High affinity nanoantibodies targeting B7H3 (CD276) for treatment of various solid tumors

Abstract

The present invention describes single domain monoclonal antibodies that specifically bind to B7H3 (also known as CD276). The single-domain monoclonal antibody comprises a camel VHH domain nano antibody and a rabbit VH domain nano antibody which are selected from a phage display library. B7H3-targeted chimeric antigen receptors (CARs) and other antibody conjugates are also described. The single-domain monoclonal antibody and the conjugate thereof can be used for diagnosing and treating solid tumors expressing B7H3.

Description

[0001] This application is a divisional application with application date of October 21, 2020, application number 202080074414.2, and titled “High-affinity nanoantibodies targeting B7H3 (CD276) for the treatment of multiple solid tumors”.

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 924,298, filed on October 22, 2019, which is incorporated herein by reference in its entirety. Technical Field

[0004] The present disclosure relates to single chain monoclonal antibodies that bind B7 homolog 3 (B7H3) with high affinity. The present disclosure further relates to uses of the monoclonal antibodies and antibody conjugates, for example, for treating solid tumors.

[0005] Government Support Statement

[0006] This invention was made with government support under Project Z01 BC010891 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Background Art

[0007] Nanobodies are the smallest known antibody antigen-binding fragments, consisting of approximately 120 amino acids, with a molecular weight of 12kD to 15kD and a size of approximately 4×2.5nm (Khodabakhsh et al., Int Rev Immunol, 37, 316-322, 2018). Most of the well-studied nanobodies are derived from the heavy chain variable region (VH), which can naturally exist in camelids (called V H H) and cartilaginous fish (V NAR) (Feng et al., Antib Ther, 2, 1-11, 2019), and exists in vivo in some human heavy chain diseases (Prelli and Frangione, J Immunol, 148, 949-952, 1992). Nanobodies can be used for medical applications or as research tools due to their small size, high solubility, good thermal stability, reversible refolding ability, and relative ease of penetrating tissues in vivo compared to conventional intact IgG (Hodabakhsh et al., International Reviews in Immunology, 37, 316-322, 2018; Wesolowski et al., Med Microbiol Immunol, 198, 157-174, 2009; Ho, Antibody Therapeutics, 1, 1-5, 2018). The first nanobody drug, caplacizumab, This is demonstrated by the approval of a humanized camelid V H H, for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) and thrombosis (Elverdi and Eskazan, Drug Des Devel Ther, 13, 1251-1258, 2019; Scully et al., N Engl J Med, 380, 335-346, 2019).

[0008] Historically, Camelidae V H H domain antibodies have been the dominant direction of research until the rise 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). In the serum of camelids, there are both conventional IgG and a large number of heavy chain-only IgG (HCAb), accounting for 45% to 75% of all serum immunoglobulins, depending on the specific species (Hodabakhsh et al., International Reviews in Immunology, 37, 316-322, 2018). HCAb consists of V H The recombinant VH fragment consists of a single antigen-binding domain, followed by the CH2 and CH3 domains, and a light chain that pairs with the VH-CH1 domain in conventional IgG. H The H domain is a functional entity under development.

[0009] Several structural features have allowed the naturally evolved camelid V H The H domain is highly soluble and stable. First, Val37 (Kabat numbering) in the human VH germline is usually V H Phe37 (or Tyr37) in the H domain (Riechmann and Muyldermans, J Immunol Methods, 231, 25-38, 1999), which makes the hydrophobic packing of the domain more compact and stable, may be the reason for V H The driving factor for the particular stability of VH (Shinozaki et al., J Biosci Bioeng, 125, 654-661, 2018). Second, the light chain contact residues G44, L45, and W47 in the human VH germline are V H E44 (or Q44), R45 (or C45) and G47 (or Ser, Leu, Phe) in H (Holt et al., Trends Biotechnol, 21, 484-490, 2003), which makes the accessible surface area more hydrophilic and less aggregated. In addition, in some V H In the H domain, W103 can be replaced by R103. H The CDR3 of the H domain is usually longer than that of human / rodent CDR3, and they usually contain a Cys in CDR3 that forms an additional disulfide bond with the Cys at the end of CDR1 (camel) or the beginning of CDR2 (llama) (Wesolovsky et al., Medical Microbiology and Immunology, 198, 157-174, 2009). This additional CDR3 disulfide bond and the typical C22-C92 disulfide bond further make the V H The H domain is more stable (T m The value range is 60-78°C) and reversibly unfolds / refolds (Holt et al., Trends in Biotechnology, 21, 484-490, 2003).

[0010] The first non-V H In 1989, H mammalian domain antibodies were screened from a cDNA expression library made from the spleen of mice immunized with lysozyme and keyhole limpet-hemocyanin, in which two mouse VH domains showed affinity for lysozyme in the range of 20 nM (Ward et al., Nature 341, 544-546, 1989), and the name "single domain antibody" (dAbs) was first proposed. HH, has promoted the idea of ​​exploring human VH domain antibodies, which are more attractive for pharmaceutical and other applications. Although both camelid domain antibodies and human domain antibodies have been intensively studied, there are some technical limitations in terms of access to resources, especially for discovering domain antibodies by immunization. To overcome the limitations of resource accessibility, several strategies have been developed to generate human or humanized VH domain antibodies. Although the affinity is not always as high as that of immunized antibodies, the original Phage display of human VH domain libraries is a validated method (Feng et al., Proc. Natl. Acad. Sci. USA, 110, E1083-1091, 2013; Tang et al., Molecular Cancer Therapy, 12, 416-426, 2013; Li et al., Proc. Natl. Acad. Sci. USA, 114, E6623-E6631, 2017). Another method for generating human domain antibodies is to use transgenic animals with human VH germline genes (Schusser et al., J. Immunol., 46, 2137-2148, 2016; Janssens et al., Proc. Natl. Acad. Sci. USA, 103, 15130-15135, 2006). Rather than isolating human domain antibodies directly, generating VH domain antibodies from immunized animals and then humanizing them is also a method for generating high affinity human-like antibodies. Summary of the invention

[0011] The present disclosure describes ten camelid single domain V proteins that specifically bind to B7H3 (also known as CD276). H H monoclonal antibody and two rabbit VH single domain antibodies. B7H3-specific camel antibodies (referred to herein as RWB12 ("B12"), RWG8 ("G8"), RWC4 ("C4"), RWB2, RWH5, RWD5, RWC3, RWG4, RWD9 and RWH1), and B7H3-specific rabbit antibodies (referred to herein as RFA1 and RFB1) bind to B7H3 with high affinity. It is also disclosed to produce chimeric antigen receptor (CAR) T cells composed of the disclosed nanobodies.

[0012] Provided herein are monoclonal antibodies that bind (e.g., specifically bind) B7H3. In some embodiments, the monoclonal antibody includes complementary determining region (CDR) sequences of nano antibodies RWB12, RWG8, RWC4, RWB2, RWH5, RWD5, RWC3, RWG4, RWD9, RWH1, RFA1, or RFB1. Also provided herein are conjugates including disclosed monoclonal antibodies. In some instances, there are provided CARs (and T cells and natural killer cells expressing CAR) including monoclonal antibodies disclosed herein, immunoconjugates (e.g., immunotoxins), multispecific antibodies (e.g., bispecific T cell conjugates), antibody-drug conjugates (ADC), antibody-nanoparticle conjugates, antibody-radioisotope conjugates (e.g., for cancer diagnosis and immune PET imaging) and fusion proteins.

[0013] The present disclosure also provides a composition comprising a B7H3-specific monoclonal antibody and a pharmaceutically acceptable carrier.

[0014] Also provided herein are nucleic acid molecules and vectors encoding B7H3-specific monoclonal antibodies, CARs, immunoconjugates (e.g., immunotoxins), multispecific antibodies, and fusion proteins disclosed herein. Further provided are isolated cells comprising nucleic acids or vectors encoding B7H3 monoclonal antibodies or CARs.

[0015] Also provided herein are methods for treating B7H3-positive cancers in subjects, and methods for inhibiting tumor growth or metastasis of B7H3-positive cancers in subjects. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a monoclonal antibody disclosed herein, or administering to the subject a therapeutically effective amount of a CAR (or CAR T cell or CAR NK cell) comprising a monoclonal antibody disclosed herein, an immunoconjugate (e.g., an immunotoxin), ADC, a multispecific antibody, an antibody-nanoparticle conjugate, or a fusion protein.

[0016] Further provided herein are methods for detecting B7H3 expression in a sample. In some embodiments, the method comprises contacting the sample with a monoclonal antibody disclosed herein, and detecting binding of the antibody to the sample.

[0017] Also provided herein are methods of diagnosing a subject with a B7H3-positive cancer. In some embodiments, the method comprises contacting a sample obtained from the subject with a monoclonal antibody disclosed herein, and detecting binding of the antibody to the sample.

[0018] The foregoing and other objects and features of the present disclosure will become more apparent from the following detailed description with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A : Schematic diagram of immunizing rabbits with recombinant B7H3, generating VH domain phage display libraries, and selecting B7H3 binders.

[0020] Figure 1B : SDS-PAGE analysis of purified B7H3-hFc. 1 μg or 5 μg of purified B7H3-hFc (non-reduced (Non.) or β-mercaptoethanol reduced (Red.)) were separated on 8% SDS-PAGE gel. Protein bands were observed by Coomassie Blue R-250 staining.

[0021] Figure 2A-2B :Titration of B7H3-hFc immune rabbit serum and confirmation of its cell binding. Figure 2A )B7H3-hFc immunized rabbit sera were titrated by protein binding ELISA. IAB-hFc is derived from the N-terminal fragment of mesothelin and is used as an hFc tag control. R31M0, R31M1, R31M2, and R31M3 represent sera from pre-immunization, 1st, 2nd, and 3rd immunization, respectively. Figure 2B ) Cell binding assay with immune serum. The shaded area represents cells stained with FITC-conjugated secondary antibody (goat-anti-rabbit) only; left curve, cells stained with pre-immune serum; right curve, cells stained with immune serum R31M3.

[0022] Figure 3A-3B :Sequence and structural modeling of B7H3 binders. Figure 3A ) Sequence alignment of B7H3 binder (A1, SEQ ID NO: 11; B1, SEQ ID NO: 12) and similar VH of rabbit anti-carboxyputrescine lysine monoclonal antibody (PDB#5DUB, SEQ ID NO: 34). CDR regions are defined by the IMGT delineation system. ( Figure 3B ) The structure modeling of A1 and B1 binders was performed using the online software SWISS-MODEL. The crystal structure of 5DUB is also shown as a control.

[0023] Figures 4A-4C :Binding properties of B7H3 conjugates. ( Figure 4A )SDS-PAGE analysis of A1 and B1 conjugates (VH-His-FLAG fusion) purified from E. coli. Two micrograms of purified B7H3-hFc (non-reduced (Non.) or β-mercaptoethanol reduced (Red.)) were separated on 8% SDS-PAGE gel. Protein bands were visualized by Coomassie Blue R-250 staining. ( Figure 4B) Protein binding affinity was measured by ELISA. 5 μg of B7H3-hFc was coated on an ELISA plate, and different concentrations of domain antibodies were incubated with the ELISA plate, and binding was detected with HRP-conjugated anti-FLAG mouse monoclonal antibody M2. Binding curves were drawn using the software GraphPad Prism, and the calculated EC50 values ​​were determined by the hyperbolic single-point binding algorithm of the software. ( Figure 4C ) Cell binding was determined by flow cytometry. 10 micrograms of domain antibody per ml was incubated with 1 million cells. Antibody binding was visualized by APC-conjugated anti-FLAG monoclonal antibody. The curve labeled "1" represents cells stained with secondary antibody only; the curve labeled "2" represents cells stained with A1 or B1 domain antibody.

[0024] 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 AKTA Explorer (GE Healthcare). The purified B7H3-Fc fusion protein was over 99% pure on SDA-PAGE gel, with molecular weights of 154 kDa and 77 kDa under non-reducing conditions (left) and reducing conditions (right), respectively. The yield of B7H3-Fc was 2 mg / L. Glypican 2 (GPC2)-Fc was used as a control protein.

[0025] Figure 6 : Eight camel V binding sites against B7H3 H Phage panning was performed on the H library. Eight V libraries made from eight camels (dromedary camels) were used. H H single domain antibody library, and three rounds of phage panning were performed against the recombinant B7H3-Fc protein. The phage titers of each round are shown in the figure. The increase in phage titers in the third round of phage panning indicates that V has high affinity for B7H3. H Enrichment of H conjugates.

[0026] Figure 7A-7B : RWC4 V H Purification of H nanobodies. Fig. 7A ) RWC4 V eluted from AKTA Explorer (GE Healthcare) H H SDS-PAGE of camel nanobody fractions. Figure 7B ) Chromatogram of nanobody elution on a nickel column (GE Healthcare) on an AKTA Explorer (GE Healthcare). The yield of RWC4 was 33.8 mg / L.

[0027] Figures 8A-8B :RWG8 V H Purification of H nanobodies. Fig. 8A ) RWG8 V eluted from AKTA Explorer (GE Healthcare) H H SDS-PAGE of camel nanobody fractions. Figure 8B ) Chromatogram of RWG8 nanobody elution on a nickel column (GE Healthcare) on an AKTA Explorer (GE Healthcare). The yield of RWG8 was 50 mg / L.

[0028] Figures 9A-9B : RWB12 V H Purification of H nanobodies. Fig.9A ) RWB12 V eluted from AKTA Explorer (GE Healthcare) H H SDS-PAGE of camel nanobody fractions. Fig. 9B ) Chromatogram of RWB12 nanobody eluted on a nickel column (GE Healthcare) on an AKTA Explorer (GE Healthcare). The yield of RWB12 was 132 mg / L.

[0029] Fig.10 : Selected B7H3-targeted V H Binding of H nanobodies to NBEB neuroblastoma cells. Binding of the B7H3-specific nanobodies was assessed by FACS analysis. Six test antibodies (RWC4, RWB12, RWG8, RWA12, RWG4 and RWD5) were able to bind to NBEB cells.

[0030] Fig.11 : Selected B7H3-targeted V H Binding of H nanobodies to A431 epidermoid carcinoma cells. The binding of the B7H3-specific nanobodies was assessed by FACS analysis. Six test antibodies (RWC4, RWB12, RWG8, RWA12, RWG4 and RWD5) were able to bind to A431 cells.

[0031] Fig.12 : Kinetics of RWC4 towards human B7H3-Fc. The association / dissociation properties of RWC4 were measured on the Octet system (Creative Biolabs) using recombinant human B7H3-Fc protein. The K of RWC4 D =3.8 nM, K of RWC4 on =2.65×10 4 , RWC4's K dis =1.01×10 -4 .

[0032] Fig.13 : Kinetics of RWG4 towards human B7H3-Fc. The association / dissociation properties of RWG4 were measured on the Octet system (Creative Biolabs) using recombinant human B7H3-Fc protein. The K of RWG4 D =6.94nM, K of RWG4 on =9.13×10 3 , K of RWG4 dis =6.34×10 -5 .

[0033] Fig.14 : T cell transfection efficiency of lentivirus expressing B7H3-targeted CAR. Transfection efficiency was measured by FACS.

[0034] Figures 15A-15D :Cytotoxicity of CAR-T cells targeting B7H3 in B7H3-positive cells. Fig.15A ) Human neuroblastoma NBEB cells. ( Fig. 15B ) Human neuroblastoma LAN-1 cells. ( Fig. 15C ) Human adenocarcinoma BXPC-3 cells. Fig.15D ) Human pancreatic cancer Miacapa2 cells. RWB12, RWG8, and RWC4 CAR-T cells were most effective in inducing specific lysis.

[0035] Figures 16A-16D :Cytotoxicity of CAR-T cells targeting B7H3 in B7H3-positive cells and B7H3-knockout cells. Fig.16A ) Human neuroblastoma IMR32 cells. ( Fig. 16B ) Mouse colon adenocarcinoma MC38-CD276 + cell.( Fig. 16C ) Human neuroblastoma IMR32-CD276 - / - cell.( Fig.16D ) Mouse colon adenocarcinoma MC38-CD276 - / - Three of the described CAR T cells (RWB12, RWG8, and RWC4) exhibited potent cytotoxicity against B7H3-positive cells, but not against B7H3-negative cells.

[0036] Figures 17A-17C : Cytotoxicity of B7H3 CAR T cells against pancreatic cancer cells. The cytotoxicity of human B7H3-targeted nanobody-derived CAR T cells (B12, G8, and C4) was evaluated using two B7H3-positive pancreatic cancer cell lines expressing luciferase: Panc-1 GFP-Luc(GL) and BxPC-3GL. Fig.17A Flow cytometry showed that both Panc-1 and BxPC-1 were B7H3 positive cell lines. Fig. 17B ) The T cell transduction efficiencies of G8, C4, and B12 CARs were 60.4%, 58.6%, and 68.4%, respectively. The transduction efficiency of unrelated CAR (CD19) T cells was 32%. ( Fig. 17C ) B7H3-targeted CAR (G8 / B12 / C4) T cells as well as irrelevant control CAR (CD19) T cells were cultured with Panc-1 GL cells or BxPC-3GL cells at different effector:target (E:T) ratios for 24 hours. Both Panc-1 GL and BxPC-3GL cells were efficiently lysed by all three B7H3-targeted CAR T cells in a dose-dependent manner, while minimal killing was observed with control CAR T cells.

[0037] Figures 18A-18D : Panc-1 mouse model treated with high-dose human B7H3-specific CAR T cells. Tumor regression in the Panc-1 xenograft mouse model after infusion of 10 million B7H3-targeted CAR T cells was evaluated. Fig.18A ) Schematic diagram of 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), mice were infused intravenously with 10 million C4, G8, or B12 CAR T cells (or control CD19 CAR T cells). Imaging was performed weekly. Fig.18B ) Representative bioluminescent images of Panc-1 tumor growth in CAR T cell-treated mice. Mice treated with 10 million B7H3-targeted CAR T cells (C4, G8, or B12) had significantly slower tumor growth compared to mice infused with control CAR T cells. ( Fig.18C )exist Fig.18B Tumor bioluminescence was quantified as photons per second in the indicated mice. Fig.18D ) Kaplan-Meier survival curves of tumor-bearing mice after treatment with 10 million C4, G8 or B12 CART cells. The results showed that when administered at a high dose (10 million), C4 CAR T cells promoted mouse survival more effectively than G8 or B12 CAR T cells, and indicated that the administration of 10 million CAR T cells was safe for mice.

[0038] Figures 19A-19D: Panc-1 mouse model treated with low-dose human B7H3-specific CAR T cells. Tumor regression in the Panc-1 xenograft mouse model following infusion of 5 million B7-H3-targeted CAR T cells was measured after tumor re-challenge. Fig.19A ) Schematic diagram of the experiment. Panc-1 xenograft mice were infused with 5 million C4 CAR T cells, B12 CAR T cells, untransduced T cells (mock), or PBS by intravenous injection 20 days after inoculation with 1 million Panc-1-Luc cells (day 0). Mice treated with C4 CAR T cells and B12 CAR T cells that did not show detectable tumors were implanted with 1 million Panc-1 cells by intravenous injection on day 35. As a control, blank mice were implanted with Panc-1 cells. Imaging was performed once a week. ( Fig.19B ) Representative bioluminescent 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 had significantly slower 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 Panc-1 tumor re-challenge, and 60% of mice previously treated with B12 CAR T cells remained tumor-free 10 weeks after treatment. Fig.19C )exist Fig.19B Tumor bioluminescence was quantified as photons per second in the mice shown in ( Fig.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 infusion.

[0039] Figures 20A-20D : Testing of B7H3-targeted CAR T cells in the IMR5 neuroblastoma mouse model. Fig. 20A ) evaluated in vitro killing of the neuroblastoma cell line IMR5 using B7H3-targeted CAR T cells. B7H3-targeted G8, B12, or C4 CAR T cells, as well as CAR T cells based on the commercial anti-human B7H3 hybridoma antibody 376.96, were cultured with IMR5 GL cells at different effector:target (E:T) ratios for 24 hours. All CAR T cells efficiently lysed IMR5 tumor cells in a dose-dependent manner compared to mock T cells; however, B12 CAR T cells were slightly more potent than the other CAR T cells tested. ( Fig. 20B) Schematic diagram of the in vivo study. IMR5 xenograft mice were intravenously infused with 5 million C4 CAR T cells, B12 CAR T cells, G8 CAR T cells, 376.96CAR T cells, or untransduced T cells (mock) 35 days after tumor inoculation. ( Fig. 20C ) Representative bioluminescent images of IMR5 tumor growth in a xenograft model. Mice treated with 5 million B12 CAR T cells showed significantly slower tumor growth compared with 376.96CAR T cells and mock T cells. C4 CAR T cells also showed modest anti-tumor activity. ( Fig.20D ) Tumor bioluminescence in treated mice as photons per second.

