WO2025146081A1 - B7-h3 binding protein - Google Patents

Abstract

Provided is a B7-H3 binding protein, and in particular a nanobody and a heavy chain antibody that specifically bind to B7-H3, as well as a vector, a cell, or a nucleic acid that encodes the antibody, and a diagnostic or therapeutic use of said antibody.

Description

B7-H3 binding protein

This international patent application claims priority to Chinese Patent Application No. 202410009195.X filed on January 3, 2024 and Chinese Patent Application No. 202411783315.8 filed on December 5, 2024, the entire contents of which are incorporated herein by reference for all purposes.

Technical Field

The present invention belongs to the field of biotechnology, and in particular relates to antigen binding proteins, heavy chain antibodies and nano antibodies, and uses thereof.

Background Art

B7-H3 (CD276) is a member of the B7 family of proteins and plays a key role in the development of cancer. As one of the targets of immune checkpoints, B7-H3 is selectively expressed in tumor cells and immune cells in the tumor microenvironment, and is involved in tumor cell proliferation and metastasis and is associated with treatment resistance.

There is a huge difference in the protein expression level of B7-H3 in normal tissues and tumor tissues. It is expressed in large quantities in a variety of tumor tissues, such as prostate cancer, pancreatic cancer, hepatocellular carcinoma, etc., but rarely expressed in normal tissues (Zhou WT, Jin WL. B7-H3/CD276: An Emerging Cancer Immunother-apy. Front Immunol. 2021; 12: 701006.). Taking advantage of this feature, targeting B7-H3 with drugs can specifically kill cancer tissues while minimizing damage to healthy cells. These characteristics make B7-H3 a promising target for cancer treatment.

Nanobody is a new type of antibody, also known as single-domain antibody, which is obtained by cloning the variable domain of the heavy chain of heavy-chain antibody (VHH) naturally lacking light chains in animals such as camelids. Compared with conventional monoclonal antibodies, nanobodies have the advantages of small molecular weight, good solubility, strong stability, weak immunogenicity, strong penetration, high specificity, simple humanization, high expression, and easy production. Nanobodies have good application prospects in the fields of biotechnology and medicine, and nanobody drugs have been approved for marketing. The application of nanobody technology to develop therapeutic antibodies against B7-H3 has good prospects.

Summary of the invention

The present disclosure provides B7-H3 binding proteins that specifically recognize and bind to B7-H3, in particular, nanobodies (also called B7-H3 single domain antibodies, or B7-H3 VHH antibodies) and heavy chain antibodies that specifically recognize and bind to B7-H3.

Therefore, in a first aspect of the present disclosure, a B7-H3 binding protein is provided. The B7-H3 binding protein according to an embodiment of the present invention can specifically target and bind to B7-H3.

In some embodiments, the binding protein comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 contained in the VHH shown in any one of SEQ ID NO: 1-13. In some embodiments, the CDR1, CDR2 and CDR3 are encoded according to Kabat, AbM, Chothia or IMGT.

In some embodiments of the B7-H3 binding protein described above, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 are encoded according to AbM, wherein:

(1) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:29, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:30, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:31;

(2) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:23, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:24, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:25;

(3) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:26, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:27, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:28;

(4) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:32, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:33, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:34;

(5) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:35, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:36, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:37;

(6) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:38, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:39, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:40;

(7) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:41, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:42, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:43;

(8) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:44, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:45, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:46;

(9) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:47, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:48, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:49;

(10) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:50, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:51, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:52;

(11) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:53, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:54, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:55;

(12) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:56, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:57, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:58; or

(13) The CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:59, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:60, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:61.

In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NO: 1-13, or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NO: 1-13.

In some embodiments of the present disclosure, the B7-H3 binding protein is monovalent, bivalent, or multivalent.

In some embodiments of the present disclosure, the B7-H3 binding protein is monospecific, bispecific, or multispecific.

