comprising:

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

1.

4. 4. The antibody of claim 1, wherein the amino acid sequence of the antibody comprises or consists of SEQ ID NO:

1.

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

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

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

8. 8. The CAR of claim 7, further comprising a hinge region, a transmembrane domain, a costimulatory signaling moiety, a signaling domain, or any combination thereof.

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

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

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

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

13. The immunoconjugate of claim 12 , wherein the effector molecule is a toxin.

14. 14. The immunoconjugate of claim 13, wherein the toxin is a Pseudomonas exotoxin or a variant thereof.

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

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

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

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

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

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

21. 21. The ADC of claim 19 or claim 20, wherein the drug is a microtubule inhibitor, an antimitotic agent and / or a cytotoxic agent.

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

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

24. 23. The multispecific antibody of claim 22, which is a trispecific antibody.

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

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

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

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

29. An antibody according to any one of claims 1 to 6, and heterologous proteins or peptides, A fusion protein comprising:

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

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

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

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

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

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

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

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

38. 37. The method of claim 36, wherein the antibody is directly labeled.

39. The method comprises: contacting the antibody with a detection antibody; detecting binding of the detection antibody to the antibody, thereby detecting expression of B7H3 in the sample; 37. The method of claim 36, further comprising:

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

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

42. 38. The composition of claim 37, wherein the antibody is directly labeled.

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

38. The composition of claim 37, further comprising:

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

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

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

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

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

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

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

51. 50. The composition of claim 49, wherein the brain cancer is neuroblastoma or glioblastoma.

52. 50. The composition of claim 49, wherein the childhood cancer is osteosarcoma, neuroblastoma, rhabdomyosarcoma, or Ewing's sarcoma.

Images