[0040] Figures 21A-21C :G8 cross-reacts with mouse B7H3 and kills mouse cancer cells. ( Fig.21A ) Anti-B7H3 nanobody binding activity to mouse antigens detected 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) and the mouse melanoma cell line B16. ( Fig. 21B ) Only G8 CAR T cells showed specific killing of mouse B7H3-positive B16 cell line. ( Fig. 21C ) Epitope mapping of anti-B7H3 nanobody and commercial antibody 376.96. A total of 48 peptides from human B7H3 protein were designed and synthesized. Each peptide consists of 18 amino acids and overlaps with the adjacent peptide by 9 amino acids. ELISA technology was used to test the binding ability of the antibody to each peptide. The numbers represent OD 450 Values. G8 and 376.96 both bind to peptides 10, 11, and 15 (SEQ ID NOs: 35-37), indicating that they bind to similar epitopes. The peptide sequences are shown in the table below. C4 and B12 may have conformational epitopes that cannot be predicted by the linearized peptide library.

[0041] Fig. 22 : Binding of B7H3-specific antibodies B12, C4, G8 and 376.96 to B7H3 protein measured by ELISA.

[0042] Sequence Listing

[0043] The nucleic acid sequences 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 defined in 37 C.FR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included in any reference to the displayed strand. The sequence listing is submitted as an ASCII text file, created on October 19, 2020, 27.8KB, and is incorporated herein by reference. In the accompanying sequence listing:

[0044] SEQ ID NO: 1 is the amino acid sequence of camelid antibody RWB12.

[0045] SEQ ID NO: 2 is the amino acid sequence of camelid antibody RWG8.

[0046] SEQ ID NO: 3 is the amino acid sequence of camelid antibody RWC4.

[0047] SEQ ID NO: 4 is the amino acid sequence of camelid antibody RWB2.

[0048] SEQ ID NO:5 is the amino acid sequence of camelid antibody RWH5.

[0049] SEQ ID NO:6 is the amino acid sequence of camelid antibody RWD5.

[0050] SEQ ID NO:7 is the amino acid sequence of camelid antibody RWC3.

[0051] SEQ ID NO: 8 is the amino acid sequence of camelid antibody RWG4.

[0052] SEQ ID NO:9 is the amino acid sequence of camelid antibody RWD9.

[0053] SEQ ID NO: 10 is the amino acid sequence of camelid antibody RWH1.

[0054] SEQ ID NO: 11 is the amino acid sequence of the rabbit antibody RFA1.

[0055] SEQ ID NO: 12 is the amino acid sequence of the rabbit antibody RFB1.

[0056] SEQ ID NO: 13 is the amino acid sequence of the extracellular domain of B7H3.

[0057] SEQ ID NOs: 14-26 are primer sequences.

[0058] SEQ ID NO:27 is the amino acid sequence of GMCSFRss.

[0059] SEQ ID NO: 28 is the amino acid sequence of the CD8α hinge region.

[0060] SEQ ID NO: 29 is the amino acid sequence of the CD8α transmembrane region.

[0061] SEQ ID NO: 30 is the amino acid sequence of 4-1BB.

[0062] SEQ ID NO:31 is the amino acid sequence of CD3ζ.

[0063] SEQ ID NO: 32 is the amino acid sequence of the self-cleaving T2A peptide.

[0064] SEQ ID NO:33 is the amino acid sequence of huEGFRt.

[0065] SEQ ID NO:34 is the amino acid sequence of the rabbit VH domain antibody 5DUB.

[0066] SEQ ID NOs: 35-37 are the amino acid sequences of B7H3 peptides. DETAILED DESCRIPTION

[0067] I. Abbreviations

[0068] ADC Antibody-drug conjugate

[0069] ADCC Antibody-dependent cell-mediated cytotoxicity

[0070] B7H3 B7 homolog 3

[0071] BBIR biotin-binding immune receptor

[0072] CAR Chimeric Antigen Receptor

[0073] CDR complementarity determining region

[0074] CTL Cytotoxic T lymphocyte

[0075] ECD extracellular domain

[0076] EGF Epidermal Growth Factor

[0077] EGFR Epidermal Growth Factor Receptor

[0078] ELISA enzyme-linked immunosorbent assay

[0079] EM effector part

[0080] FACS fluorescence activated cell sorting

[0081] GMCSFRss Granulocyte-macrophage colony-stimulating factor receptor signal sequence

[0082] hFc human Fc

[0083] huEGFRt human truncated epidermal growth factor receptor

[0084] IC50 Inhibitory concentration 50

[0085] Ig immunoglobulin

[0086] KO knockout

[0087] NK Natural Killer Cells

[0088] PBD Pyrrolobenzodiazepine

[0089] PE Pseudomonas exotoxin

[0090] PET Positron Emission Tomography

[0091] TM Transmembrane

[0092] VH Variable Weight

[0093] VL Variable Lightweight

[0094] II. Terminology and Methodology

[0095] Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in: Benjamin Lewin, Genes X, Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, Wiley-VCH, Vol. 16, 2008; and other similar references.

[0096] 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 single or multiple antigens and can be considered equivalent to the phrase "at least one antigen". As used herein, the term "comprising" means "including". It should also be understood that, unless otherwise stated, any and all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weight or molecular mass values ​​are approximate and are provided for descriptive purposes. Although many methods and materials similar or equivalent to the methods and materials described herein can be used, particularly suitable methods and materials are described herein. In the event of a conflict, the present specification (including term explanations) shall prevail. In addition, the materials, methods, and examples are illustrative only and not restrictive. In order to facilitate viewing of various embodiments, the following term explanations are provided:

[0097] 4-1BB: A co-stimulatory molecule expressed by lymphocytes and other cells (including natural killer cells) activated by T cell receptors (TCR). The connection of 4-1BB induces a signal cascade, leading to cytokine production, expression of anti-apoptotic molecules, and enhancement of immune response. The exemplary amino acid sequence of 4-1BB is shown in SEQ ID NO: 30 herein.

[0098] Administration: Provide or administer an agent to a subject by any effective route, such as a monoclonal antibody, CAR, or a cell expressing CAR provided herein. Exemplary administration routes include, but are not limited to, oral routes, injection routes (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraprostatic, and intratumoral), sublingual routes, rectal routes, transdermal routes, intranasal routes, vaginal routes, and inhalation routes.

[0099] Antibody: A polypeptide ligand that contains at least one variable region that recognizes and binds (e.g., specifically recognizes and specifically binds) an epitope of an antigen. Mammalian immunoglobulin molecules are composed of heavy (H) chains and light (L) chains, each with a variable region, called a variable heavy (V H ) area and variable lightness (V L ) area. H Area and V L The heavy chain domains are together responsible for binding to the antigen recognized by the antibody. There are five major classes (or isotypes) of mammalian immunoglobulins that 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 similar in function to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0100] The variable region of an antibody comprises a "framework" region and a hypervariable region, called a "complementarity determining region" or "CDR". The CDR is primarily responsible for binding to the epitope of an antigen. The framework region of an antibody is used to position and align the CDR in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, USD Department of Health and Human Services, 1991; “Kabat” numbering scheme), Chothia et al. (see 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; “Chotia” numbering scheme), Kunik et al. (see Kunik et al., PLoS Comput Biology 274, 927-948, 1997; “Chotia” numbering scheme), Biol) 8:e1002388, 2012; and Kunik et al., Nucleic Acids Res 40 (web server issue): W521-524, 2012; "Paratome CDR") and ImMunoGeneTics (IMGT) databases (see Lefranc, Nucleic Acids Res 29:207-9, 2001; "IMGT" numbering scheme). Kabat, Paratome and IMGT databases are maintained online.

[0101] "Single domain antibody" refers to an antibody with a single domain (variable domain) that is capable of specifically binding to an antigen or antigenic epitope in the absence of additional antibody domains. Single domain antibodies include, for example, V H Domain antibody, V NAR Antibodies, Camelidae V H H antibody and V L Domain antibody. V NAR Antibodies are produced by cartilaginous fishes, such as the nurse shark, wobegon shark, spiny dogfish, and bamboo shark. H H antibodies are produced by a variety of species, including camels, llamas, alpacas, dromedaries, and guanacos, which produce heavy chain antibodies that naturally lack light chains.

[0102] A "monoclonal antibody" is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence for a single antibody has been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art. Monoclonal antibodies include humanized monoclonal antibodies.

[0103] "Chimeric antibodies" have framework residues from one species (eg, human) and CDRs (typically responsible for antigen binding) from another species.

[0104] A "humanized" antibody is an immunoglobulin that includes a human framework region and one or more CDRs of a non-human (e.g., mouse, rabbit, rat, shark or synthetic) immunoglobulin. The non-human immunoglobulin that provides the CDR is called a "donor," and the human immunoglobulin that provides the framework is called an "acceptor." In one embodiment, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. The constant region does not have to be present, but if present, they must be substantially identical to the human immunoglobulin constant region, i.e., at least about 85-90% identical, such as about 95% or higher percentages identical. Therefore, all parts of the humanized immunoglobulin, possibly except the CDR, are substantially identical to the corresponding parts of the natural human immunoglobulin sequence. Humanized antibodies bind to the same antigen as the donor antibody that provides the CDR. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions.

[0105] Antibody-drug conjugate (ADC): A molecule that includes an antibody (or antigen-binding fragment of an antibody) conjugated to a drug (e.g., a cytotoxic agent). ADCs can be used to specifically target drugs to cancer cells through specific binding of the antibody to tumor antigens expressed on the cell surface. Exemplary drugs used with ADCs include anti-microtubule agents (e.g., maytansines, auristatin E, and auristatin F) and interchain crosslinkers (e.g., pyrrolobenzodiazepines; PBDs). In some cases, the ADC is a bispecific ADC, which consists of two monoclonal antibodies or antigenic fragments thereof, each directed against a different antigen or epitope, and conjugated to a drug. In one example, the agent attached to the antibody is 700DX (IR700, Li-cor, Lincoln, NE), can then be used with near-infrared NIR light to kill cancer cells to which the antibody is bound (photoimmunotherapy; see, e.g., US 8,524,239 and 10,538,590). For example, amino-reactive IR700 can be covalently bound to the antibody using the NHS ester of IR700.

[0106] Antimicrotubule agent: A drug that stops cell growth by stopping mitosis. Antimicrotubule agents, also called "antimitotic agents," are used to treat cancer.

[0107] B7 homolog 3 (B7H3): An immune checkpoint molecule expressed by certain types of solid tumors. This protein is a member of the B7 superfamily of co-stimulatory molecules. B7H3 is also known as CD276.

[0108] B7H3-positive cancer: A cancer that expresses or overexpresses B7H3. Examples of B7H3-positive cancers include, but are not limited to, liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer (e.g., neuroblastoma or glioblastoma), pediatric cancer (e.g., osteosarcoma, neuroblastoma, rhabdomyosarcoma, or Ewing's sarcoma), melanoma, and mesothelioma (see, e.g., Seaman et al., Cancer Cell 31(4):501-505, 2017).

[0109] Binding affinity: The affinity of an antibody for an antigen. In one embodiment, affinity is calculated by modifying the Scatchard method described by Frankel 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 by surface plasma reference. An antibody that "specifically binds" an antigen (such as B7H3) is an antibody that binds to the antigen with high affinity and does not significantly bind to other unrelated antigens.

[0110] In some examples, a monoclonal antibody (e.g., an anti-B7H3 single domain antibody provided herein) specifically binds to a target (e.g., B7H3) with a binding constant that is at least 10 greater than the binding constant for other molecules in a sample or subject. 3 M -1 , 10 4 M -1 or 10 5 M -1 In some examples, the antibody (e.g., a monoclonal antibody) has an equilibrium constant (Kd) of 1 μM or less, such as 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 binds a target (e.g., B7H3) with a binding affinity of at least about 1×10 -6 M, at least about 0.5×10 -6 M, at least about 1×10 -7 M, at least about 0.5×10-7 M, at least about 1×10 -8 M, at least about 0.5×10 -8 M, at least about 1×10 -9 M, at least about 0.5×10 -9 M, or at least about 0.1×10 -9 In certain embodiments, the dissociation constant (Kd) of a specific binding agent for its target is ≤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, for example, from 10 -6 M to 10 -10 M, for example, 10 -7 M to 10 -9 M). In some examples, binding affinity is measured using the Octet system (Creative Biolabs) based on biolayer interferometry (BLI) technology. In some examples, Kd is measured using surface plasmon resonance assays using BIAcores-2000 or BIAcores-3000 (BIAcore, Piscataway, NJ).

[0111] Bispecific antibody: A recombinant protein comprising antigen-binding fragments of two different monoclonal antibodies, thereby being able to bind to two different antigens. In some embodiments, bispecific antibodies are used for cancer immunotherapy by simultaneously targeting, for example, 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 comprising antigen-binding fragments of at least two different monoclonal antibodies (e.g., two, three, or four different monoclonal antibodies).

[0112] Brain cancer or tumor: A type of cancer or tumor that develops from brain tissue. Brain cancers include, but are not limited to, neuroblastoma, medulloblastoma, glioma, glioblastoma, meningioma, pituitary adenoma, astrocytoma, choroid plexus carcinoma, ependymoma, and pineoblastoma.

[0113] Breast cancer: A cancer that forms in breast tissue, usually in the ducts and lobules. Types of breast cancer include, for example, ductal carcinoma in situ, invasive ductal carcinoma, triple-negative breast cancer, inflammatory breast cancer, metastatic breast cancer, medullary carcinoma, tubular carcinoma, and mucinous carcinoma. Triple-negative breast cancer refers to a 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 HER2 / neu-negative breast cancer.

[0114] Chemotherapeutic agent: Any chemical agent that has a therapeutic effect in treating a disease characterized by abnormal cell growth. Such diseases include tumors, neoplasms and cancers as well as diseases characterized by hyperplastic growth, such as psoriasis. In one embodiment, a chemotherapeutic agent is an agent used to treat a B7H3-positive tumor. In one embodiment, a chemotherapeutic agent is a radioactive compound. One skilled in the art can readily determine the chemotherapeutic agent to use (see, e.g., Slapak and Kufe, Princes of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th ed.; Perry et al., Chemotherapy, Chapter 17 in Abeloff, Clinical Oncology, 2nd ed., 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed., St. Louis, Mosby-Year Book, 1995; Fischer, 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 agent to treat cancer. An example is the administration of an antibody that binds to B7H3 in combination with a radioactive or chemical compound. In one example, the chemotherapeutic agent is a biologic, such as a therapeutic antibody (e.g., a therapeutic monoclonal antibody), such as an 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).

[0115] Chimeric antigen receptor (CAR): a chimeric molecule comprising an antigen binding portion (e.g., scFv or single domain antibody) and a signaling domain, such as a signaling domain of a T cell receptor (e.g., CD3ζ). Typically, CAR consists of an antigen binding portion, a transmembrane domain, and an endodomain. The endodomain typically includes a signaling chain having an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3ζ or FcεRIγ. In some cases, the endodomain also includes an intracellular portion of at least one additional costimulatory domain, such as CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, and / or DAP10. In some instances, CAR is multispecific (e.g., bispecific) or bicistronic. Multispecific CAR is a single CAR molecule consisting of at least two antigen binding domains (e.g., scFv and / or single domain antibody), each of which binds to different antigens or different epitopes on the same antigen (see, e.g., US2018 / 0230225). For example, bispecific CAR refers to a single CAR molecule with two antigen binding domains, each of which binds to different antigens. Bicistronic CAR refers to two complete CAR molecules, each of which comprises an antigen binding moiety that binds to different antigens. In some cases, the bicistronic CAR construct expresses two complete CAR molecules connected by cutting a joint. T cells or NK cells expressing bispecific or bicistronic CARs can bind to cells expressing two antigens for which the binding moiety is directed (see, for example, Qin et al., Blood 130: 810, 2017; and WO / 2018 / 213337).

[0116] Colon cancer: A type of cancer that develops in the colon or rectum. The most common type of colon cancer (also called "colorectal cancer") is colorectal adenocarcinoma, which accounts for about 95% of all colon cancers. Adenocarcinomas develop in the cells lining the colon and / or rectum. Other types of colorectal cancer include gastrointestinal carcinoids, metastatic colorectal cancer, primary colorectal lymphoma (a type of non-Hodgkin lymphoma), gastrointestinal stromal tumors (classified as sarcomas, arising from interstitial cells of Cajal), leiomyosarcomas (arising from smooth muscle cells), and colorectal melanoma.

[0117] Complementarity Determining Region (CDR): A region of highly variable amino acid sequences that defines the binding affinity and specificity of an antibody. The light and heavy chains of mammalian immunoglobulins each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3. Single domain antibodies contain three CDRs, referred to herein as CDR1, CDR2, and CDR3.

[0118] Conjugate: In the context of the present disclosure, a "conjugate" is an antibody or antibody fragment (e.g., an antigen binding fragment) covalently linked to an effector molecule or a second protein (e.g., a second antibody). The effector molecule can be, for example, a drug, a toxin, a therapeutic agent, a detectable marker, a protein, a nucleic acid, a lipid, a nanoparticle, a photon absorber, a carbohydrate, or a recombinant virus. Antibody conjugates are often referred to as "immunoconjugates". When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is often referred to as an "antibody-drug conjugate" or "ADC". Other antibody conjugates include, for example, multispecific (e.g., bispecific or trispecific) antibodies and chimeric antigen receptors (CARs).

[0119] Conservative variant: A protein containing conservative amino acid substitutions that do not substantially affect or reduce the affinity of a protein (e.g., an antibody to B7H3). For example, a monoclonal antibody that specifically binds to B7H3 may include up to about 1, up to about 2, up to about 5, and up to about 10, or up to about 15 conservative substitutions and specifically binds to a B7H3 polypeptide. The term "conservative variant" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the antibody specifically binds to B7H3. Non-conservative substitutions are substitutions that reduce activity or binding to B7H3.

[0120] Conservative amino acid substitution tables for functionally similar amino acids are well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0121] 1) Alanine (A), serine (S), threonine (T);

[0122] 2) Aspartic acid (D), glutamic acid (E);

[0123] 3) Asparagine (N), glutamine (Q);

[0124] 4) Arginine (R), Lysine (K);

[0125] 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0126] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0127] Contact: Direct physical association; includes both solid and liquid forms.

[0128] Cytotoxic agent: Any drug or compound that kills cells.

[0129] Cytotoxicity: Toxicity of a molecule (e.g., an immunotoxin) to the cells of its intended target, rather than to cells in the rest of the organism. In contrast, the term "toxicity" refers to the toxicity of an immunotoxin to cells other than the cells to which the targeting portion of the immunotoxin is intended, and the term "zootoxicity" refers to the toxicity of an immunotoxin to an animal, i.e., the toxicity of the immunotoxin to cells other than the cells to which the immunotoxin is intended.

[0130] Degenerate variants: Polynucleotides encoding polypeptides, including sequences that are degenerate due to the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, as long as the amino acid sequence of the polypeptide is unchanged, all degenerate nucleotide sequences are included.

[0131] Diagnosis: Determining the presence or nature of a pathological condition (e.g., B7H3-positive cancer). Diagnostic methods differ in sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (the percentage of true positives). The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the false positive rate is defined as the proportion of disease-free patients who test positive. Although a particular diagnostic method may not be able to definitively diagnose a condition, it is sufficient that the method provides a positive indication that helps with diagnosis. "Prognosis" is the probability of the development (e.g., severity) of a pathological condition such as cancer.

[0132] Diagnostic tumor imaging: Conjugating antibodies and their derivatives to positron-emitting radionuclides for positron emission tomography (PET) is a process generally referred to as immuno-PET. Although full-length antibodies make good immuno-PET agents, their biological half-lives require waiting several days before imaging, resulting in increased non-target radiation doses. Smaller single-domain antibodies or nanobodies have biological half-lives suitable for same-day imaging.

[0133] Drug: Any compound used to treat, improve or prevent a disease or condition in a subject. In some embodiments herein, the drug is an anticancer agent, such as a cytotoxic agent, such as an antimitotic agent or an antimicrotubule agent.

[0134] Effector molecule: The portion of a chimeric molecule that is intended to produce a desired effect on the cells targeted by the chimeric molecule. Effector molecules are also referred to as effector moieties (EMs), 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-stranded or double-stranded DNA, and oligonucleotides that form triple chains. Alternatively, the molecule attached to the targeting moiety (e.g., anti-B7H3 antibody) can be an encapsulation system, such as a liposome or micelle, which contains a therapeutic composition (e.g., a drug), a nucleic acid (e.g., an antisense nucleic acid), or another therapeutic moiety that can be shielded from direct exposure to the circulatory system. Methods for preparing liposomes attached to antibodies are well known to those skilled in the art (see, 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 and include radioisotopes (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), fluorophores, chemiluminescent agents and enzymes.