In some embodiments of the present disclosure, the immunoglobulin single variable domain contained in the B7-H3 binding protein comprises a heavy chain framework region, and at least a portion of the heavy chain framework region is from at least one of a mouse antibody, a human antibody, a primate antibody, and a mutant thereof. In some embodiments of the present disclosure, at least a portion of the heavy chain framework region contained in the B7-H3 binding protein is from a human antibody, preferably, the immunoglobulin single variable domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% identity with the amino acid sequence shown in any one of SEQ ID NO: 14-20, more preferably, the immunoglobulin single variable domain comprises or consists of an amino acid sequence shown in any one of SEQ ID NO: 14-20.

In some embodiments of the present disclosure, the B7-H3 binding protein is a heavy chain antibody. In some preferred embodiments, the heavy chain antibody further comprises human IgG Fc. In some more preferred embodiments, the heavy chain antibody further comprises human IgG1 Fc. In some specific embodiments, the human IgG1 Fc comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% identity with the amino acid sequence shown in SEQ ID NO: 69, more preferably, the human IgG1 Fc comprises or consists of an amino acid sequence as shown in SEQ ID NO: 69. In some specific embodiments of the present disclosure, the heavy chain antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% identity with the amino acid sequence shown in any one of SEQ ID NO: 70-82 and SEQ ID NO: 62-68. In some embodiments of the present disclosure, the heavy chain antibody comprises or consists of the amino acid sequence shown in any one of SEQ ID NO:70-82 and SEQ ID NO:62-68.

In some embodiments of the present disclosure, the B7-H3 binding protein is a nanobody. In some embodiments, the nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% identity with the amino acid sequence shown in any one of SEQ ID NO: 1-13. In some preferred embodiments, the nanobody comprises an amino acid sequence shown in any one of SEQ ID NO: 1-13 or consists of an amino acid sequence shown in any one of SEQ ID NO: 1-13. In some embodiments, the nanobody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% identity with the amino acid sequence shown in any one of SEQ ID NO: 14-20. In some preferred embodiments, the nanobody comprises an amino acid sequence shown in any one of SEQ ID NO: 14-20 or consists of an amino acid sequence shown in any one of SEQ ID NO: 14-20.

The Nanobody with the amino acid sequence shown in SEQ ID NO:1 corresponds to clone C23 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:2 corresponds to clone A13 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:3 corresponds to clone A2 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:4 corresponds to clone A3 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:5 corresponds to clone A77 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:6 corresponds to clone A83 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:7 corresponds to clone B102 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:8 corresponds to clone B91 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:9 corresponds to clone C184 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:10 corresponds to clone C357 of the present disclosure; The Nanobody with the amino acid sequence shown in SEQ ID NO:11 corresponds to the clone C59 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:12 corresponds to the clone D16 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:13 corresponds to the clone A14 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:14 corresponds to the clone VHH21 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:15 corresponds to the clone VHH22 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:16 corresponds to the clone VHH23 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:17 corresponds to the clone VHH24 of the present disclosure. The Nanobody with the amino acid sequence shown in SEQ ID NO:16 corresponds to the clone VHH23 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:17 corresponds to the clone VHH24 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:18 corresponds to the clone VHH25 of the present disclosure; the Nanobody with the amino acid sequence shown in SEQ ID NO:19 corresponds to the clone VHH26 of the present disclosure; and the Nanobody with the amino acid sequence shown in SEQ ID NO:20 corresponds to the clone VHH27 of the present disclosure.

In a second aspect of the present disclosure, a fusion protein is provided, which comprises the B7-H3 binding protein described in the first aspect of the present disclosure.

In the third aspect of the present disclosure, a nucleic acid molecule is provided, which encodes the B7-H3 binding protein described in the first aspect of the present disclosure or the fusion protein described in the second aspect of the present disclosure. In some preferred embodiments, the nucleic acid molecule is DNA.