[0135] Epitope: Antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic (elicit a specific immune response). Antibodies specifically bind to specific epitopes on a polypeptide, such as B7H3.

[0136] Framework region: The amino acid sequence inserted between CDRs. The framework region of an immunoglobulin molecule includes a variable light framework region and a variable heavy framework region.

[0137] Fusion protein: A protein that contains at least portions of two different (heterologous) proteins.

[0138] Heterologous: Derived from a single genetic source or species.

[0139] Immune response: The response of an immune system cell (such as a B cell, T cell, or monocyte) to a stimulus. In one embodiment, the immune response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, the immune response is a T cell response, such as a CD4 + Response or CD8 + In another embodiment, the immune response is a B cell response and produces specific antibodies.

[0140] Immunoconjugate: covalent attachment of an effector molecule to an antibody or a functional fragment thereof. The effector molecule may be, for example, a detectable marker, a photon absorber (e.g., IR700) or a toxin (to form an immunotoxin, such as an immunotoxin comprising a pseudomonas exotoxin or a variant thereof). Specific non-limiting examples of toxins include, but are not limited to, abrin, ricin, pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin or its modified toxins, or other agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that usually cause death through hepatotoxicity. However, PE and DT can be modified to a form used as an immunotoxin by removing the natural targeting components of the toxin (e.g., domain Ia of PE and B chain of DT) and replacing them with different targeting moieties (such as antibodies). In one embodiment, an antibody is bound to an effector molecule. In another embodiment, the antibody bound to the effector molecule is further bound to a lipid or other molecule, such as to extend its half-life in vivo. The connection may be performed chemically or recombinantly. In one embodiment, the connection is chemical, wherein the reaction between the antibody portion and the effector molecule produces a covalent bond formed between the two molecules, thereby forming one molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the effector molecule. Because immunoconjugates are initially prepared from two molecules with different functions, such as antibodies and effector molecules, they are sometimes also referred to as "chimeric molecules". Therefore, as used herein, the term "chimeric molecule" refers to a targeting portion, such as a ligand or antibody, conjugated (coupled) to an effector molecule. The term "conjugated" or "connected" refers to making two polypeptides into one continuous polypeptide molecule.

[0141] Immunoliposomes: Liposomes with antibodies or antibody fragments conjugated to their surface. Immunoliposomes can carry cytotoxic agents or other drugs to cells targeted by antibodies, such as tumor cells.

[0142] Interstrand cross-linker: A cytotoxic drug that covalently binds between two DNA strands, thereby preventing DNA replication and / or transcription.

[0143] Isolated: An "isolated" biological component (e.g., a nucleic acid, protein (including antibodies), or organelle) that 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 (e.g., a cell) in which the component naturally occurs. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids.

[0144] Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule (such as an antibody or protein) to facilitate detection of that molecule. Specific non-limiting examples of labels include fluorescent tags, enzymatic bonds, and radioactive isotopes. In one example, a "labeled antibody" refers to the incorporation of another molecule into an antibody. For example, a label is a detectable marker, such as a polypeptide that incorporates a radiolabeled amino acid or is attached to a biotinyl moiety that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of polypeptide labels include, but are not limited to, the following: 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 phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotin groups, predetermined polypeptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pair sequences, binding sites of secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, the labels are attached via spacer arms of various lengths to reduce potential steric hindrance.

[0145] Linker: In some cases, a linker is a peptide within an antibody binding fragment (e.g., an Fv fragment) that is used to indirectly bind a variable heavy chain to a variable light chain. A "linker" may also refer to a peptide used to connect a targeting moiety (e.g., an antibody) to an effector molecule (e.g., a cytotoxin or a detectable marker). The terms "conjugation," "joining," "binding," or "linking" refer to making two polypeptides into a continuous polypeptide molecule, or covalently linking a radionuclide or other molecule to a polypeptide, such as an antibody. The connection may be chemically or recombinantly performed. "Chemical" refers to a reaction between an antibody moiety and an effector molecule such that a covalent bond is formed between the two molecules to form one molecule.

[0146] Liver cancer: Any type of cancer that develops in the liver tissue. The most common type of liver cancer is hepatocellular carcinoma (HCC), which forms in liver cells. Other types of liver cancer include cholangiocarcinoma, which forms in the bile ducts; hepatic angiosarcoma, which is a rare liver cancer that starts in the blood vessels of the liver; and hepatoblastoma, which is a very rare liver cancer that most often develops in children.

[0147] Lung cancer: Any cancer that develops in the lungs. Most cancers that begin in the lungs are malignant tumors. The two main types of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Subtypes of NSCLC include adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.

[0148] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Typically, operably linked DNA sequences are contiguous and, when necessary to link two protein coding regions, in the same reading frame.

[0149] Ovarian cancer: Cancer that develops in the tissue of the ovaries. Most ovarian cancers are epithelial ovarian cancers (cancer that begins in cells on the surface of the ovaries) or malignant germ cell tumors (cancer that begins in egg cells). Another type of ovarian cancer is stromal cell cancer, which begins in cells that release hormones and connect different structures of the ovaries.

[0150] Pancreatic cancer: A disease in which malignant cells are found in pancreatic tissue. Based on the cell of origin of the cancer, pancreatic tumors can be exocrine tumors or neuroendocrine tumors. The vast majority (~94%) of pancreatic cancers are exocrine tumors. Exocrine cancers include, for example, adenocarcinomas (the most common type of exocrine tumor), acinar cell carcinomas, intraductal papillary mucinous neoplasms (IPMNs), and mucinous cystadenocarcinomas. In some instances, pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). Pancreatic neuroendocrine tumors, also called islet cell tumors, are classified according to the type of hormones they produce. Exemplary neuroendocrine tumors include gastrinomas, glucagonomas, insulinomas, somatostatinomas, VIPomas (vasoactive intestinal polypeptide), and non-functioning islet cell tumors.

[0151] Pediatric cancer: A type of cancer that occurs in children between the ages of 0 and 14. Major types of pediatric cancer include, for example, neuroblastoma, acute lymphoblastic leukemia (ALL), embryonal rhabdomyosarcoma (ERMS), alveolar rhabdomyosarcoma (ARMS), Ewing's sarcoma, desmoplastic small round cell tumor (DRCT), osteosarcoma, brain and other CNS tumors (such as neuroblastoma and medulloblastoma), Wilm's tumor, non-Hodgkin lymphoma, and retinoblastoma.

[0152] Pharmaceutically acceptable carrier: The pharmaceutically acceptable carrier used is a conventional carrier. "Remington: The Science and practice of Pharmacy", University of the Sciences in Philadelphia, editor, Lippincott Williams and Wilkins (Lippincott, Williams, & Wilkins), Philadelphia, Pennsylvania, 21st edition (2005), describes compositions and formulations suitable for drug delivery of antibodies and other compositions disclosed herein. In general, the nature of the carrier will depend on the specific mode of administration adopted. For example, parenteral formulations generally include injectable fluids, which include pharmaceutically and physiologically acceptable fluids, such as water, saline, balanced salt solutions, aqueous glucose solutions, glycerol, etc. as vehicles. For solid compositions (such as powders, pills, tablets or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0153] Photoimmunotherapy: A targeted cancer therapy that utilizes antigen-specific antibody-photoabsorber conjugates that can be activated by near-infrared light to kill targeted cells. Photon absorbers are often based on phthalocyanine dyes, such as near-infrared (NIR) phthalocyanine dyes (e.g., 700DX, also known as IR700). The antibody (e.g., a B7H3-specific antibody) binds to an appropriate cell surface antigen (e.g., B7H3), and the photoactivatable dye induces lethal cell membrane damage after NIR light irradiation. NIR light irradiation (e.g., 690 nm) can induce highly selective necrotic cancer cell death within minutes without damaging adjacent cells (e.g., see U.S. Application No. 2018 / 0236076).

[0154] Prevent, treat, or alleviate disease: "Preventing" a disease means inhibiting the overall development of the disease. "Treatment" refers to therapeutic intervention to improve the signs or symptoms of a disease or pathological condition after it has begun to develop, such as reducing tumor burden or decreasing the size of metastases. "Allowing" refers to reducing the number or severity of signs or symptoms of a disease (e.g., cancer).

[0155] Purification: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein in its natural environment within a cell. In one embodiment, the preparation is purified so that the protein or peptide accounts for at least 50% of the total peptide or protein content of the preparation. Substantially purified refers to purification from other proteins or cellular components. A substantially purified protein has a purity of at least 60%, 70%, 80%, 90%, 95%, or 98%. Thus, in a specific, non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components.

[0156] Pyrrolobenzodiazepine (PBD): A class of sequence-selective DNA minor groove binding cross-linkers originally discovered in Streptomyces species. PBDs are more effective than systemic chemotherapy drugs. The mechanism of action of PBDs is related to their ability to form adducts in the minor groove of DNA, thereby interfering with DNA processing. In the context of the present disclosure, PBDs include naturally occurring and isolated PBDs, chemically synthesized naturally occurring PBDs, and chemically synthesized non-naturally occurring PBDs. PBDs also include monomeric PBDs, dimeric PBDs, and hybrid PBDs (for review, see Gerratana, Med Res Rev 32(2):254-293, 2012).

[0157] Recombinant: A recombinant nucleic acid or protein is a nucleic acid or protein that has a sequence that does not occur naturally or that is produced by artificially combining two otherwise separate sequence fragments. This artificial combination is usually accomplished by chemical synthesis or by artificial manipulation of isolated nucleic acid fragments (e.g., by genetic engineering techniques).

[0158] Sample (or biological sample): A biological sample obtained from a subject containing genomic DNA, RNA (including mRNA), protein, or a combination thereof. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, biopsy, fine needle aspirates, surgical specimens, and autopsy material. In one example, the sample comprises a tumor biopsy.

[0159] Sequence identity: Similarity between amino acid or nucleic acid sequences is expressed as the similarity between the sequences, also known as sequence identity. Sequence identity is usually measured as a percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologues or variants of polypeptides or nucleic acid molecules will have a higher sequence identity when aligned using standard methods.

[0160] Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described in the following references: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, PNAS 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 73:237, 1988. 5:151, 1989; Corpet et al., Nucleic Acids Res. 16:10881, 1988; and Pearson and Lipman, Proc. Acad. Sci. USA. 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, consider in detail sequence alignment methods and homology calculations.

[0161] NCBI Basic Local Alignment Search Tool (BLAST) (Alshur et al., Journal of Molecular Biology)

[0162] 215:403, 1990) is available from a number of sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Maryland) and the Internet for use with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Instructions on how to use this program to determine sequence identity are available at the NCBI website on the Internet.

[0163] Homologs and variants of antibodies that specifically bind to a B7H3 polypeptide are typically characterized by having at least about 75%, such as at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity in a full-length alignment with the antibody amino acid sequence, using NCBI Blast 2.0, with blastp for gaps set to default parameters. For comparisons of amino acid sequences greater than about 30 amino acids, the Blast 2 sequence function is used, using the default BLOSUM62 matrix set to default parameters (gap existence cost of 11, gap cost per residue of 1). When aligning short peptides (less than about 30 amino acids), the Blast 2 sequence function should be used for alignment, using the PAM30 matrix, set to default parameters (gap creation penalty of 9, gap extension penalty of 1). When evaluated by this method, proteins with higher similarity to the reference sequence will show an increase in percentage identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity. When comparing sequence identities less than the entire sequence, homologs and variants typically have at least 80% sequence identity within 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 appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible to obtain highly significant homologs beyond the provided ranges.

[0164] Small molecule: A molecule generally having a molecular weight of less than about 1000 Daltons, or in some embodiments, less than about 500 Daltons, wherein the molecule is capable of modulating the activity of a target molecule to some measurable degree.

[0165] Subject: Living multicellular vertebrate organisms, including humans, and animal subject categories, including human and non-human mammals.

[0166] Synthetic: Produced by artificial means in a laboratory. For example, synthetic nucleic acids or proteins (such as antibodies) can be chemically synthesized in a laboratory.

[0167] Therapeutically effective amount: The amount of a specific substance sufficient to achieve the desired effect in a subject being treated. For example, this can be the amount necessary to prevent or inhibit tumor growth. In one embodiment, a therapeutically effective amount is the amount necessary to eliminate, reduce tumor size, or prevent tumor metastasis, such as by reducing 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 by reducing 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%, compared to the size / volume / number before treatment. When administered to a subject, the dose typically used will reach a concentration in the target tissue (e.g., in a tumor) that has been shown to achieve the desired in vitro effect.

[0168] 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 often cause death through hepatotoxicity. However, PE and DT can be modified to a form useful as an immunotoxin by removing the natural targeting component of the toxin (e.g., domain Ia of PE or chain B of DT) and replacing it with a different targeting moiety (e.g., an antibody).

[0169] Vector: A nucleic acid molecule introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence that enables it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. In some embodiments, the vector is a viral vector, such as a lentiviral vector.

[0170] III. B7H3-specific single-domain monoclonal antibodies (“nanobodies”)

[0171] Nanobodies include Camelidae V H H. Cartilaginous fish V NAR and human VH single domain antibodies. Rabbit monoclonal antibodies can recognize a variety of epitopes, including epitopes with poor immunogenicity in mice and humans. The present disclosure describes immunizing rabbits with recombinant B7H3 protein and generating a phage-displayed VH single domain library. After three rounds of phage panning, two binders (referred to as RFA1 and RFB1, respectively) were selected. Both binders expressed well in E. coli with a yield of 2 mg / L

[0172] (RFA1) and 10 mg / L (RFB1). Rabbit nanobodies showed antigen-dependent binding to B7H3-positive tumor cell lines (IMR32, MC38-B7H3+, A431, and NBEB) but not B7H3 knockout cell lines (IMR32-B7H3 KO, MC38-B7H3 KO). The present disclosure also describes the binding of eight different camel V H Ten B7H3-specific camel V isolated from the H library H H Nanobodies. The selected Nanobodies were able to bind to B7H3 expressing cells, such as neuroblastoma cells, epidermoid carcinoma cells, and pancreatic tumor cells.

[0173] Provided below are the amino acid sequences of ten camelid and two rabbit single domain antibodies. The CDR sequences determined using the Kabat, IMGT and Paratome methods are underlined, bolded and italicized, respectively. The table lists the amino acid positions of CDR1, CDR2 and CDR3 of each antibody determined using Kabat, IMGT or Paratome. Those skilled in the art can easily determine CDR boundaries using alternative numbering schemes (e.g., Chothia numbering schemes).

[0174] RWB12 (SEQ ID NO: 1)

[0175]

[0176]

[0177] RWG8 (SEQ ID NO: 2)

[0178]

[0179]

[0180] RWC4 (SEQ ID NO: 3)

[0181]

[0182]

[0183] RWB2 (SEQ ID NO:4)

[0184]

[0185]

[0186] RWH5 (SEQ ID NO: 5)

[0187]

[0188]

[0189] RWD5 (SEQ ID NO:6)

[0190]

[0191]

[0192] RWC3 (SEQ ID NO:7)

[0193]

[0194]

[0195] RWG4 (SEQ ID NO: 8)

[0196]

[0197]

[0198] RWD9 (SEQ ID NO:9)

[0199]

[0200]

[0201] RWH1 (SEQ ID NO: 10)

[0202]

[0203]

[0204] RFA1 (SEQ ID NO: 11)

[0205]

[0206]

[0207] RFB1 (SEQ ID NO: 12)

[0208]

[0209]

[0210] Provided herein are monoclonal antibodies that bind (eg, specifically bind) B7H3 (eg, cell surface or soluble B7H3). In some embodiments, the monoclonal antibody is a single domain antibody, eg, a VH single domain antibody.

[0211] In some embodiments, a single domain monoclonal antibody comprises at least a portion of an amino acid sequence as set forth herein in any one of SEQ ID NOs: 1-12, e.g., one or more (e.g., all three) CDR sequences from any of the following 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, e.g., 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 specific 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.

[0212] 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, respectively; 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. 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.

[0213] 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, respectively; residues 26-33, 51-58, and 97-106 of SEQ ID NO:2; or residues 27-35, 47-61, and 97-106 of SEQ ID NO:2. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:2.

[0214] In some embodiments, the CDR1, CDR2, and CD3 sequences of the antibody comprise residues 31-35, 50-66, and 97-114 of SEQ ID NO:3, respectively; residues 26-33, 51-58, and 97-115 of SEQ ID NO:3; or residues 26-35, 50-61, and 98-114 of SEQ ID NO:3. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:3.

[0215] 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, respectively; residues 26-33, 51-57, and 96-110 of SEQ ID NO:4; or residues 27-35, 47-60, and 96-109 of SEQ ID NO:4. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:4. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:4.

[0216] 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, respectively; residues 26-28, 46-53, and 90-110 of SEQ ID NO:5; or residues 27-30, 43-55, and 90-110 of SEQ ID NO:5. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:5. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:5.

[0217] 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, respectively; residues 26-33, 51-57, and 96-112 of SEQ ID NO:6; or residues 27-35, 47-60, and 96-111 of SEQ ID NO:6. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:6. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:6.

[0218] 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, respectively; residues 26-33, 51-57, and 96-112 of SEQ ID NO:7; or residues 27-35, 47-60, and 96-111 of SEQ ID NO:7. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7. In specific examples, the amino acid sequence of the antibody comprises or consists of SEQ ID NO:7.

[0219] 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, respectively; 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. 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.

[0220] 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, respectively; 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. 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.

[0221] 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, respectively; 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. 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.

[0222] 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, respectively; residues 25-32, 50-56, and 93-104 of SEQ ID NO: 11; or residues 26-34, 46-59, and 93-105 of SEQ ID NO: 11. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11 or 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.

[0223] 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, respectively; residues 26-33, 51-57, and 94-107 of SEQ ID NO: 12; or residues 27-35, 47-60, and 94-108 of SEQ ID NO: 12. In some examples, the amino acid sequence of the antibody is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12 or 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.

[0224] In some embodiments, the antibody is a humanized antibody or a chimeric antibody.

[0225] Also provided herein is a chimeric antigen receptor (CAR) comprising a single domain monoclonal antibody disclosed herein. In some embodiments, CAR also includes 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 includes a CD8α hinge region, the transmembrane domain includes a CD8α transmembrane domain, the costimulatory signaling moiety includes a 4-1BB signaling moiety, and / or the signaling domain includes a CD3ζ signaling domain.

[0226] Also provided herein are B7H3-specific antibodies modified to enable them to be used with a universal CAR system. In some embodiments, the B7H3-specific antibody is fused to a component of a specific binding pair. In some instances, the antibody is fused to a leucine zipper or biotin.

[0227] Further provided herein are cells expressing B7H3-specific CARs. In some instances, the cells are T lymphocytes, such as CTLs, or natural killer cells. CARs and cells expressing CARs are further described in Part IV.

[0228] Also provided herein are immunoconjugates comprising a single domain antibody disclosed herein and an effector molecule. In some embodiments, the effector molecule is a toxin, such as, but not limited to, a Pseudomonas exotoxin or a variant thereof, such as PE38. In other embodiments, the effector molecule is a detectable marker, such as, but not limited to, a fluorophore, an enzyme, or a radioisotope. In other embodiments, the effector molecule is a photon absorber, such as IR700. Immunoconjugates comprising photon absorbers can be used for photoimmunotherapy. Immunoconjugates are further described in Section V.

[0229] Further provided herein are antibody-drug conjugates (ADCs), including drugs conjugated to single domain antibodies disclosed herein. In some embodiments, the drug is a small molecule, such as an anti-microtubule agent, an anti-mitotic agent, and / or a cytotoxic agent. ADCs are further described in Section VI.

[0230] Also provided herein are multispecific antibodies, including single domain antibodies disclosed herein and at least one additional monoclonal antibody or its antigen binding fragment. 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 its antigen binding fragment specifically binds to a component of a T cell receptor or a natural killer (NK) cell activation receptor. Multispecific antibodies are further described in Section VII.

[0231] Further provided herein are antibody-nanoparticle conjugates, including nanoparticles conjugated to single domain antibodies disclosed herein. In some embodiments, the nanoparticles include polymer nanoparticles, nanospheres, nanocapsules, liposomes, dendrimers, polymer micelles, or vesicles. In some embodiments, the nanoparticles include cytotoxic agents. Antibody-nanoparticle conjugates are further described in Section VIII.

[0232] 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.

[0233] Further provided herein are nucleic acid molecules encoding antibodies, CARs, immunoconjugates, multispecific antibodies or fusion proteins disclosed herein. In some embodiments, the nucleic acid molecules are operably linked to a promoter. Also provided herein are vectors comprising the disclosed nucleic acid molecules. Further provided are isolated cells comprising nucleic acid molecules as vectors disclosed herein.

[0234] Also provided herein is a nucleic acid construct expressing CAR and truncated human EGFR (huEGFRt). In some embodiments, the nucleic acid comprises in the 5' to 3' direction: nucleic acid encoding the first granulocyte-macrophage colony stimulating factor receptor signal sequence (GMCSFRss); nucleic acid encoding the B7H3 specific single domain monoclonal antibody disclosed herein; nucleic acid encoding the extracellular hinge region; nucleic acid encoding the transmembrane domain; nucleic acid encoding the intracellular co-stimulatory domain; nucleic acid encoding the intracellular signaling domain; nucleic acid encoding the self-cleaved 2A peptide; nucleic acid encoding the second GMCSFRss; and nucleic acid encoding the truncated human epidermal growth factor receptor (huEGFRt). In some examples, the nucleic acid further includes a human elongation factor 1 alpha (EF1 alpha) promoter sequence 5' encoding the nucleic acid of the first GMCSFRss. In some examples, the hinge region includes the CD8 alpha hinge region. In some examples, the transmembrane domain includes the CD8 alpha transmembrane domain. In some examples, the costimulatory signaling portion includes a 4-1BB signaling portion. In some examples, the signaling domain includes a CD3ζ signaling domain. In some examples, the amino acid sequence of the B7H3-specific antibody includes any one of SEQ ID NO: 1-12. Also provided herein is a vector comprising a nucleic acid construct. In some embodiments, the vector is a lentiviral vector.

[0235] Further provided herein are isolated cells that co-express a B7H3-specific CAR disclosed herein and huEGFRt. In some examples, the cell is a CTL or NK cell.

[0236] The present disclosure further provides a composition comprising a pharmaceutically acceptable carrier and a single domain monoclonal antibody, CAR, isolated cells (e.g., cells expressing CAR, such as CAR T cells or CAR NK cells), immunoconjugates, ADCs, multispecific antibodies, antibody-nanoparticle conjugates, or fusion proteins disclosed herein. Compositions and uses thereof are further described in Part IX.

[0237] IV. Chimeric Antigen Receptor (CAR)

[0238] The disclosed nano antibodies can also be used to produce CAR (also referred to as chimeric T cell receptors, artificial T cell receptors or chimeric immune receptors) and / or engineered to express CAR cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. Typically, CAR includes a binding portion, an extracellular hinge and a spacer element, a transmembrane region, and an inner domain that performs a signal transduction function (Cartellieri et al., Journal of Biomedicine and Biotechnology (JBiomed Biotechnol) 2010: 956304, 2010; Dai et al., Journal of the National Cancer Institute (J Natl Cancer Inst) 108 (7): djv439, 2016). In many cases, the binding portion is an antigen binding fragment of a monoclonal antibody (such as scFv or a single domain antibody). The spacer / hinge region typically includes 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 CAR. For example, the endodomain can be composed of a signal transduction chain with ITAM, such as CD3ζ or FcεRIγ. In some cases, the endodomain also includes 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.

[0239] CTL, NK cells (or other immune cells) expressing CAR can be used to target specific cell types, such as B7H3 positive tumor cells. Therefore, nano antibodies disclosed herein can be used to transform CTL or NK cells expressing CAR containing B7H3-specific monoclonal antibodies, so as to target the transformed CTL or NK cells to tumor cells expressing B7H3. Transformed T cells have been used for adoptive therapy of certain types of cancer (see, e.g., Parker et al., "Molecular Therapy" (Mol Ther) 15 (4): 825-833, 2007). Using T cells expressing CAR is more common than standard CTL-based immunotherapy, because CTL expressing CAR is not restricted by HLA, and therefore can be used for any patient with a tumor expressing a target antigen.

[0240] The present disclosure also encompasses multispecific (e.g., bispecific) or bicistronic CARs. In some embodiments, multispecific or bispecific CARs include nanoantibodies (e.g., any of RWB12, RWG8, RWC4, RWB2, RWH5, RWD5, RWC3, RWG4, RWD9, RWH1, RFA1, and RFB1) specific to B7H3 and monoclonal antibodies specific to different antigens (e.g., T cell antigens). Similarly, bicistronic CARs include two CAR molecules expressed from the same construct, one of which is a B7H3 targeting CAR, and the second CAR targets a second antigen. For example, see Qin et al., Blood 130: 810, 2017; and WO / 2018 / 213337.

[0241] Therefore, CARs including B7H3-specific antibodies (e.g., any nanobody disclosed herein) are provided herein. Also provided herein are isolated nucleic acid molecules and vectors encoding CARs (including bispecific CARs and bicistronic CARs), and host cells expressing CARs, bispecific CARs, or bicistronic CARs, such as CTL cells or NK cells. CTL or NK cells expressing CARs composed of B7H3-specific monoclonal antibodies can be used to treat cancers expressing B7H3. In some embodiments herein, CAR is a bispecific CAR. In other embodiments herein, CAR is a bicistronic CAR.

[0242] In some embodiments, CAR includes a signal peptide sequence, for example, the N-terminus of the antigen binding domain. The signal peptide sequence can be any suitable signal peptide sequence, such as a signal sequence from a granulocyte-macrophage colony stimulating factor receptor (GMCSFR), an immunoglobulin light chain κ, or IL-2. Although the signal peptide sequence can promote the expression of CAR on the cell surface, the presence of a signal peptide sequence in the expressed CAR is not necessary for CAR to function. When CAR is expressed on the cell surface, the signal peptide sequence may be cut off from CAR. Therefore, in some embodiments, CAR lacks a signal peptide sequence.

[0243] In some embodiments, the CAR disclosed herein is expressed from a construct (e.g., a lentiviral vector) that also expresses a truncated form of human EGFR (huEGFRt). CAR and huEGFRt are separated by a self-cleaving peptide sequence (e.g., T2A) such that after expression in a transduced cell, CAR is cleaved from huEGFRt.

[0244] In some embodiments disclosed herein, the CAR construct encodes the following amino acid sequence in the N-terminal to C-terminal direction:

[0245] GMCSFRss:MLLVTSLLCELPHPAFLLIP(SEQ ID NO:27)

[0246] NdeI:HM

[0247] Antigen binding: B7H3-specific antibodies (e.g., Nanobodies disclosed herein)

[0248] SpeI:TS

[0249] CD8α hinge: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 28)

[0250] CD8αTM:IYIWAPLAGTCGVLLLSLVIT(SEQ ID NO:29)

[0251] 4-1BB:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:30)CD3ζ:

[0252] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

[0253] GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:31)

[0254] T2A:EGRGSLLTCGDVEENPGP(SEQ ID NO:32)

[0255] GMCSFRss:MLLVTSLLCELPHPAFLLIP(SEQ ID NO:27)

[0256] huEGFRt:

[0257] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTTHTPPLDPQELDI

[0258] LKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSSLGLRSLK

[0259] EISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPE

[0260] GCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITC

[0261] TGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM(SEQ ID NO:33)

[0262] The human epidermal growth factor receptor consists of four extracellular domains, a transmembrane domain, and three intracellular domains. The EGFR domains are in the following order from N-terminus to C-terminus: domain I-domain II-domain III-domain IV-transmembrane (TM) domain-near membrane domain-tyrosine kinase domain-C-terminal tail. Domain I and domain III are leucine-rich domains involved in ligand binding. Domain II and domain IV are cysteine-rich domains that do not contact EGFR ligands. Domain II mediates the formation of homodimers or heterodimers with similar domains of other EGFR family members, and domain IV can form a disulfide bond with domain II. The EGFR TM domain passes through the cell membrane once and may play a role in protein dimerization. The intracellular domain includes 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).

[0263] A truncated form of human EGFR, referred to herein as "huEGFRt", includes only domain III, domain IV, and TM domains. Thus, huEGFRt lacks domain I, domain II, and all three intracellular domains. huEGFRt cannot bind to EGF and does not have signaling activity. However, the molecule retains the ability to bind to specific EGFR-specific monoclonal antibodies, such as FDA-approved cetuximab (PCT Publication No. WO 2011 / 056894, which is incorporated herein by reference).

[0264] Transduction of T cells (or NK cells) with a construct (e.g., a lentiviral vector) encoding huEGFRt and a tumor antigen-specific CAR disclosed herein can be performed using the labeled EGFR monoclonal antibody cetuximab (ERBITUX TM )Select transduced T cells.For example, cetuximab can be labeled with biotin, and commercially available (e.g., Miltenyi Biotec) anti-biotin magnetic beads can be used to select transduced T cells. The co-expression of huEGFRt can also track adoptively transferred CAR-expressing T cells (or NK cells) in vivo. In addition, the combination of cetuximab and huEGFRt-expressing T cells induces the cytotoxicity of ADCC effector cells, thereby providing a mechanism for eliminating transduced T cells in vivo (Wang et al., Blood 118 (5): 1255-1263, 2011), such as at the end of treatment.

[0265] Also provided herein is a B7H3-specific monoclonal antibody (e.g., a nanobody disclosed herein) that has been modified to enable it to be used with a universal CAR system. A universal CAR system has been developed to improve the flexibility of CAR and expand its application to other antigens. Currently, for each patient receiving CAR T cell therapy, autologous T cells must be cultivated, amplified, and modified to express antigen-specific CARs. This process is long and expensive, which limits its use. Universal CAR is based on a system in which the signaling components of CAR are separated from the antigen-binding portion of the molecule, but are combined together using a "key" system. For example, the biotin-binding immune receptor (BBIR) CAR consists of an intracellular T cell signaling domain fused to an extracellular domain containing avidin. Then, biotinylated antigen-specific (such as B7H3-specific) monoclonal antibodies can bind to BBIR to guide T cells to tumor antigen-expressing cells. Another example is a separated universal programmable (SUPRA) CAR system. In the SUPRA system, CAR includes an intracellular signaling domain fused to an extracellular leucine zipper, which is paired with an antigen-specific monoclonal antibody fused to a homologous leucine zipper. For a review of the general CAR system, see, for example, Zhao et al., J Hematol Oncol 11(1):132, 2018; and Cho et al., Cell 173:1426-1438, 2018. In some embodiments herein, a B7H3-specific monoclonal antibody is fused to a component of a specific binding pair. In some instances, the monoclonal antibody is fused to a leucine zipper or biotin.

[0266] Another type of universal CAR can be generated using a sortase. A sortase is a prokaryotic enzyme that modifies surface proteins by recognizing and cutting a carboxyl-terminal sorting signal. The sortase catalyzes the transpeptide reaction between the sortase recognition motif and the sortase receptor motif. Therefore, antigen-specific CARs can be produced by contacting an antigen-specific antibody fused to a sortase recognition motif with a portion of a CAR molecule including an intracellular signaling domain, a transmembrane region, and an extracellular portion (comprising a sortase receptor motif). In the presence of a sortase, the two components are covalently attached to form a complete antigen-specific CAR. Therefore, in some embodiments herein, B7H3-specific monoclonal antibodies are modified to include a sortase recognition motif (see, e.g., PCT Publication No. WO 2016 / 014553).

[0267] V. Immunoconjugates

[0268] 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 that has a specific biological activity against a specific target molecule or a cell bearing a target molecule. Those skilled in the art will appreciate that therapeutic agents may include various drugs (e.g., vinblastine, daunomycin, etc.), cytotoxins (e.g., natural or modified Pseudomonas exotoxin or diphtheria toxin), encapsulating agents containing pharmacological compositions (e.g., liposomes), radioactive agents (e.g., 125 I. 32 P. 14 C. 3 H and 35 S), photon absorbers (e.g. IR700), and other labels, targeting moieties and ligands.

[0269] The choice of a particular therapeutic agent depends on the particular target molecule or cell, and the desired biological effect. Thus, for example, the therapeutic agent may be a cytotoxin designed to cause the death of a particular target cell (e.g., a tumor cell). Conversely, where it is desired to induce a non-lethal biological response, the therapeutic agent may be conjugated to a non-lethal pharmacological agent or to a liposome containing a non-lethal pharmacological agent.

[0270] Using the therapeutic agents and antibodies described herein, one skilled in the art can easily construct multiple clones containing functionally equivalent nucleic acids (e.g., nucleic acids with different sequences but encoding the same effector portion or antibody sequence). Therefore, the present disclosure provides nucleic acids encoding antibodies and their conjugates and fusion proteins.

[0271] The effector molecule can be connected to the antibody of interest using any number of methods known to those skilled in the art. Covalent and non-covalent attachment methods can be used. The procedure for attaching the effector molecule to the antibody varies according to the chemical structure of the effector molecule. Polypeptides generally contain a variety of functional groups; for example, carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which can be used to react with suitable functional groups on the antibody so that the effector molecule binds. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. Derivatization can involve attaching any of many known linker molecules. The linker can be any molecule for connecting the antibody to the effector molecule. The linker can form a covalent bond with the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched carbon linkers, heterocyclic carbon linkers or peptide linkers. In the case where the antibody and the effector molecule are polypeptides, the linker can be connected to the α-carbon amino and carboxyl groups of the constituent amino acids or to the terminal amino acids through their side groups (e.g., through a disulfide bond with cysteine).

[0272] In some cases, when the immunoconjugate reaches its target site, it is desirable to release the effector molecule from the antibody. Therefore, in these cases, the immunoconjugate will contain a bond that is cleavable near the target site. Cleavage of the linker to release the effector molecule from the antibody can be promoted by enzymatic activity or by conditions to which the immunoconjugate is subjected inside the target cell or near the target site.

[0273] Given that a large number of methods have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (e.g., enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, one skilled in the art will be able to determine the appropriate method for attaching a given agent to an antibody or other polypeptide.

[0274] The antibodies disclosed herein can be derivatized or linked to another molecule (e.g., another peptide or protein). Typically, the antibody or portion thereof is derivatized so that binding to the target antigen is not adversely affected by derivatization or labeling. For example, an antibody can be functionally linked (by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other molecular entities, examples of which are another antibody (e.g., a bispecific antibody or a diabody), a detection agent, a photon absorber, a pharmaceutical agent, and / or a protein or peptide that mediates the association of an antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0275] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, e.g., to produce bispecific antibodies). Suitable cross-linking agents include heterobifunctional cross-linkers, having two distinct reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are commercially available.

[0276] The antibody can be conjugated to a detectable marker; for example, a detectable marker that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic inspection, and laparoscopy). Specifically, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic bonds, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) can also be used. The antibody or antigen binding fragment can also be conjugated to an enzyme that can be used for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When the antibody or antigen binding fragment is conjugated to a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a recognizable reaction product. For example, when the horseradish peroxidase reagent is present, the addition of hydrogen peroxide and diaminobenzidine will result in a colored reaction product that can be detected visually. The antibody or antigen binding fragment can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or a fluorescent marker.

[0277] Antibodies can be labeled with magnetic agents such as gadolinium. Antibodies can also be labeled with lanthanides such as europium and dysprosium and manganese. Paramagnetic particles such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be labeled with a predetermined polypeptide epitope (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag) that is recognized by a secondary reporter molecule. In some embodiments, the labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0278] Antibodies can also be labeled with radiolabeled amino acids. Radiolabels can be used for diagnostic and therapeutic purposes. For example, radiolabels can be used to detect the expression of the target antigen by x-ray, emission spectroscopy or other diagnostic techniques. Examples of polypeptide labels 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.

[0279] The antibodies disclosed herein may also be conjugated to a photon absorber. In some embodiments, the photon absorber is a phthalocyanine dye, such as, but not limited to, 700DX (also known as "IR700"). Antibody-photoabsorber conjugates can be used in photoimmunotherapy.

[0280] Antibodies can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups or carbohydrate groups. These groups can be used to improve the biological properties of antibodies, such as extending serum half-life or enhancing tissue binding.

[0281] Toxins can be used together with the monoclonal antibodies described herein to produce immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxin and its subunits, and botulinum toxins A to F. These toxins are easily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, Missouri). Toxins contemplated also include variants of toxins described herein (see, e.g., U.S. Patents 5,079,163 and 4,689,401). In one embodiment, the toxin is a Pseudomonas exotoxin (PE) (U.S. Patent 5,602,095). As used herein, "Pseudomonas exotoxin" refers to full-length natural (naturally occurring) PE or modified PE. Such modifications may include, but are not limited to, elimination of domain Ia, multiple 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).

[0282] PE used with the monoclonal antibodies described herein can include native sequences, cytotoxic fragments of native sequences, and conservatively modified variants of native PE and its cytotoxic fragments. Cytotoxic fragments of PE include cytotoxic fragments with or without subsequent proteolytic processing or other processing in target cells. Cytotoxic fragments of PE include PE40, PE38 and PE35. For additional description of PE and its variants, see, e.g., U.S. Patent Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; 5,854,044; U.S. Patent Application Publication No. 2015 / 0099707; PCT Publication Nos. WO 99 / 51643 and WO 2014 / 052064; Pai et al., Proc. Acad. Sci. USA 88:3358-3362, 1991; Kondo et al., J. Biol. Chem. 263:9470-9475, 1988; Pastan et al., Acta Biochimica et Biophysica Sin. 1333:C1-C6, 1997.

[0283] Also contemplated herein are 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 (see, e.g., Weldon et al., Blood 113(16):3792-3800, 2009; Onda et al., Proc. Natl. Acad. Sci. USA 105(32):11311-11316, 2008; and PCT Publication Nos. WO 2007 / 016150, WO 2009 / 032954, and WO 2011 / 032022, incorporated herein by reference).

[0284] In some instances, PE is a variant resistant to lysosomal degradation, such as PE-LR (Weldon et al., Blood 113(16):3792-3800, 2009; PCT Publication No. WO 2009 / 032954). In other instances, PE is a variant designated as PE-LR / 6X (PCT Publication No. WO 2011 / 032022). In other instances, the PE variant is a PE with reduced immunogenicity. In yet another instance, PE is a variant designated as PE-LR / 8M (PCT Publication No. WO 2011 / 032022).

[0285] Modification of PE can occur in any previously described variants, including cytotoxic fragments of PE (e.g., PE38, PE-LR, and PE-LR / 8M). The modified PE can include any substitution, such as substitution of one or more amino acid residues within one or more T cell epitopes and / or B cell epitopes of PE, or deletion of one or more T cell and / or B cell epitopes (see, e.g., U.S. Patent Application Publication No. 2015 / 0099707).

[0286] It is contemplated that PE also includes PE in a deimmunized form, such as a version with domain II deleted (e.g., PE24). Deimmunized forms of PE are described in, for example, PCT Publications WO 2005 / 052006, WO 2007 / 016150, WO 2007 / 014743, WO 2007 / 031741, WO 2009 / 32954, WO 2011 / 32022, WO 2012 / 154530, and WO 2012 / 170617.

[0287] The antibodies described herein can also be used to target any number of different diagnostic or therapeutic compounds to cells expressing B7H3 on their surfaces. Therefore, the antibodies disclosed herein can be attached directly or via a linker to a drug that will be delivered directly to cells expressing cell surface B7H3. This can be used for treatment, diagnosis or research purposes. Therapeutic agents include compounds such as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioactive isotopes, photon absorbers, lipids, carbohydrates or recombinant viruses. Nucleic acid therapy and diagnostic moieties include antisense nucleic acids, derivative oligonucleotides for covalent crosslinking with single-stranded or double-stranded DNA, and oligonucleotides that form triple chains.

[0288] Alternatively, the molecule linked to the antibody can be an encapsulation system, such as a nanoparticle, liposome or micelle, containing a therapeutic composition (e.g., a drug), a nucleic acid (e.g., an antisense nucleic acid), or another therapeutic moiety that is preferably shielded from direct exposure to the circulatory system. Methods for preparing liposomes attached to antibodies 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).

[0289] The antibodies described herein may also be covalently or non-covalently linked to a detectable label. Detectable labels suitable for such uses include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. Available labels include magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, etc.), radioactive labels (e.g., 3 H.125 I. 35 S. 14 C or 32 P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and colorimetric labels, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

[0290] Methods for detecting such labels are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme labels are usually detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, while colorimetric labels are detected by simply observing the colored label.