In the fourth aspect of the present disclosure, an expression vector comprising the nucleic acid molecule described in the third aspect of the present disclosure is provided. As described above, the nucleic acid molecule encodes the B7-H3 binding protein described in the first aspect of the present disclosure or the fusion protein described in the second aspect of the present disclosure. Therefore, the expression vector introduced into the host cell according to the embodiment of the present invention can express the B7-H3 binding protein described in the first aspect of the present disclosure or the fusion protein described in the second aspect of the present disclosure under conditions suitable for protein expression. In some embodiments of the present disclosure, the expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

In a fifth aspect of the present disclosure, a cell comprising the nucleic acid molecule described in the third aspect of the present disclosure or the expression vector described in the fourth aspect of the present disclosure is provided. In some embodiments of the present disclosure, the cell is obtained by introducing the expression vector described in the fourth aspect of the present disclosure into a host cell. In some embodiments of the present disclosure, the cell is a prokaryotic cell or a eukaryotic cell. In some embodiments of the present disclosure, the cell is a mammalian cell, such as a CHO cell.

In the sixth aspect of the present disclosure, a conjugate is provided. In some embodiments of the present disclosure, the conjugate comprises the B7-H3 binding protein described in the first aspect of the present disclosure or the fusion protein described in the second aspect of the present disclosure, and further comprises a therapeutic agent, a diagnostic agent, or an imaging agent conjugated to the B7-H3 binding protein or the fusion protein. In some embodiments, there is a joint between the B7-H3 binding protein described in the first aspect of the present disclosure or the fusion protein described in the second aspect of the present disclosure and the therapeutic agent, the diagnostic agent, or the imaging agent. In some embodiments, the B7-H3 binding protein described in the first aspect of the present disclosure or the fusion protein described in the second aspect of the present disclosure is conjugated to the therapeutic agent, the diagnostic agent, or the imaging agent through a joint. In some embodiments, the joint comprises a cleavable joint or a non-cleavable joint. In some embodiments, the linker is selected from MC (6-maleimidocaproyl), Val-Cit (valine-citrulline), PABC (p-amino-benzyloxycarbonyl), DMEA (dimethylethylamine), Val-Cit-PABC, MC-Val-Cit-PABC, MC-Val-Cit-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), MC-GGFG, AcBut (4-(4-acetylphenoxy)-butyric acid) and AcBut-dimethylhydrazide, preferably MC-GGFG. In some embodiments of the present disclosure, the therapeutic agent is a small molecule cytotoxic drug. In some embodiments, the therapeutic agent is selected from topoisomerase inhibitors, microtubule inhibitors, antibiotics, DNA synthesis inhibitors, RNA polymerase II inhibitors and RNA spliceosome inhibitors, preferably topoisomerase inhibitors. In some embodiments, the therapeutic agent is selected from Exitecan (DX8951), MMAE, MMAF, duocarmycin, DM1, DM4, SN-38, Dxd, calicheamicin, doxorubicin and PBDs (benzodiazepines), preferably Exitecan. In some embodiments, the part formed by the connection of the linker and the therapeutic agent has the following structure:

The conjugate according to the sixth aspect of the present disclosure can be targeted and act on target cells containing B7-H3 under the guidance of the B7-H3 binding protein or fusion protein.

In the seventh aspect of the present disclosure, a composition is provided, the composition comprising the B7-H3 binding protein described in the first aspect of the present disclosure, the fusion protein described in the second aspect of the present disclosure, the nucleic acid described in the third aspect of the present disclosure, the expression vector described in the fourth aspect of the present disclosure, the recombinant cell described in the fifth aspect of the present disclosure, and/or the conjugate described in the sixth aspect of the present disclosure. In some embodiments of the present disclosure, the composition is a pharmaceutical composition. In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

In the eighth aspect of the present disclosure, provided is the use of the B7-H3 binding protein described in the first aspect of the present disclosure, the fusion protein described in the second aspect of the present disclosure, the nucleic acid described in the third aspect of the present disclosure, the expression vector described in the fourth aspect of the present disclosure, the recombinant cell described in the fifth aspect of the present disclosure, the conjugate described in the sixth aspect of the present disclosure, and/or the composition described in the seventh aspect of the present disclosure in the preparation of a medicament for preventing, treating or alleviating B7-H3-related diseases.