[0291] VI. Antibody Drug Conjugates (ADCs)

[0292] ADCs are compounds composed of a tumor antigen-specific antibody (e.g., a single domain antibody or antigen-binding fragment of an immunoglobulin) and a drug (usually a cytotoxic agent, such as an anti-microtubule agent or a cross-linking agent). Because ADCs are able to specifically target cancer cells, the drug can be more effective than drugs used in standard chemotherapy. IC values ​​for the most common cytotoxic drugs currently used with ADCs are 50 100 to 1000 times stronger than conventional chemotherapy drugs. Common cytotoxic drugs include anti-microtubule agents such as maytansines and auristatins (e.g., auristatin E and auristatin F). Other cytotoxins used with ADCs include pyrrolobenzodiazepines (PBDs), which covalently bind to the minor groove of DNA to form interchain crosslinks. In many cases, the antibody to drug ratio of ADCs is 1:2 to 1:4 (Bander, Clinical Advances in Hematology & Oncology 10(8; Suppl 10):3-7, 2012).

[0293] The antibody and drug can be linked by a cleavable or non-cleavable linker. However, in some cases, it is desirable to have a linker that is stable in circulation, thereby preventing systemic release of the cytotoxic drug, which could result in significant off-target toxicity. Non-cleavable linkers prevent release of the cytotoxic agent before the ADC is internalized by the target cell. Once in the lysosome, cleavage of the antibody by lysosomal proteases allows the release of the cytotoxic agent (Bender, Clinical Advances in Hematology and Oncology 10(8; Suppl 10):3-7, 2012).

[0294] One method for site-specific and stable conjugation of drugs to monoclonal antibodies is through glycan engineering. Monoclonal antibodies have a conserved N-linked oligosaccharide chain at the Asn297 residue of each heavy chain CH2 domain (Qasba et al., Biotechnol Prog 24:520-526, 2008). Using mutant β1,4-galactosyltransferase (Y289L-Gal-T1; U.S. Patent Application Publication Nos. 2007 / 0258986 and 2006 / 0084162, incorporated herein by reference), 2-keto-galactose is transferred to the free GlcNAc residue on the antibody heavy chain, thereby performing conjugation chemistry.

[0295] Oligosaccharide chains attached to monoclonal antibodies can be divided into three groups based on the terminal galactose residues - 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 can convert the antibodies to the IgG-G0 glycoform. Mutant β1,4-galactosyltransferase is able to transfer 2-keto-galactose or 2-azido-galactose from their respective UDP derivatives to the GlcNAc residues on the IgG-G1 and IgG-G0 glycoforms. Chemical treatment on the transfer sugar enables a variety of molecules to be conjugated to monoclonal antibodies through the glycan residues (Kaspar et al., Advances in Biotechnology 24:520-526, 2008).

[0296] ADC is provided herein, including drugs (e.g., cytotoxic agents) conjugated to monoclonal antibodies that bind (e.g., specifically bind) B7H3. In some embodiments, the drug is a small molecule. In some instances, the drug is a cross-linking agent, an anti-microtubule agent and / or an anti-mitotic agent, or any cytotoxic agent suitable for mediating killing of tumor cells. Exemplary cytotoxic agents include, but are not limited to, PBD, auristatin, maytansine, dolastatin, calicheamicin, nemorubicin and its derivatives, PNU-159682, anthracycline, vinca alkaloids, taxanes, trichothecenes, CC1065, camptothecin, elinafad, compretin, dolastatin, duokamycin, enediyne, geldanamycin, indoline benzodiazepine dimer, puromycin, tubulysin, hemisteroidal hormone, splice inhibitor or pradinolide, and stereoisomers, isosteres, analogs and derivatives with cytotoxic activity.

[0297] In some embodiments, the ADC comprises a pyrrolobenzodiazepine (PBD). The natural product anthramycin (a PBD) was first reported in 1965 (Leimgruber et al., Journal of the American Chemical Society (JAm Chem Soc), 87:5793-5795, 1965; Leimgruber et al., Journal of the American Chemical Society, 87:5791-5793, 1965). Since then, many PBDs have been reported, including naturally occurring and synthetic analogs (Geratan, Medical Research Review 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 recognize and bind specific DNA sequences and have been shown to be useful as cytotoxic agents. PBD dimers have been conjugated to antibodies, and the resulting ADCs have been shown to have anticancer properties (see, e.g., US2010 / 0203007). Exemplary attachment sites on PBD dimers include five-membered pyrrole rings, tethers between PBD units, and N10-C11 imine groups (see WO 2009 / 016516; US2009 / 304710; US2010 / 047257; US2009 / 036431; US2011 / 0256157; and WO 2011 / 130598).

[0298] In some embodiments, ADC comprises an antibody conjugated to one or more maytansine molecules. Maytansines are derivatives of maytansine, and are mitotic inhibitors that work by inhibiting tubulin polymerization. Maytansine was originally isolated from the East African shrub Maytansine (U.S. Patent number 3,896,111). Subsequently, it was found that some microorganisms also produced maytansines, such as maytansinol and C-3 maytansinol esters (U.S. Patent number 4,151,042). Synthetic maytansinoids are disclosed, for example, in the following 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.

[0299] In some embodiments, the ADC includes an antibody conjugated to dolastatin or auristatin or an analog or derivative thereof (see U.S. Pat. Nos. 5,635,483; 5,780,588; 5,767,237; and 6,124,431). Auristatin is a derivative of the marine mollusk compound dolastatin-10. Dolastatin and auristatin 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. Pat. No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob Agents and 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-methyl valine-valine-aplysia isoleucine-aplysia proline-phenylalanine), MMAE (monomethyl auristatin E or N-methyl valine-valine-aplysia isoleucine-aplysia proline-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other auristatins (see, e.g., U.S. Publication No. 2013 / 0129753).

[0300] In some embodiments, the ADC comprises an antibody conjugated to one or more calicheamicin molecules. Antibiotics of the calicheamicin family and their analogs are capable of producing double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al., Cancer Res 53:3336-3342, 1993; Lode et al., Cancer Res 58:2925-2928, 1998). Exemplary methods for preparing ADCs with calicheamicin drug moieties are described in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.

[0301] In some embodiments, ADC comprises anthracycline. Anthracycline is an antibiotic compound with cytotoxic activity. It is believed that anthracycline can kill cells by a variety of different mechanisms, including embedding drug molecules into the DNA of cells, thereby inhibiting DNA-dependent nucleic acid synthesis; inducing the production of free radicals, which then react with cell macromolecules to damage cells; and / or the interaction of drug molecules with cell membranes. Non-limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, daunorubicin, doxorubicin, epirubicin, nemorubicin, valrubicin and mitoxantrone, and their derivatives. For example, PNU-159682 is an effective metabolite (or derivative) of nemorubicin (Quintieri et al., Clinical Cancer Research (Clin Cancer Res) 11 (4): 1608-1617, 2005). Nemorubicin is a semisynthetic analog of doxorubicin with a 2-methoxymorpholinyl group on the glycosidic amino group of doxorubicin (Grandi et al., Cancer Treat Rev 17:133, 1990; Ripamonti et al., Br J Cancer 65:703-707, 1992).

[0302] In some embodiments, the ADC may further include a linker. In some instances, the linker is a bifunctional or multifunctional moiety that can be used to connect one or more drug moieties to an antibody to form an ADC. In some embodiments, an ADC is prepared using a linker having a reactive functional group for covalently attaching a drug and an antibody. For example, a cysteine ​​thiol of an antibody can form a bond with a reactive functional group of a linker or a drug-linker intermediate to prepare an ADC.

[0303] In some instances, the linker has a functional group that can react with the free cysteine ​​present on the antibody to form a covalent bond. Exemplary linkers with such reactive functional groups include maleimide, haloacetamide, α-haloacetyl, activated esters (such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters), anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0304] In some instances, the joint has a functional group that can react with the electrophilic group present on the antibody. Examples of such electrophilic groups include, but are not limited to, aldehydes and ketone carbonyls. In some cases, the heteroatom of the joint reactive functional group can react with the electrophilic group on the antibody and form a covalent bond with the antibody unit. Non-limiting examples include hydrazides, oximes, amino, hydrazine, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0305] In some instances, the linker is a cleavable linker that promotes drug release. Examples of cleavable linkers include acid-labile linkers (e.g., containing hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive linkers), photolabile linkers, and disulfide-containing linkers (Chari et al., Cancer Research 52: 127-131, 1992; U.S. Pat. No. 5,208,020).

[0306] The ADCs disclosed herein can be used alone to treat B7H3-positive cancers, or in combination with another therapeutic agent and / or in combination with any standard therapy for treating cancer (e.g., surgical resection of the tumor, chemotherapy, or radiation therapy) to treat B7H3-positive cancers.

[0307] VII. Multispecific Antibodies

[0308] 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. Therefore, bispecific antibodies bind to two different antigens, while trispecific antibodies bind to three different antigens. Multispecific antibodies can be used for cancer immunotherapy by simultaneously targeting, for example, CTL (e.g., 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 produce multispecific (e.g., bispecific or trispecific) antibodies targeting both B7H3 and CTL or both B7H3 and NK cells, thereby providing a method for treating cancers expressing B7H3.

[0309] Bispecific T cell engagers (BiTEs) are a type of bispecific monoclonal antibody that fuses a first monoclonal antibody (such as a scFv or single domain antibody) that targets a tumor antigen (such as B7H3) and a second antibody that binds to T cells (such as CD3 on T cells). In some embodiments herein, one of the binding moieties of the BiTE is specific for B7H3.

[0310] Bispecific killer cell engagers (BiKEs) are a type of bispecific monoclonal antibody that fuses a first monoclonal antibody (such as a scFv or single domain antibody) that targets a tumor antigen (such as B7H3) and a second scFv that binds to a NK cell activating receptor (such as CD16).

[0311] Provided herein are multispecific (e.g., trispecific or bispecific) monoclonal antibodies comprising B7H3-specific monoclonal antibodies. In some embodiments, the multispecific monoclonal antibodies further comprise monoclonal antibodies that specifically bind to a T cell receptor component (e.g., CD3). In other embodiments, the multispecific monoclonal antibodies further comprise monoclonal antibodies that specifically bind to NK cell activation receptors (e.g., CD16, Ly49, or CD94). Also provided herein are isolated nucleic acid molecules and vectors encoding multispecific antibodies, as well as host cells comprising the nucleic acid molecules or vectors. Multispecific antibodies comprising B7H3-specific antibodies can be used to treat cancers expressing B7H3. Therefore, provided herein are methods for treating subjects with cancer, i.e., selecting subjects with B7H3-expressing cancers, and administering to the subjects a therapeutically effective amount of multispecific antibodies targeting B7H3.

[0312] VIII. Antibody-Nanoparticle Conjugates

[0313] The monoclonal antibodies disclosed herein can be conjugated with a variety of different types of nanoparticles to combine with B7H3 expressed on the surface of tumor cells through antibodies, and cytotoxic agents or other anticancer agents are delivered directly to tumor cells. The use of nanoparticles reduces off-target side effects, and can also improve the bioavailability of drugs and reduce the drug dosage required to achieve therapeutic effects. Nanoparticle formulations can be customized to be suitable for drugs carried or encapsulated in nanoparticles. For example, hydrophobic molecules can be incorporated into the core of nanoparticles, and hydrophilic drugs can be carried in aqueous cores protected by polymer shells or lipid shells. Examples of nanoparticles include, but are not limited to, nanospheres, nanocapsules, liposomes, dendrimers, polymer micelles, vesicles, and polymer nanoparticles (Fay and Scott, "Immunotherapy" (Immunotherapy) 3 (3): 381-394, 2011).

[0314] Liposomes are common nanoparticle types for drug delivery. Antibodies conjugated to liposomes are commonly referred to as "immunoliposomes". The liposome component of immunoliposomes is generally a lipid vesicle of one or more concentric phospholipid bilayers. In some cases, phospholipids are composed of a hydrophilic head group and two hydrophobic chains, so that hydrophobic drugs and hydrophilic drugs can be encapsulated. Conventional liposomes are rapidly removed from circulation by macrophages of the reticuloendothelial system (RES). In order to produce long-circulating liposomes, the composition, size and charge of the liposomes can be adjusted. The surface of the liposome can also be modified, for example, with glycolipids or sialic acid. For example, the addition of polyethylene glycol (PEG) significantly prolongs the circulation half-life. Liposomes for use as drug delivery agents, including liposomes for preparing immunoliposomes, have been described in the art (see, e.g., Paszko and Senge, Curr Med Chem 19(31):5239-5277, 2012; Immordino et al., Int J Nanomedicine 1(3):297-315, 2006; U.S. Patent Application Publication Nos. 2011 / 0268655; 2010 / 00329981).

[0315] Niosomes are vesicles based on nonionic surfactants, similar in structure to liposomes. The vesicle membrane consists only of nonionic surfactants, such as polyglyceryl alkyl ethers or N-palmitoyl glucosamine. Vesicles range from small unilamellar particles to large multilamellar particles. These nanoparticles are monodisperse, water-soluble, chemically stable, have low toxicity, are biodegradable and non-immunogenic, and improve the bioavailability of encapsulated drugs.

[0316] Dendrimers include a family of branched polymer complexes. These nanoparticles are water-soluble, biocompatible, and sufficiently non-immunogenic for human use. Typically, a dendrimer consists of an initiator core, which is surrounded by a layer of a selected polymer (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 in a variety of therapeutic and diagnostic applications, including for delivery of DNA, RNA, bioimaging contrast agents, and chemotherapeutic agents.

[0317] Polymer micelles consist of aggregates of amphiphilic copolymers (composed of hydrophilic and hydrophobic monomeric units) assembled into a hydrophobic core surrounded by a hydrophilic polymer corona exposed to an aqueous environment. In many cases, the polymer used to prepare polymer micelles is a heterobifunctional copolymer composed of a hydrophilic block of PEG, poly(vinyl pyrrolidone) and a hydrophobic poly(L-lactide) or poly(L-lysine) forming the core of the particle. Polymer micelles can be used to carry poorly soluble drugs. These nanoparticles have been used to encapsulate many anticancer drugs, including doxorubicin and camptothecin. Cationic micelles have also been developed to carry DNA or RNA molecules.

[0318] Polymer nanoparticles include nanospheres and nanocapsules. Nanospheres are composed of a solid matrix of polymers, while nanocapsules contain an aqueous core. The selected formulation usually depends on the solubility of the therapeutic agent to be carried / encapsulated; drugs with poor water solubility are more easily encapsulated in nanospheres, while water-soluble and unstable drugs (such as DNA and proteins) are more easily encapsulated in nanocapsules. The polymers used to produce these nanoparticles include, for example, poly(acrylamide), poly(esters), poly(alkyl cyanoacrylates), poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and poly(D,L-lactic-co-glycolic acid) (PLGA).

[0319] According to standard methods known in the art, the antibody can be conjugated to suitable nanoparticles. For example, the conjugation can be covalent or non-covalent. In some embodiments, where the nanoparticle is a liposome, the antibody is attached to a spatially stable long-circulating liposome via a PEG chain. The coupling of an antibody or antibody fragment to a liposome can also involve a thioester bond, such as by the reaction of a thiol and a maleimide group. Cross-linking agents can be used to generate sulfhydryl groups to attach the antibody to the nanoparticle (Pasco and Senger, Contemporary Medicinal Chemistry 19 (31) 5239-5277, 2012).

[0320] IX. Compositions and Methods of Use

[0321] The compositions provided herein include one or more disclosed monoclonal antibodies that bind (e.g., specifically bind) to B7H3 in a carrier. Compositions comprising ADC, CAR (and CTL or other cells comprising CAR), multispecific (e.g., bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes, and immunoconjugates are also provided. The composition can be prepared into a unit dosage form for administration to a subject. The amount and time of administration are determined by the treating clinician to achieve the desired result. Antibodies, ADC, CAR, cells expressing CAR, multispecific antibodies, antibody-nanoparticle conjugates, immunoliposomes, or immunoconjugates can be formulated for systemic or local (e.g., intratumoral) administration. In one example, the antibody is formulated for parenteral administration, such as intravenous administration.

[0322] The composition for administration may include a solution of an antibody, ADC, CAR, a cell expressing CAR (e.g., CTL), a multispecific (e.g., bispecific or trispecific) antibody, an antibody-nanoparticle conjugate, an immunoliposome, or an immunoconjugate in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers may be used, such as buffered saline, etc. These solutions are sterile and generally free of unwanted substances. These compositions may be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances as needed to approach physiological conditions, such as pH regulators and buffers, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The antibody concentrations in these formulations may vary greatly, and will be selected primarily based on the amount of body fluid, viscosity, body weight, etc., according to the selected specific mode of administration and the needs of the subject.

[0323] Typical pharmaceutical compositions for intravenous administration include about 0.1 to 10 mg of antibody (or ADC, CAR, multispecific antibody, antibody-nanoparticle conjugate or immunoconjugate) per subject per day. A dosage of 0.1 to about 100 mg per subject per day can be used, especially if the agent is applied to a hidden site rather than the circulatory system or lymphatic system, such as a body cavity or an organ lumen. The actual method of preparing the composition that can be applied is known or obvious to those skilled in the art and is described in such publications as Remington: The Science and Practice of Pharmacy, University of the Sciences in Philadelphia, editor, Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, 21st edition (2005).

[0324] The monoclonal antibodies disclosed herein can also be administered by other routes, including by inhalation, oral, topical or intraocular administration. In some examples, the monoclonal antibody (or its conjugate) is administered by a fine needle.

[0325] Although antibodies (or other therapeutic molecules) are also provided as sterile solutions of known concentrations, they can be provided in lyophilized form and rehydrated with sterile water before administration. The antibody solution is then added to an infusion bag containing 0.9% sodium chloride (USP) and, in some cases, administered at a dose of 0.5 to 15 mg / kg body weight. TMSince its approval, the art has a lot of experience in administering antibody drugs that have been listed in the United States. Antibodies, ADCs, CARs (or cells expressing CARs), multispecific (such as bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes or immunoconjugates can be administered by slow infusion rather than intravenous push or high-dose push. In one example, a higher loading dose is administered, followed by a maintenance dose at a lower level. For example, an initial loading dose of 4 mg / kg can be infused over a period of about 90 minutes, and if the previous dose is well tolerated, a maintenance dose of 2 mg / kg is infused over 30 minutes each week for 4-8 weeks.

[0326] Controlled release parenteral formulations can be made into implants, oily injections or microparticle systems. For a comprehensive overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: formulation, processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, Pennsylvania, (1995). Microparticle systems include, for example, microspheres, microparticles, microcapsules, nanocapsules, nanospheres and nanoparticles. Microcapsules contain therapeutic proteins such as cytotoxins or drugs as cores. In microspheres, the therapeutic agent is dispersed throughout the particles. Particles, microspheres and microcapsules less than about 1 μm are usually referred to as nanoparticles, nanospheres and nanocapsules, respectively. The diameter of capillaries is about 5 μm, so only nanoparticles can be administered intravenously. The diameter of microparticles is usually about 100 μm and can be administered subcutaneously or intramuscularly. See, e.g., 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, eds., Marcel Dekker, Inc., New York, NY, pp. 315-339, (1992).

[0327] Polymers can be used for ion controlled release of antibody-based compositions disclosed herein. Various degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26: 537-542, 1993). For example, block copolymer polaxamer 407 exists as a viscous and flowing liquid at low temperatures, but forms a semisolid gel at body temperature. It has been shown to be an effective medium for the preparation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9: 425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44 (2): 58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112: 215-224, 1994). On the other hand, liposomes are used for controlled release of lipid-encapsulated drugs and drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, Pennsylvania (1993)). Many additional systems for the controlled delivery of therapeutic proteins are known (see U.S. Pat. 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).

[0328] A. Treatment methods

[0329] Antibodies, compositions, CARs (and cells expressing CARs, such as CTLs), ADCs, multispecific (e.g., bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes, and immunoconjugates disclosed herein can be administered to slow or inhibit the growth of tumor cells or inhibit the metastasis of tumor cells, such as B7H3-positive solid tumors. In these applications, the therapeutically effective amount of the composition is an amount administered to a subject sufficient to inhibit the growth, replication, or metastasis of cancer cells or to inhibit the signs or symptoms of cancer. Suitable subjects may include subjects diagnosed with solid tumors expressing B7H3, such as, but not limited to, liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer (e.g., neuroblastoma or glioblastoma), pediatric cancer (e.g., osteosarcoma, neuroblastoma, rhabdomyosarcoma, or Ewing's sarcoma), melanoma, or mesothelioma.

[0330] Provided herein is a method for treating a subject's B7H3-positive cancer by administering to the subject a therapeutically effective amount of a B7H3-specific antibody, immunoconjugate, CAR (or a cell expressing CAR), ADC, multispecific (e.g., bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome or composition disclosed herein. Also provided herein is a method for inhibiting tumor growth or metastasis of a subject's B7H3-positive cancer by administering to the subject a therapeutically effective amount of a B7H3-specific antibody, immunoconjugate, CAR (e.g., a cell expressing CAR), ADC, multispecific (e.g., bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome or composition disclosed herein. In some embodiments, the B7H3-positive cancer is liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer (e.g., neuroblastoma or glioblastoma), pediatric cancer (e.g., osteosarcoma, neuroblastoma, rhabdomyosarcoma or Ewing's sarcoma), melanoma or mesothelioma.

[0331] The therapeutically effective amount of the B7H3-specific monoclonal antibody, ADC, CAR (e.g., CTL expressing CAR), multispecific (e.g., bispecific or trispecific) antibody, immunoconjugate, immunoliposome or composition disclosed herein will depend on the severity of the disease, the type of disease and the overall condition of the patient's health. The therapeutically effective amount of an antibody-based composition is an amount that can provide subjective relief of symptoms or objectively identifiable improvements noted by a clinician or other qualified observer.

[0332] In one example, the B7H3-specific antibodies provided herein are conjugated to IR700, and photoimmunotherapy is used to treat B7H3-positive cancer. For example, such a method may include administering a therapeutically effective amount of one or more B7H3-specific antibody-IR700 conjugates to a subject with B7H3-positive cancer, wherein the B7H3-specific antibody specifically binds to B7H3 on cancer cells. After administration of the conjugate, the subject is exposed to light at a wavelength of 660 to 740 nm (e.g., 660 to 710 nm, e.g., 680 nm) and at least 1 Jcm -2 In some examples, the dose of at least 1 J cm -2 (e.g. at least 1 J cm -2 , at least 4J cm -2 , at least 10 J cm -2 , at least 50 J cm -2 or at least 100 J cm -2 ) at a dose of 660 to 740 nm (such as 660 to 710 nm, for example 680 nm) to irradiate the B7H3-positive cancer, thereby treating the tumor in the subject. In some examples, multiple rounds of treatment are performed, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 treatment cycles. In a specific example, for example when administered intravenously, the therapeutically effective dose of the B7H3-specific antibody-IR700 conjugate is at least 0.5 milligrams per 60 kilograms (mg / kg), at least 5 mg / 60kg, at least 10 mg / 60kg, at least 20 mg / 60kg, at least 30 mg / 60kg, at least 50 mg / 60kg, such as 0.5 to 50 mg / 60kg, such as 1 mg / 60kg, 2 mg / 60kg, 5 mg / 60kg, 20 mg / 60kg or 50 mg / 60kg. In another example, the therapeutically effective dose of the B7H3-specific antibody-IR700 conjugate is 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, e.g., when administered intratumorally or intraperitoneally. In one example, when administered as a topical solution, the therapeutically effective dose of the B7H3-specific antibody-IR700 conjugate is at least 1 μg / ml, such as at least 500 μg / ml, such as 20 μg / ml to 100 μg / ml, such as 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.

[0333] Administration of 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 anticancer agents or therapeutic treatments (e.g., surgical removal of tumors). Any suitable anticancer agent may be administered in combination with antibodies, compositions, and immunoconjugates disclosed herein. Exemplary anticancer agents include, but are not limited to, chemotherapeutics, such as mitotic inhibitors, alkylating agents, antimetabolites, embedded antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g., anti-androgens), and anti-angiogenic agents. Other anticancer treatments include radiotherapy and other antibodies (e.g., biologics) that specifically target cancer cells.

[0334] Non-limiting examples of alkylating agents include nitrogen mustards (eg, mechlorethamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkyl sulfonates (eg, busulfan), nitrosoureas (eg, carmustine, lomustine, semustine, streptozotocin, or dacarbazine).

[0335] Non-limiting examples of antimetabolites include folate analogs (eg, methotrexate), pyrimidine analogs (eg, 5-FU or cytarabine), and purine analogs (eg, mercaptopurine or thioguanine).

[0336] Non-limiting examples of natural products include vinca alkaloids (e.g., vinblastine, vincristine, or vindesine), epipodophyllotoxins (e.g., etoposide or teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (e.g., L-asparaginase).

[0337] Non-limiting examples of admixtures include platinum coordination complexes (eg, cis-diamine-dichloroplatinum II, also known as cisplatin), substituted ureas (eg, hydroxyurea), methylhydrazine derivatives (eg, procarbazine), and adrenocortical hormone inhibitors (eg, mitotane and aminoglutethimide).

[0338] Non-limiting examples of hormones and antagonists include adrenocortical steroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and medroxyprogesterone acetate), estrogens (e.g., diethylstilbestrol and ethinyl estradiol), antiestrogens (e.g., tamoxifen), and androgens (e.g., testosterone propionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include doxorubicin, melphalan, Ara-C, BiCNU, busulfan, CCNU, carboplatin, cisplatin, cyclophosphamide, daunorubicin, DTIC, 5-FU, fludarabine, hydroxyurea, idarubicin, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel (or other taxanes such as docetaxel), vinblastine, vincristine, VP-16, while some of the newer drugs include gemcitabine (Gemzar), Herceptin, irinotecan (Irinotecan, CPT-11), rosidertin injection, navelbine, rituximab STI-571, taxotere, topotecan (and mesityl), Xeloda (capecitabine), Zevelin, and calcitriol.

[0339] Non-limiting examples of immunomodulators that can be used include AS-10 (Wyeth-Ayerst Labs), bropiridamine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), Imreg (Imreg, New Orleans, Louisiana), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0340] 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, and immunoliposomes include therapeutic monoclonal antibodies, e.g., 3F8, Abagovomab, Adecatumumab, Afutuzumab, Alacizumab, Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox), Apolizumab, Arcitumomab, Bavituximab, Bectumomab, Belimumab, Besilesomab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Capromab pendetide, Catumaxomab, CC49, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan), conatumumab, dacetuzumab, detumomab, ecomeximab, eculizumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab, galiximab, gemtuzumab ozogamicin, girentuximab, glembatumumab vedotin, ibritumomabtiuxetan), Igovomab, Imciromab, Intetumumab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumab mertansine), Lucatumumab, Lumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab tafenatox, Naptumomabestafenatox, Necitumumab, Nimotuzumab, Nofetumomab merpentan, Ofatumumab, Olaratumab, Oportuzumab monatox), Oregovomab, Panitumumab, Pemtumomab, Pertuzumab, Pintumomab, Pritumumab, Ramucirumab, Rilotumumab, Rituximab, Robatumumab, Satumomab pendetide, Sibrotuzumab, Sonepcizumab, Tacatuzumabtetraxetan, Tapitumomabpaptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, trastuzumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab, volociximab, votumumab, and zalutumumab. In some examples, the therapeutic antibody specifically binds to and antagonizes PD-1 or PD-L1, such as atezolizumab, MPDL3280A, BNS-936558 (nivolumab), pembrolizumab, pidilizumab, CT011, AMP-224, AMP-514, MEDI-0680, BMS-936559, BMS935559, M EDI-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.

[0341] In some instances, the other therapeutic agent administered is a T cell agonist, such as an agonist of 4-1BB (CD137), OX40 and / or GITR. In one example, the other 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 instances, the other therapeutic agent administered is a 4-1BB agonist, such as a 4-1BB agonist antibody, such as mAb. Specific agonist mAbs that can be used for the disclosed method include PF-05082566 (Utolu monoclonal antibody) and BMS-663513 (Urelu monoclonal antibody). In one example, 4-1BB agonist is 4-1BB ligand (4-1BBL), such as natural 4-1BBL (such as human 4-1IBBL) or streptavidin 4-1BBL (SA-4-1BBL) complex. In some examples, the other therapeutic agent applied is GITR (glucocorticoid-induced tumor necrosis factor (TNF) receptor, or TNFRSF18) agonist, such as GITR agonist antibody, such as mAb. Specific GITR agonist mAbs that can be used with the disclosed method include DTA-1, TRX518, MK-4166, MK-1248, AMG 228, INCAGN01876, GWN323 (from Novartis (Novartis)), CK-302 (from Checkpoint Therapeutics (Checkpoint Therapeutics)) and BMS-986156. In one example, the GITR agonist is a GITR ligand (GITRL), such as a natural GITRL or a multivalent GITR ligand fusion protein. In one example, the GITR agonist is MEDI1873, a hexameric GITRL molecule with human IgG1 Fc domains. In some examples, the other therapeutic agent administered is an immunotherapy. Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Elster Laboratories), bropiridamine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Gene Research Institute), IL-2 (Certus or Hoffman-La Roche), human immunoglobulin (Chet Bio), Imreg (from Imreg in New Orleans, Louisiana), SK&F 106528 and TNF (tumor necrosis factor; Genentech).

[0342] In one example, the additional therapy is surgical treatment, such as surgical removal of the cancer or a portion thereof. Another example of treatment is radiation therapy, such as administration of radioactive material or energy (e.g., external beam radiation therapy) to the tumor site prior to surgical removal to help eradicate or shrink the tumor.

[0343] B. Diagnosis and testing methods

[0344] Provided herein are methods for detecting B7H3 proteins in vitro or in vivo. For example, the disclosed monoclonal antibodies can be used for in vivo tumor imaging. In order to use the disclosed antibodies as in vivo diagnostic reagents, the antibodies are labeled with a detectable portion, such as a radioisotope, a fluorescent marker, or a positron-emitting radionuclide. As an example, the monoclonal antibodies disclosed herein can be conjugated with positron-emitting radionuclides for positron emission tomography (PET); this diagnostic process is generally referred to as immuno-PET. Although full-length antibodies can be made into good immuno-PET reagents, their biological half-life requires waiting for a few days before imaging, which increases the associated non-target radiation dose. The biological half-life of smaller single-domain antibodies / nanoantibodies is suitable for same-day imaging.

[0345] In other cases, B7H3 expression is detected in a biological sample. The sample can be any sample, including but not limited to tissues from biopsy samples, autopsy samples, and pathological samples. Biological samples also include tissue sections, for example, frozen sections for histological purposes. Biological samples also include body fluids, such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. In some instances, the sample is a serum sample containing exosomes. The biological sample is typically obtained from a mammal, such as a human or a non-human primate.

[0346] Provided herein is a method for determining whether a subject has a B7H3-positive cancer by contacting a sample of the subject with a B7H3-specific monoclonal antibody disclosed herein and detecting binding of the antibody to the sample. An increase in 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.

[0347] In another embodiment, a method for confirming a diagnosis of B7H3-positive cancer in a subject is provided by contacting a sample from a subject diagnosed with a B7H3-positive cancer with a B7H3-specific monoclonal antibody disclosed herein, and detecting binding of the antibody to the sample. An increase in binding of the antibody to the sample compared to binding of the antibody to a control sample confirms a diagnosis that the subject has a B7H3-positive cancer.

[0348] In some examples of the disclosed methods, the monoclonal antibody is directly labeled.

[0349] In other examples, the method further comprises contacting the sample with a second antibody (detection antibody) that specifically binds to the monoclonal antibody; and detecting binding of the second antibody. An increase in binding of the second antibody to the sample compared to binding of the second antibody to a control sample detects that the subject has a B7H3-positive cancer or confirms a diagnosis that the subject has a B7H3-positive cancer.

[0350] In some instances, the cancer is liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer (e.g., neuroblastoma or glioblastoma), pediatric cancer (e.g., osteosarcoma, neuroblastoma, rhabdomyosarcoma, or Ewing's sarcoma), melanoma, or mesothelioma.

[0351] In some instances, the control sample is a sample from a subject who does not have cancer. In certain instances, the sample is a blood sample or a tissue sample.

[0352] In some embodiments of the diagnostic and detection methods, the antibody that binds (e.g., specifically binds) B7H3 is directly labeled with a detectable marker. In another embodiment, the antibody that binds (e.g., specifically binds) B7H3 (the first antibody) is unlabeled, and a second antibody or other molecule that can bind to 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 to a first antibody of a particular class and category is selected. For example, if the first antibody is human IgG, the secondary antibody can be anti-human IgG. Other molecules that can bind to the antibody include, but are not limited to, protein A and protein G, both of which are commercially available.

[0353] 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, β-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 luminophoric material is luminol; a non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125 I. 131 I. 35 S or 3 H.

[0354] In an alternative embodiment, B7H3 in a biological sample can be determined by a competitive immunoassay that utilizes a B7H3 protein standard labeled with a detectable substance and an unlabeled antibody that specifically binds to B7H3. In this assay, the biological sample, the labeled B7H3 protein standard, and the antibody that specifically binds to B7H3 are combined, and the amount of the labeled B7H3 protein standard that binds to the unlabeled antibody is determined. The amount of B7H3 in the biological sample is inversely proportional to the amount of the labeled B7H3 protein standard that binds to the antibody that specifically binds to B7H3.

[0355] The immunoassays and methods disclosed herein can be used for a variety of purposes. In one embodiment, the specifically bound antibody can be used to detect B7H3 produced in cell culture. In another embodiment, the antibody can be used to detect the amount of B7H3 in a biological sample (e.g., a tissue sample or a blood sample or a serum sample). In some instances, B7H3 is cell surface B7H3. In other instances, the B7H3 protein is soluble (e.g., in a cell culture supernatant or in a body fluid sample, such as a blood sample or a serum sample).

[0356] In one embodiment, a kit for detecting B7H3 in a biological sample (e.g., a blood sample or a tissue sample) is provided. For example, in order to confirm a cancer diagnosis in a subject, a biopsy can be performed to obtain a tissue sample for histological examination. The kit for detecting a polypeptide generally comprises a monoclonal antibody that specifically binds to B7H3, such as any monoclonal antibody disclosed herein. In another embodiment, the antibody is labeled (e.g., with a fluorescent marker, a radioactive marker, or an enzyme marker).

[0357] In one embodiment, the kit includes explanatory materials that disclose methods for using the combined B7H3 antibody. The explanatory materials can be written in electronic form (e.g., a computer floppy disk or CD) or can be visual (e.g., a video file). The kit can also include additional components to facilitate the specific application of the kit design. Thus, for example, the kit can additionally include methods for detecting markers (e.g., enzyme substrates for enzyme markers, filter sets for detecting fluorescent markers, suitable second markers, such as secondary antibodies, etc.). The kit can additionally include buffers and other conventional reagents for implementing specific methods. Such kits and appropriate contents are well known to those skilled in the art.

[0358] In one embodiment, the diagnostic kit includes an immunoassay. Although the details of the immunoassay may vary depending on the specific format used, the method of detecting B7H3 in a biological sample generally includes the step of contacting the biological sample with an antibody that specifically reacts with B7H3 under immunoreactive conditions. The antibody is allowed to specifically bind under immunoreactive conditions to form an immune complex, thereby directly or indirectly detecting the presence of the immune complex (bound antibody).

[0359] The antibodies disclosed herein can also be used in immunoassays, such as but not limited to radioimmunoassay (RIA), ELISA or immunohistochemical assays. The antibodies can also be used for fluorescence activated cell sorting (FACS). FACS uses multiple color channels, low angle and obtuse light scattering detection channels, impedance channels and other more complex detection levels to separate or sort cells (see U.S. Patent No. 5,061,620). As disclosed herein, any monoclonal antibody that binds B7H3 can be used in these assays. Therefore, the antibodies can be used in conventional immunoassays, including but not limited to ELISA, RIA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation.

[0360] The following examples are provided to illustrate certain specific features and / or embodiments. These examples should not be construed as limiting the disclosure to the specific features or embodiments described.

[0361] Example

[0362] Example 1: Materials and Methods

[0363] This example describes the materials and experimental procedures used for the studies in Example 2.

[0364] Cell lines

[0365] 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 (balance air). The neuroblastoma cell line NBEB was cultured in RPMI-1640 medium with the same supplements as DMEM. The medium was refreshed twice a week.

[0366] Protein expression and purification

[0367] 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 accomplished using a protein A column (GE Healthcare). Rabbit VH domain antibodies were expressed in E. coli as a VH-His-FLAG fusion. The 6x His tag was used for affinity purification on a nickel column (GE Healthcare) and the FLAG tag was used for protein binding assays by ELISA and cell binding assays by flow cytometry.

[0368] Construction of DNA oligonucleotide and rabbit VH phage library

[0369] To amplify the rabbit VH cDNA fragment, forward and reverse primers annealing to the 5′ and 3′ ends of the VH cDNA were synthesized according to the literature (Peng et al., J Mol Biol, 429, 2954-2973, 2017). The primers are listed in Table 1, and the underlined nucleotides correspond to the SfiI restriction enzyme site.

[0370] Table 1: Primers used to construct rabbit VH phage library

[0371]

[0372]

[0373] Use Invitrogen TM SuperScript TMRabbit VH cDNA was synthesized from total RNA isolated from immune spleens using the IV first strand synthesis kit according to the manufacturer's instructions (ThermoFisher, Cat.#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 and digested with restriction enzyme SfiI (NEB, Cat.#R0123S) and then ligated to pComb3x plasmid pre-digested with the same enzyme. Ten micrograms of the ligated product were used to transform 0.6 ml of E. coli TG1 competent cells (Lucigen, Cat.#60502-2) by electroporation according to the manufacturer's instructions. The transformed TG1 cells were recovered at 37°C for 45 min with shaking at 150 rpm, then inoculated into 1 L of 2XYT medium and cultured at 37°C for another 1 h with shaking at 250 rpm. 10 Helper phage M13KO7 (NEB, Cat. #N0315S) was added to the cell culture and cultured for another 4 hours at 37°C. The cell culture was centrifuged at 3300 g for 30 minutes to precipitate cell debris, and the supernatant containing phage particles was collected and mixed with 3 / 10 volume of PEG 8000 / NaCl solution (20% PEG in 2.5 M NaCl, autoclaved before use). The phage / PEG solution mixture was incubated on ice for 4 hours and centrifuged at 3300 g for 30 minutes. The final phage pellet was resuspended in 100 ml of PBS buffer containing 20% ​​glycerol, aliquoted in 1 ml volumes and stored at -80°C.

[0374] Phage panning method

[0375] Phage panning was performed using fixed B7H3-hFc protein. In order to exclude hFc tag conjugates, IAB-hFc controls were also fixed in parallel. ELISA plates (96-well plates) were coated with 50 μl / well of B7H3-hFc and IAB-hFc proteins (100 μg / mL in PBS) and cultured for 1 hour at 37°C. After pouring the coated protein solution, the plate and phage solution were pre-blocked by mixing with PBS buffer containing 2% BSA and cultured for 30 minutes at 37°C. After pouring the blocking buffer, the pre-blocked phage solution was added to the IAB-hFc plate, and the hFc conjugate was consumed by culturing for 1 hour at 37°C. Thereafter, the unbound phage solution was transferred to the B7H3-hFc plate and cultured for 1 hour at 37°C. B7H3-specific phage binders were eluted from the plates by incubation with pH 2.0 citrate buffer and immediately neutralized with pH 8.0 Tris-HCl buffer. The eluted output phages were re-amplified by re-infection of fresh TG1 cells, and the re-amplified phages were 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 subjected to monoclonal phage ELISA to identify B7H3-specific binders.

[0376] Phage ELISA

[0377] ELISA plates (96-well plates) were coated with B7H3-hFc and IAB-hFc tag controls. After blocking with PBS buffer containing 2% BSA, 50 microliters of pre-blocked phage solution were added to the plates and incubated at 37°C for 30 minutes. After the plates were washed twice with PBS buffer containing 0.05% Tween 20, phage binding was detected by HRP-conjugated anti-M13 antibody (Sinobiological, Cat. #11973-MM05T-H).

[0378] For ELISA of bound antibodies, different concentrations of antibodies (1:2 serial dilutions starting from 100 μg / mL) were incubated on B7H3-hFc coated plates as described above, and antibody binding was detected by HRP-conjugated anti-FLAG mouse monoclonal antibody M2 (Sigma, Cat. #A8592).

[0379] Flow cytometry

[0380] Cells were harvested by separation with trypsin-EDTA (Thermo Fisher Scientific, Cat. #25200114), centrifuged to form a pellet, and resuspended in ice-cold PBS. One million cells per ml were cultured with 10 μg / mL of B7H3 domain antibody. Antibody binding was detected by APC-conjugated anti-FLAG mouse monoclonal antibody (Biolegend, Cat. 637308). Fluorescence associated with live cells was measured using FACS Calibur (BD Biosciences, Franklin Lakes, NJ).

[0381] Statistical analysis

[0382] All statistical analyses were performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA).

[0383] Example 2: Generation of rabbit nanobodies against B7H3 by protein immunization and phage display

[0384] This example describes the selection and characterization of two B7H3-specific rabbit single domain VH monoclonal antibodies.

[0385] Preparation of recombinant B7H3 protein

[0386] The extracellular domain (ECD) of B7H3 (NP_001019907, amino acids 29-466; SEQ ID NO: 13) was fused to human IgG1Fc and expressed in a secretory manner in HEK293 cells. After purification on a protein A column, the purity was checked by running on SDS-PAGE (Figure 1). Probably due to glycosylation and B7H3 having six N-glycosylation sites, the theoretical size of reduced B7H3-hFc is about 75kD, and the apparent migration position on the gel is about 100kD. The transient expression level of B7H3-hFc is extremely low, about 0.5mg / L.

[0387] Immunization of rabbits with recombinant B7H3-hFc

[0388] 100 μg 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 3 immunizations at 14-day intervals, the titer of anti-B7H3-hFc was measured by ELISA using IAB-hFc as hFc control ( Figure 2AIAB is a fragment of mesothelin (Q13421, amino acids 296-359) (Kanebo et al., Journal of Biochemistry, 284, 3739-3749, 2009). The sera from the second (M2) and third (M3) immunizations clearly showed increased binding of B7H3-hFc compared to the IAB-hFc tag control. The cell binding activity of the polyclonal sera was examined by flow cytometry ( Figure 2B The final immunized polyclonal sera clearly showed cell binding to 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 background binding of the pre-immunized sera was extremely low. The results of ELISA and flow cytometry showed that B7H3 has good immunogenicity in rabbits, although the protein is highly conserved, especially between humans and rabbits.

[0389] Screening of B7H3-specific binders

[0390] After confirming successful immunization, the spleen of the immunized rabbit was taken and the VH gene fragment was cloned using degenerate primers (Peng et al., Journal of Molecular Biology, 429, 2954-2973, 2017), and then ligated with the phage display vector pComb3x. TG1 competent cells were transformed by electroporation using 10 μg of the ligated product, and a 7×10 9 VH library of individual clones. Library phage production and subsequent antigen panning were performed at 37°C.

[0391] Panning was performed on immobilized B7H3-hFc. Both B7-H6-hFc and IAB-hFc were coated on 96-well ELISA plates, and B7H3-specific phage particles were enriched by pre-adsorption on IAB-hFc coated plates and then captured on B7H3-hFc coated plates. After three rounds of panning, monoclonal phage ELISA was performed to identify B7H3-specific binders. Of the 96 randomly picked clones, 41 were B7H3-specific binders, and sequencing analysis identified two representative binders, termed RFA1 and RFB1 ( Figure 3A ). Both binders have very similar germline sequences that are also similar to the VH from a rabbit anti-carboxyputrescine lysine mAb (deposited in the GenBank structural database, PDB#5DUB).

[0392] Structural modeling of A1 and B1 using online tools suggests that they may have similar CDR1 and CDR2 loop conformations, which are also similar to the crystal structure of rabbit anti-carboxyputrescine lysine VH (PDB#5DUB), but their CDR3 loops appear different ( Figure 3B ).

[0393] Binding properties of B7H3 binders

[0394] The VH coding sequences of binders A1 and B1 were fused to a His-FLAG tag at their C-termini and cloned into an E. coli expression vector. The soluble VH domain was purified by one-step Ni affinity chromatography according to the laboratory protocol (Feng et al., Antibody Therapeutics, 2, 1-11, 2019). The purified yields were 2 mg / L (A1) and 10 mg / L fermentation (B1), respectively. The purity was high due to separation on SDS-PAGE ( Figure 4A The protein binding affinities of RFA1 and RFB1 were measured by ELISA, and the calculated EC50 values ​​were 403 nM (A1) and 189 nM (B1) ( Figure 4B ), which is relatively low but common for VH-only antibodies. Cell binding capacity was also tested by flow cytometry ( Figure 4C ), which showed that the A1 and B1 conjugates had good cell binding ability to B7H3-positive cell lines (IMR32, MC38-B7H3+, A431 and NBEB), but not to the B7H3 knockout cell lines IMR32-B7H3 KO and MC38-B7H3KO.

[0395] Therapeutic applications

[0396] Rabbits are an excellent resource for generating excellent monoclonal antibodies for use as research tools, diagnostic and therapeutic purposes (Weber et al., Journal of Experimental and Molecular Medicine, 49, e305, 2017). There are several major advantages to using rabbit antibodies. First, rabbits are phylogenetically farther from humans than mice, so conserved proteins that are poorly immunogenic in mice may be more immunogenic in rabbits (Popkov et al., Journal of Molecular Biology, 325, 325-335, 2003). Second, rabbit monoclonal antibodies generally have high affinity and specificity, with an affinity range of 20-200 pM (Weber et al., Journal of Experimental and Molecular Medicine, 49, e305, 2017; Landry et al., Journal of Immunological Methods, 417, 86-96, 2015). Third, rabbit monoclonal antibodies can be successfully humanized (Zhang and Huo, MAbs, 9, 419-429, 2017), so immunogenicity should not be an obstacle to their therapeutic application. Despite their many advantages, only a few rabbit monoclonal antibodies have been studied for clinical application.

[0397] Although all-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 developed. A recent group of studies showed that high-affinity rabbit VH domain antibodies can be produced by low-temperature (i.e., 16°C) phage display methods (Shinozaki et al., "Sci Rep", 7, 5794, 2017), which will greatly accelerate the research of rabbit VH domain antibodies. However, low-temperature phage display methods tend to enrich a large portion of unstable and difficult-to-express binders, which may require great efforts to improve their physicochemical properties, especially expression and thermal stability (Shinozaki et al., "Journal of Bioscience and Bioengineering", 125, 654-661, 2018). The current study investigated the possibility of using high-temperature phage display methods to screen for thermally stable and well-expressed binders.

[0398] As a proof of concept, B7H3 was selected as a target. B7H3 is overexpressed in many cancer types and can inhibit T cell activation, so B7H3 is considered to be an important immune checkpoint member of the B7 and CD28 families (Picarda et al., Clinical Cancer Research, 22, 3425-3431, 2016). It is also widely overexpressed in many solid tumors, making it a therapeutic target (Seeman et al., Cancer Cell, 31, 501-515e508, 2017). The extracellular domain of B7H3 is expressed in 293F cells, although the expression level is very low (<0.5 mg / L). Although B7H3 is highly conserved in humans, mice and rabbits, the recombinant B7H3-hFc protein is sufficiently immunogenic in rabbits, as shown by immune polyclonal sera that can bind to the recombinant protein and B7H3-positive cells. Rabbit VH phage library particles were prepared and panned using high temperature (37°C) phage display. Two representative binders with moderate affinity to B7H3 protein and B7H3-positive cancer cells were obtained. The RFA1 and RFB1 binders had good cell binding ability to B7H3-positive cells, but not to B7H3 knockout cells. This study showed that rabbit VH domain antibodies with moderate affinity and expression levels can be produced at conventional temperature (37°C) by phage display. Considering the important role of B7H3 in regulating T cell function, the two B7H3 domain antibodies generated can be used for cancer immunotherapy applications.

[0399] Example 3: Isolation of camel nanobodies targeting B7H3 (CD276) by phage display

[0400] This example describes the selection of ten camel V H H nanobodies and characterized their binding properties. Also described are nanobodies composed of V H Chimeric antigen receptor composed of H nanobodies.

[0401] Eight camel Vs targeting B7H3 binders H Phage panning on H library

[0402] B7H3-Fc fusion protein was generated to select B7H3 binders. Recombinant B7H3-Fc fusion protein was expressed in HEK-293 cells and purified on a protein A column (GE Healthcare) using AKTA Explorer (GE Healthcare). As shown on the SDA-PAGE gel, the purified B7H3-Fc fusion protein was more than 99% pure 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.

[0403] Using eight Vs made from eight camels (dromedaries) H H single domain antibody library, three rounds of phage panning were performed on the recombinant B7H3-Fc protein. The phage titer of each round is shown in Figure 6 The increase in phage titer in the third round of phage panning indicated that V with high affinity for B7H3 was enriched. H H binders. The phage binding to B7H3-Fc was also evaluated by ELISA; the results are shown in Table 2. All selected phages could bind to B7H3-Fc but not to the IgG control.

[0404] Table 2: Evaluation of selected phage binding to B7H3-Fc by ELISA

[0405] Bacteriophage B7H3-Fc IgG RWA12 2.50985 0.1147 RWB2 2.54615 0.0777 RWH5 2.88595 0.06665 RWB12 2.4812 0.1637 RWG8 2.5186 0.10125 RWD5 2.4972 0.0685 RWC3 2.422 0.08885 RWG4 2.1349 0.10345 RWD9 2.43255 0.07875 RWC4 2.6597 0.07775 RWH1 2.75015 0.13615

[0406] The selected B7H3-specific phages were tested for binding to monkey, mouse, rat and human B7H3 by ELISA. As shown in Table 3, 9 selected phages can bind to monkey B7H3, 5 phages can bind to mouse B7H3, 9 phages can bind to rat B7H3, and 8 phages can bind to human B7H3 (bold numbers indicate positive binding).

[0407] Table 3: Cross-species binding of selected B7H3 phages tested by ELISA

[0408]

[0409]

[0410] V H H purification and binding

[0411] Purification of V from selected phage H The purification of RWC4, RWG8 and RWB12 nanobodies is shown in Figures 7-9. H H camel nanobody fractions were subjected to SDS-PAGE (see Fig. 7A , 8A and 9A). The chromatogram of the nanobody eluted from a nickel column (GE Healthcare) on an AKTA Explorer (GE Healthcare) is shown in Figure 7B , 8B and 9B. The yields of RWC4, RWG8, and RWB12 were 33.8 mg / L, 50 mg / L, and 132 mg / L, respectively.

[0412] Measurement of selected V by ELISA HThe binding of H nanobodies to hB7H3-Fc, hB7H3-His, mouse B7H3-His, monkey B7H3-His and rat B7H3-His fusion proteins. The results are shown in Tables 4 and Fig. 22 Except for RWB2, V H H can bind to B7H3-Fc and B7H3-His fusion proteins. RWG8 exhibits cross-reactivity to mouse B7H3 ( Fig. 22 ). In addition, RWA12 showed cross-reactivity with PBS and PD-L1; the rest of V H H had no cross-reactivity to PDL1 (Table 5).

[0413] Table 4: Measurement of selected V by ELISA H H-binding B7H3 fusion protein

[0414]

[0415] Table 5: Cross-reactivity of selected B7H3 binders to PD-L1 measured by ELISA

[0416]

[0417]

[0418] Selected B7H3 targeting V H Binding of H to cells expressing B7H3

[0419] Evaluation of selected B7H3 targeting V by FACS analysis H Binding of H nanobody to NBEB neuroblastoma cells ( Fig.10 ). Six of the tested antibodies (RWC4, RWB12, RWG8, RWA12, RWG4, and RWD5) were able to bind to NBEB cells.

[0420] Evaluation of selected B7H3 targeting V by FACS analysis H Binding of H nanobody to A431 epidermoid cancer cells ( Fig.11 ). Six of the tested antibodies (RWC4, RWB12, RWG8, RWA12, RWG4 and RWD5) were able to bind to A431 cells.

[0421] The ability of five B7H3-targeted antibodies (RWC4, RWB12, RWG8, RWG4 and RWD5) to bind to MC38-CD276+ and MC38-CD276KO cells was also evaluated. All antibodies tested bound to B7H3-positive cells but not to B7H3-negative cells (see Table 6).

[0422] Table 6: 5 types of VH Summary of cell binding ability of H

[0423]

[0424] Binding kinetics

[0425] The association / dissociation properties of RWC4 and RWG4 were measured using recombinant human B7H3-Fc protein or recombinant mouse B7H3-His protein on the Octet system (Creative Biolabs, Inc.).

[0426] The kinetics of RWC4 binding to human B7H3-Fc are shown in Fig.12 and are summarized in Table 7. K of RWC4 against human B7H3-Fc D 3.8×10 -9 .

[0427] Table 7: Kinetics of RWC4 against human B7H3-Fc

[0428] nM <![CDATA[K D (M)]]> <![CDATA[k on (1 / Ms)]]> <![CDATA[k dis (1 / s)]]> 660 <![CDATA[3.8×10 -9 ]]> <![CDATA[2.65×10 4 ]]> <![CDATA[1.01×10 -4 ]]> 132 <![CDATA[3.8×10 -9 ]]> <![CDATA[2.65×10 4 ]]> <![CDATA[1.01×10 -4 ]]> 26.4 <![CDATA[3.8×10 -9 ]]> <![CDATA[2.65×10 4 ]]> <![CDATA[1.01×10 -4 ]]>

[0429] The kinetics of RWG4 against human B7H3-Fc were shown Fig.13 and are summarized in Table 8. K of RWG4 against human B7H3-Fc D is 6.94×10 -9 .

[0430] Table 8: Kinetics of RWG4 against human B7H3-Fc

[0431] nM <![CDATA[K D (M)]]> <![CDATA[k on (1 / Ms)]]> <![CDATA[k dis (1 / s)]]> 660 <![CDATA[6.94×10 -9 ]]> <![CDATA[9.13×10 3 ]]> <![CDATA[6.34×10 -5 ]]> 528 <![CDATA[6.94×10 -9 ]]> <![CDATA[9.13×10 3 ]]> <![CDATA[6.34×10 -5 ]]> 264 <![CDATA[6.94×10 -9 ]]> <![CDATA[9.13×10 3 ]]> <![CDATA[6.34×10 -5 ]]> 198 <![CDATA[6.94×10 -9 ]]> <![CDATA[9.13×10 3 ]]> <![CDATA[6.34×10 -5 ]]> 66 <![CDATA[6.94×10 -9 ]]> <![CDATA[9.13×10 3 ]]> <![CDATA[6.34×10 -5 ]]> 33 <![CDATA[6.94×10 -9 ]]> <![CDATA[9.13×10 3 ]]> <![CDATA[6.34×10 -5 ]]>

[0432] Expression of B7H3 on various cancer cell lines

[0433] B7H3 expression was assessed by FACS in several cancer cell lines. Expression was detected on 14 cell lines, but not on two cell lines with B7H3 (CD276) knockout (see Table 9).

[0434] Table 9: Summary of cells expressing B7H3

[0435] Cell line (cancer type) B7H3 expression A431 (human epidermoid carcinoma) + NBEB (human neuroblastoma) + IMR5 (human neuroblastoma) + MC-38-CD276+ (murine colon adenocarcinoma cells) + MC-38-CD276KO (murine colon adenocarcinoma cells) - IMR32 (human neuroblastoma) + IMR32-CD276KO (human neuroblastoma) - H9 (Human Skin T Lymphocytes) + EKVX (human lung adenocarcinoma) + LAN-1 (human neuroblastoma) + H2269 (human melanoma) + C55 + Miaca2 + OVCAR8 (human ovarian cancer) + BXPC-3 (adenocarcinoma) +

[0436] Generation of B7H3-targeted CAR

[0437] First, PCR amplification of the nanobody sequence was performed, the plasmid Pwpt and the backbone of the PCR product were digested with NdeI and SpeI, respectively, and the digested backbone was connected to the digested PCR product. After transformation, bacteria were selected on ampicillin plates.

[0438] The T cell transfection efficiency of lentivirus expressing B7H3-targeting CAR was measured by FACS. The results are shown in Fig.14 and Table 10.

[0439] Table 10: Transfection efficiency of CAR-T cells

[0440]

[0441]

[0442] Cytotoxicity of CAR-T cells targeting B7H3

[0443] The cytotoxicity of CAR-T cells targeting B7H3 against B7H3-positive cells and B7H3-knockout cells was tested. FIG15 shows the cytotoxicity of CAR-T cells targeting B7H3 against B7H3-positive cells and B7H3-knockout cells. Fig.15A ), human neuroblastoma LAN-1 cells ( Fig. 15B ), human adenocarcinoma BXPC-3 cells ( Fig. 15C ) and human pancreatic cancer Miacapa2 cells ( Fig.15D ) Results of a cytotoxicity assay. In this assay, RWB12, RWG8, and RWC4 CAR-T cells were most effective in inducing specific lysis.

[0444] In the second assay, the cytotoxicity of CAR-T cells targeting B7H3 in B7H3-positive cells and B7H3-knockout cells was evaluated. Figure 16 shows the cytotoxicity of CAR-T cells targeting B7H3 in B7H3-positive cells and B7H3-knockout cells using human neuroblastoma IMR32 cells ( Fig.16A ), mouse colon adenocarcinoma MC38-CD276 + cell( Fig. 16B ), human neuroblastoma IMR32-CD276 - / - cell( Fig. 16C ) and mouse colon adenocarcinoma MC38-CD276 - / - cell( Fig.16D ) Results of cytotoxicity assays. Three CAR T cells (RWB12, RWG8, and RWC4) showed potent cytotoxicity against B7H3-positive cells but not against B7H3-negative cells.

[0445] Example 4: B7H3-targeted CAR T cells kill pancreatic tumor cells in vitro and in vivo

[0446] The in vitro cytotoxicity of human B7H3-targeting nanobody-derived CAR T cells (RWB12, RWG8, and RWC4, referred to herein as B12, G8, and C4) was evaluated using two luciferase-expressing B7H3-positive pancreatic cancer cell lines: Panc-1 GFP-Luc (GL) and BxPC-3GL. First, flow cytometry was performed to confirm that both Panc-1 and BxPC-1 cells expressed B7H3 ( Fig.17A ). The transduction efficiency of the lentiviral constructs expressing each CAR targeting B7H3 and CAR targeting CD19 was also determined by flow cytometry. Fig. 17B As shown, 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 with an irrelevant CAR (CD19) was 32%. To evaluate cytotoxicity, CAR T cells targeting B7H3 (C4, G8 and B12) and control (CD19) CAR T cells were cultured with Panc-1 GL cells or BxPC-3GL cells at different effector: target (E: T) ratios. Both Panc-1 GL and BxPC-3GL 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 show that nanobody-based CAR T cells targeting B7H3 are able to effectively lyse B7H3-positive pancreatic cancer cell lines in vitro.

[0447] B7H3-targeted CAR T cells were further evaluated in the Panc-1 mouse xenograft model. One study used a high dose of CAR T cells (10 million), while the second study used a low dose of CAR T cells (5 million). A schematic diagram of the experimental design for the high dose study is shown in Fig.18A One million Panc-1 GFP / Luc tumor cells were implanted intravenously into NSG mice to establish the tumor model. After 20 days (day 0), mice were infused with 10 million C4, G8, or B12 CAR T cells (or control CD19 CAR T cells) intravenously and imaged weekly. Representative bioluminescent images of Panc-1 tumor growth are shown in Fig.18B Compared with infusion of control CAR T cells, mice treated with 10 million B7H3-targeted CAR T cells (C4, G8, or B12) had significantly slower tumor growth, which can be seen in mice treated with CAR T cells ( Fig.18C ) was demonstrated by a reduction in tumor bioluminescence measured in photons per second. The survival rate of mice treated with B7H3-targeted CAR T cells was also determined. Fig.18DKaplan-Meier survival curves of tumor-bearing mice after treatment with 10 million C4, G8 or B12 CAR T cells are shown. The results show that when administered at a high dose (10 million), C4 CAR T cells are more effective in promoting mouse survival than G8 or B12 CAR T cells, and indicate that the administration of 10 million CAR T cells is safe for mice.

[0448] The second in vivo study evaluated the infusion of a lower dose of B7H3-specific CAR T cells in mice bearing Panc-1 tumors after tumor rechallenge. Fig.19A As shown. Panc-1 xenograft mice were infused intravenously 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 with 1 million Panc-1-Luc cells (day 0). Mice treated with C4 CAR T cells and B12CAR T cells that did not show detectable tumors were implanted with 1 million Panc-1 cells intravenously on day 35. As a control, blank mice were implanted with Panc-1 cells. Imaging was performed weekly. Representative bioluminescent images of Panc-1 tumor growth in CAR T cell-treated mice are shown in Fig.19B Mice treated with 5 million C4 CAR T cells or 5 million B12 CAR T cells had significantly slower tumor growth compared with mice given 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 Panc-1 tumor rechallenge, while 60% of mice previously treated with B12 CAR T cells remained tumor-free 10 weeks after treatment. Quantification of tumor bioluminescence is shown in Fig.19C The survival rate of mice treated with B7H3-targeted CAR T cells was also determined. Fig.19D Kaplan-Meier survival curves of tumor-bearing mice after treatment are shown. 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 infusion.

[0449] Example 5: B7H3-targeted CAR T cells kill neuroblastoma tumor cells in vitro and in vivo

[0450] The in vitro cytotoxicity of B7H3-targeted CAR T cells against the neuroblastoma cell line IMR5 was tested. The study compared CAR T cells produced using the B7H3-targeted nanobodies disclosed herein with CAR T cells based on the commercial anti-human B7H3 hybridoma antibody 376.96 (Du et al., Cancer Cells 35(2):221-237, 2019). G8, B12, C4, and 376.96 CAR T cells were cultured with IMR5 GL cells at different effector: target (E:T) ratios for 24 hours. Compared with mock T cells, all CAR T cells effectively lysed IMR5 tumor cells in a dose-dependent manner; however, B12 CAR T cells were slightly more effective than the other tested CAR T cells ( Fig. 20A ).

[0451] Next, CAR T cells were evaluated in the IMR5 xenograft model (see Fig. 20B Schematic diagram in ). IMR5 xenograft mice were intravenously infused with 5 million C4CAR T cells, B12 CAR T cells, G8 CAR T cells, 376.96CAR T cells, or untransduced T cells (mock) 35 days after tumor inoculation (day 0). Representative bioluminescent images of IMR5 tumor growth in the xenograft model are shown in Fig. 20C , and quantification of tumor bioluminescence is shown in Fig.20D In mice treated with 5 million B12 CAR T cells, tumor growth was significantly slowed compared with 376.96CAR T cells and mock T cells. C4 CAR T cells also showed modest anti-tumor activity.

[0452] Example 6: Cross-reactivity of G8 to mouse B7H3

[0453] 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 mouse melanoma cell line B16. The in vitro cytotoxicity of CAR targeting B7H3 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 ( Fig. 21B ).

[0454] Example 7: Epitope mapping of B7H3 nanobody

[0455] Epitope mapping of anti-B7H3 nanobodies and commercial antibody 376.96 was performed. A total of 48 peptides from human B7H3 protein were designed and synthesized. Each peptide consists of 18 amino acids and overlaps with the adjacent peptide by 9 amino acids. ELISA technology was used to test the ability of the antibody to bind to each peptide ( Fig. 21C ). The results suggest that G8 and 376.96 bind to similar epitopes, as they both bind to 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.

[0456] This application also relates to the following aspects.

[0457] 1. A single domain monoclonal antibody that specifically binds to B7H3, wherein the antibody comprises the complementary 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.

[0458] 2. An antibody according to claim 1, wherein the CDR sequences are defined using the Kabat, IMGT or Paratome numbering schemes, or a combination of the Kabat, IMGT and Paratome numbering schemes.

[0459] 3. The antibody according to item 1 or 2, wherein the CDR1, CDR2 and CD3 sequences respectively comprise:

[0460] residues 31-35, 50-66 and 97-114 of SEQ ID NO:3;

[0461] residues 26-33, 51-58, and 97-115 of SEQ ID NO:3; or

[0462] Residues 26-35, 50-61 and 98-114 of SEQ ID NO:3.

[0463] 4. The antibody according to item 1 or 2, wherein the CDR1, CDR2 and CD3 sequences respectively comprise:

[0464] residues 31-35, 50-66 and 97-118 of SEQ ID NO: 1;

[0465] residues 26-33, 51-58, and 97-119 of SEQ ID NO: 1; or

[0466] Residues 27-35, 47-62 and 98-118 of SEQ ID NO:1.

[0467] 5. The antibody according to item 1 or 2, wherein the CDR1, CDR2 and CD3 sequences respectively comprise:

[0468] residues 31-35, 50-66 and 97-105 of SEQ ID NO:2;

[0469] residues 26-33, 51-58, and 97-106 of SEQ ID NO:2; or

[0470] Residues 27-35, 47-61 and 97-106 of SEQ ID NO:2.

[0471] 6. The antibody according to item 1 or 2, wherein the CDR1, CDR2 and CD3 sequences respectively comprise:

[0472] residues 31-35, 50-65, and 96-110 of SEQ ID NO:4;

[0473] residues 26-33, 51-57, and 96-110 of SEQ ID NO:4; or

[0474] Residues 27-35, 47-60 and 96-109 of SEQ ID NO:4.

[0475] 7. The antibody according to item 1 or 2, wherein the CDR1, CDR2 and CD3 sequences respectively comprise:

[0476] residues 27-30, 45-61 and 90-109 of SEQ ID NO:5;

[0477] residues 26-28, 46-53, and 90-110 of SEQ ID NO:5; or

[0478] Residues 27-30, 43-55 and 90-110 of SEQ ID NO:5.

[0479] 8. The antibody according to item 1 or 2, wherein the CDR1, CDR2 and CD3 sequences respectively comprise:

[0480] residues 31-35, 50-65, and 96-111 of SEQ ID NO:6;

[0481] residues 26-33, 51-57, and 96-112 of SEQ ID NO:6; or

[0482] Residues 27-35, 47-60 and 96-111 of SEQ ID NO:6.

[0483] 9. The antibody according to item 1 or 2, wherein the CDR1, CDR2 and CD3 sequences respectively comprise:

[0484] residues 31-35, 50-65, and 96-111 of SEQ ID NO:7;

[0485] residues 26-33, 51-57, and 96-112 of SEQ ID NO:7; or

[0486] Residues 27-35, 47-60 and 96-111 of SEQ ID NO:7.

[0487] 10. The antibody according to item 1 or 2, wherein the CDR1, CDR2 and CD3 sequences respectively comprise:

[0488] residues 31-35, 50-65, and 96-111 of SEQ ID NO:8;

[0489] residues 26-33, 51-57, and 96-112 of SEQ ID NO:8; or

[0490] Residues 27-35, 47-60 and 96-111 of SEQ ID NO:8.

[0491] 11. The antibody according to item 1 or 2, wherein the CDR1, CDR2 and CD3 sequences respectively comprise:

[0492] residues 31-35, 50-65, and 96-111 of SEQ ID NO:9;

[0493] residues 26-33, 51-57, and 96-112 of SEQ ID NO:9; or

[0494] Residues 27-35, 47-60 and 96-111 of SEQ ID NO:9.

[0495] 12. The antibody according to item 1 or 2, wherein the CDR1, CDR2 and CD3 sequences respectively comprise:

[0496] residues 31-35, 50-65 and 96-113 of SEQ ID NO:10;

[0497] residues 26-33, 51-57, and 96-114 of SEQ ID NO:10; or

[0498] Residues 27-35, 47-60 and 97-114 of SEQ ID NO:10.

[0499] 13. The antibody according to item 1 or 2, wherein the CDR1, CDR2 and CD3 sequences respectively comprise:

[0500] residues 30-34, 50-64 and 93-105 of SEQ ID NO: 11;

[0501] residues 25-32, 50-56 and 93-104 of SEQ ID NO: 11; or

[0502] Residues 26-34, 46-59 and 93-105 of SEQ ID NO:11.

[0503] 14. The antibody according to item 1 or 2, wherein the CDR1, CDR2 and CD3 sequences respectively comprise:

[0504] residues 32-35, 51-65 and 94-107 of SEQ ID NO:12;

[0505] residues 26-33, 51-57, and 94-107 of SEQ ID NO:12; or

[0506] Residues 27-35, 47-60 and 94-108 of SEQ ID NO:12.

[0507] 15. An antibody according to any one of items 1-14, 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.

[0508] 16. The antibody according to 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.

[0509] 17. The antibody according to any one of items 1-14, wherein the antibody is a humanized antibody.

[0510] 18. The antibody according to any one of items 1-14, wherein the antibody is a chimeric antibody.

[0511] 19. A chimeric antigen receptor (CAR) comprising the antibody of any one of items 1-18.

[0512] 20. The CAR according to claim 19, further comprising a hinge region, a transmembrane domain, a co-stimulatory signaling portion, a signaling domain, or any combination thereof.

[0513] 21. The CAR according to claim 20, wherein:

[0514] The hinge region includes a CD8α hinge region;

[0515] The transmembrane domain comprises a CD8α transmembrane domain;

[0516] The co-stimulatory signaling moiety comprises a 4-1BB signaling moiety; and / or

[0517] The signaling domain comprises a CD3ζ signaling domain.

[0518] 22. An isolated cell expressing the CAR described in any one of items 19-21.

[0519] 23. The isolated cell according to item 22, which is a cytotoxic T lymphocyte (CTL) or a natural killer (NK) cell.

[0520] 24. An immunoconjugate comprising the antibody of any one of items 1-18 and an effector molecule.

[0521] 25. The immunoconjugate of claim 24, wherein the effector molecule is a toxin.

[0522] 26. An immunoconjugate according to item 25, wherein the toxin is Pseudomonas exotoxin or a variant thereof.

[0523] 27. An immunoconjugate according to claim 26, wherein the Pseudomonas exotoxin variant is PE38.

[0524] 28. An immunoconjugate according to item 24, wherein the effector molecule is a photon absorber.

[0525] 29. The immunoconjugate of claim 24, wherein the effector molecule is a detectable marker.

[0526] 30. An immunoconjugate according to claim 29, wherein the detectable label comprises a fluorophore, an enzyme or a radioisotope.

[0527] 31. An antibody-drug conjugate (ADC) comprising a drug conjugated to the antibody of any one of items 1-18.

[0528] 32. The ADC of claim 31, wherein the drug is a small molecule.

[0529] 33. The ADC according to item 31 or 32, wherein the drug is an anti-microtubule agent, an anti-mitotic agent and / or a cytotoxic agent.

[0530] 34. A multispecific antibody comprising the antibody of any one of items 1-18 and at least one additional monoclonal antibody or antigen-binding fragment thereof.

[0531] 35. The multispecific antibody according to item 34, which is a bispecific antibody.

[0532] 36. The multispecific antibody according to item 34, which is a trispecific antibody.

[0533] 37. A multispecific antibody according to any one of items 34-36, wherein at least one additional monoclonal antibody or antigen-binding fragment thereof specifically binds to a component of a T cell receptor or a natural killer (NK) cell activation receptor.

[0534] 38. An antibody-nanoparticle conjugate comprising a nanoparticle conjugated to the antibody of any one of items 1-18.

[0535] 39. The antibody-nanoparticle conjugate of item 38, wherein the nanoparticle comprises a polymer nanoparticle, a nanosphere, a nanocapsule, a liposome, a dendrimer, a polymer micelle or a vesicle.

[0536] 40. The antibody-nanoparticle conjugate of item 38 or 39, wherein the nanoparticle comprises a cytotoxic agent.

[0537] 41. A fusion protein comprising the antibody described in any one of items 1-18 and a heterologous protein or peptide.

[0538] 42. A fusion protein according to item 41, wherein the heterologous protein is an Fc protein or a leucine zipper.

[0539] 43. An isolated nucleic acid molecule encoding the antibody described in any one of items 1-18, the CAR described in any one of items 19-21, the immunoconjugate described in any one of items 24-30, the multispecific antibody described in any one of items 34-37, or the fusion protein described in item 41 or item 42.

[0540] 44. An isolated nucleic acid molecule according to item 43, which is operably linked to a promoter.

[0541] 45. A vector comprising the nucleic acid molecule of item 43 or item 44.

[0542] 46. ​​An isolated host cell comprising the nucleic acid molecule described in item 44 or the vector described in item 45.

[0543] 47. A composition comprising a pharmaceutically acceptable carrier and the antibody described in any one of items 1-18, the CAR described in any one of items 19-21, the separated cells described in any one of items 22, 23 and 46, the immunoconjugate described in any one of items 24-30, the ADC described in any one of items 31-33, the multispecific antibody described in any one of items 34-37, the antibody-nanoparticle conjugate described in any one of items 38-40, or the fusion protein described in item 41 or item 42.

[0544] 48. A method for detecting B7H3 expression in a sample, comprising:

[0545] contacting the sample with the antibody of any one of items 1-18; and

[0546] The binding of the antibody to the sample is detected, thereby detecting the expression of B7H3 in the sample.

[0547] 49. A method of diagnosing a subject with a B7H3-positive cancer, comprising:

[0548] contacting a sample obtained from the subject with the antibody of any one of items 1-18; and

[0549] Binding of the antibody to the sample is detected, thereby diagnosing the subject as having a B7H3-positive cancer.

[0550] 50. A method according to claim 48 or 49, wherein the antibody is directly labeled.

[0551] 51. The method according to item 48 or item 49, further comprising:

[0552] contacting the antibody with a detection antibody, and

[0553] The binding of the detection antibody to the antibody is detected, thereby detecting the expression of B7H3 in the sample or diagnosing the subject as having a B7H3-positive cancer.

[0554] 52. A method according to any one of items 48-51, wherein the sample is obtained from a subject suspected of having a B7H3-positive cancer.

[0555] 53. A method according to any one of items 48-52, wherein the sample is a tumor biopsy.

[0556] 54. A method for treating B7H3-positive cancer in a subject, comprising administering to the subject the antibody described in any one of items 1-18, the CAR described in any one of items 19-21, the separated cells described in any one of items 22-23, the immunoconjugate described in any one of items 24-30, the ADC described in any one of items 31-33, the multispecific antibody described in any one of items 34-37, the antibody-nanoparticle conjugate described in any one of items 38-40, the fusion protein described in item 41 or item 42, or the composition described in item 47.

[0557] 55. A method for inhibiting tumor growth or metastasis of a subject's B7H3-positive cancer, comprising administering to the subject an antibody as described in any one of items 1-18, a CAR as described in any one of items 19-21, an isolated cell as described in any one of items 22-23, an immunoconjugate as described in any one of items 24-30, an ADC as described in any one of items 31-33, a multispecific antibody as described in any one of items 34-37, an antibody-nanoparticle conjugate as described in any one of items 38-40, a fusion protein as described in item 41 or item 42, or a composition as described in item 47.

[0558] 56. A method according to claim 54 or claim 55, wherein the B7H3-positive cancer is a solid tumor.

[0559] 57. The method of claim 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.

[0560] 58. A method according to claim 57, wherein the liver cancer is hepatocellular carcinoma.

[0561] 59. The method of claim 57, wherein the brain cancer is neuroblastoma or glioblastoma.

[0562] 60. The method of claim 57, wherein the pediatric cancer is osteosarcoma, neuroblastoma, rhabdomyosarcoma, or Ewing's sarcoma.

[0563] In view of the many possible embodiments to which the principles of the disclosed subject matter can be applied, it should be recognized that the illustrated embodiments are merely examples of the present disclosure and should not be considered to limit the scope of the present disclosure. Instead, the scope of the present disclosure is defined by the following claims. Therefore, we claim all that falls within the scope and spirit of these claims.

Claims

1. A single-domain monoclonal antibody that specifically binds to B7H3, wherein the antibody comprises the complementary determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 3, and wherein the CDR1, CDR2 and CDR3 sequences are respectively as shown below: 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. 2 . The antibody according to claim 1 , wherein the amino acid sequence of the antibody is at least 90% identical to SEQ ID NO: 3, and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:

3. 3 .

3. The antibody according to claim 1, wherein the amino acid sequence of the antibody is shown in SEQ ID NO:

3. The antibody according to claim 1 , wherein the antibody is a humanized antibody. The antibody according to claim 1 , wherein the antibody is a chimeric antibody.

6. A chimeric antigen receptor (CAR) comprising the antibody of claims 1-5.

7. The CAR according to claim 6 further comprises a hinge region, a transmembrane domain, a co-stimulatory signaling portion and a signaling domain.

8. The CAR according to claim 7, wherein: The hinge region includes a CD8α hinge region; The transmembrane domain comprises a CD8α transmembrane domain; The costimulatory signaling moiety comprises a 4-1BB signaling moiety; and The signaling domain comprises a CD3ζ signaling domain.

9. An isolated cell expressing the CAR of any one of claims 6-8.

10. The isolated cell according to claim 9, which is a cytotoxic T lymphocyte (CTL) or a natural killer (NK) cell.

11. An immunoconjugate comprising the antibody according to any one of claims 1 to 5 and an effector molecule.

12. The immunoconjugate of claim 11, wherein the effector molecule is a toxin.

13. The immunoconjugate of claim 12, wherein the toxin is Pseudomonas exotoxin or a variant thereof.

14. The immunoconjugate of claim 13, wherein the Pseudomonas exotoxin variant is PE38.

15. The immunoconjugate of claim 11, wherein the effector molecule is a photon absorber.

16. The immunoconjugate of claim 11, wherein the effector molecule is a detectable label.

17. The immunoconjugate of claim 16, wherein the detectable label comprises a fluorophore, an enzyme, or a radioisotope.

18. An antibody-drug conjugate (ADC) comprising a drug conjugated to the antibody of any one of claims 1-5.

19. The ADC of claim 18, wherein the drug is a small molecule.

20. The ADC according to claim 18 or 19, wherein the drug is an anti-microtubule agent, an anti-mitotic agent and / or a cytotoxic agent.

21. A multispecific antibody comprising the antibody of any one of claims 1-5 and at least one additional monoclonal antibody or antigen-binding fragment thereof.

22. The multispecific antibody according to claim 21, which is a bispecific antibody. The multispecific antibody according to claim 21 , which is a trispecific antibody.

24. The multispecific antibody of any one of claims 21-23, wherein the at least one additional monoclonal antibody or antigen-binding fragment thereof specifically binds to a component of a T cell receptor or a natural killer (NK) cell activation receptor.

25. An antibody-nanoparticle conjugate comprising a nanoparticle conjugated to the antibody of any one of claims 1-5.

26. The antibody-nanoparticle conjugate of claim 25, wherein the nanoparticle comprises a polymer nanoparticle, a nanosphere, a nanocapsule, a liposome, a dendrimer, a polymer micelle or a vesicle.

27. The antibody-nanoparticle conjugate of claim 25 or 26, wherein the nanoparticle comprises a cytotoxic agent.

28. A fusion protein comprising the antibody according to any one of claims 1 to 5 and a heterologous protein or peptide. The fusion protein of claim 28 , wherein the heterologous protein is an Fc protein or a leucine zipper.

30. An isolated nucleic acid molecule encoding the antibody of any one of claims 1-5, the CAR of any one of claims 6-8, the immunoconjugate of any one of claims 11-17, the multispecific antibody of any one of claims 21-24, or the fusion protein of claim 28 or claim 29.

31. The isolated nucleic acid molecule of claim 30, operably linked to a promoter.

32. A vector comprising the nucleic acid molecule of claim 30 or claim 31.

33. An isolated host cell comprising the nucleic acid molecule of claim 31 or the vector of claim 32.

34. A composition comprising a pharmaceutically acceptable carrier and an antibody as described in any one of claims 1-5, a CAR as described in any one of claims 6-8, an isolated cell as described in any one of claims 9, 10 and 33, an immunoconjugate as described in any one of claims 11-17, an ADC as described in any one of claims 18-20, a multispecific antibody as described in any one of claims 21-24, an antibody-nanoparticle conjugate as described in any one of claims 25-27, or a fusion protein as described in claim 28 or claim 29.

35. A method for detecting B7H3 expression in a sample for non-diagnostic purposes, comprising: contacting the sample with the antibody of any one of claims 1-5; and The binding of the antibody to the sample is detected, thereby detecting the expression of B7H3 in the sample.

36. The method of claim 35, wherein the antibody is directly labeled.

37. The method of claim 35, wherein: The method further comprises: contacting the antibody with a detection antibody, and The binding of the detection antibody to the antibody is detected, thereby detecting the expression of B7H3 in the sample.

38. Use of the antibody of any one of claims 1-5, the CAR of any one of claims 6-8, the isolated cell of any one of claims 9-10, the immunoconjugate of any one of claims 11-17, the ADC of any one of claims 18-20, the multispecific antibody of any one of claims 21-24, the antibody-nanoparticle conjugate of any one of claims 25-27, the fusion protein of claim 28 or claim 29, or the composition of claim 34 for the preparation of a medicament for use in a method for treating B7H3-positive cancer in a subject.

39. Use of the antibody of any one of claims 1-5, the CAR of any one of claims 6-8, the isolated cell of any one of claims 9-10, the immunoconjugate of any one of claims 11-17, the ADC of any one of claims 18-20, the multispecific antibody of any one of claims 21-24, the antibody-nanoparticle conjugate of any one of claims 25-27, the fusion protein of claim 28 or claim 29, or the composition of claim 34 for the preparation of a drug for use in a method of inhibiting tumor growth or metastasis of a B7H3-positive cancer in a subject.

40. The use of claim 38 or claim 39, wherein the B7H3-positive cancer is a solid tumor.

41. The use according to claim 40, 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, melanoma or mesothelioma.

42. The use according to claim 41, wherein the liver cancer is hepatocellular carcinoma.

43. The use according to claim 41, wherein the brain cancer is neuroblastoma or glioblastoma.

44. The use of claim 38 or claim 39, wherein the B7H3-positive cancer is a pediatric cancer.

45. The use according to claim 44, wherein the pediatric cancer is osteosarcoma, neuroblastoma, rhabdomyosarcoma or Ewing's sarcoma.

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