Also provided is the use of the combination of the B7-H3 binding protein described in the first aspect of the disclosure, the fusion protein described in the second aspect of the disclosure, the nucleic acid described in the third aspect of the disclosure, the expression vector described in the fourth aspect of the disclosure, the recombinant cell described in the fifth aspect of the disclosure, the conjugate described in the sixth aspect of the disclosure, and/or the composition described in the seventh aspect of the disclosure and another agent in the preparation of a drug for preventing, treating or alleviating a disease associated with B7-H3. In some embodiments, the other agent is an immunotherapeutic agent or a chemotherapeutic agent.

In the ninth aspect of the present disclosure, provided is the B7-H3 binding protein described in the first aspect of the present disclosure, the fusion protein described in the second aspect of the present disclosure, the nucleic acid described in the third aspect of the present disclosure, the expression vector described in the fourth aspect of the present disclosure, the recombinant cell described in the fifth aspect of the present disclosure, the conjugate described in the sixth aspect of the present disclosure, and/or the composition described in the seventh aspect of the present disclosure, which are used for preventing, treating or alleviating diseases associated with B7-H3.

In the tenth aspect of the present disclosure, a method for preventing, treating or alleviating a B7-H3-related disease in a subject is provided. In some embodiments, the method comprises administering to the subject the B7-H3 binding protein described in the first aspect of the present disclosure, the fusion protein described in the second aspect of the present disclosure, the nucleic acid described in the third aspect of the present disclosure, the expression vector described in the fourth aspect of the present disclosure, the recombinant cell described in the fifth aspect of the present disclosure, the conjugate described in the sixth aspect of the present disclosure, and/or the composition described in the seventh aspect of the present disclosure.

In some embodiments of the eighth aspect to the tenth aspect of the present disclosure, the B7-H3-related disease or condition is a B7-H3-mediated disease or condition. In some embodiments, the B7-H3-mediated disease or condition is a tumor. In some embodiments, the B7-H3-mediated disease or condition is a solid tumor and/or a hematological tumor. In some specific embodiments, the B7-H3-mediated disease or condition is one or more tumors selected from the group consisting of lung cancer, breast cancer, prostate cancer, pancreatic cancer, colorectal cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer and kidney cancer. In some specific embodiments, the B7-H3 mediated disease or condition is one or more tumors selected from the group consisting of: adrenal tumors, AIDS-related cancers, alveolar soft part sarcomas, astrocytic tumors, bone cancer, brain and spinal cord cancer, metastatic brain tumors, B cell cancers, cancer, carotid body tumors, chondrosarcomas, chordomas, benign fibrous histiocytomas of the skin, desmoplastic small round cell tumors, ependymomas, Ewing's tumors, extraosseous myxoid chondrosarcomas, osteogenesis imperfecta, fibrous dysplasia of bone, gallbladder cancer or bile duct cancer, gestational trophoblastic disease, germ cell tumors , head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, leukemia, liposarcoma/malignant lipoma, lymphoma, medulloblastoma, meningioma, multiple endocrine neoplasms, multiple myeloma, myelodysplastic syndrome, neuroblastoma, papillary thyroid carcinoma, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, melanocytoma, pituitary tumor, posterior uveal melanoma, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell cancer, gastric cancer, synovial sarcoma, testicular cancer, thymoma, and thyroid metastatic cancer. In a preferred embodiment, the B7-H3 mediated disease or condition is lung cancer.

In the eleventh aspect of the present disclosure, a kit for detecting B7-H3 or cells containing B7-H3 is provided. In some embodiments, the kit comprises the B7-H3 binding protein of the first aspect of the present disclosure or the conjugate of the sixth aspect of the present disclosure.

In the twelfth aspect of the present disclosure, provided is use of the B7-H3 binding protein of the first aspect of the present disclosure or the conjugate of the sixth aspect of the present disclosure in preparing a kit for detecting B7-H3 or cells containing B7-H3.

In the thirteenth aspect of the present disclosure, provided is the B7-H3 binding protein of the first aspect of the present disclosure or the conjugate of the sixth aspect of the present disclosure, which is used for detecting B7-H3 or cells containing B7-H3.

In the fourteenth aspect of the present disclosure, a method for detecting B7-H3 or a cell containing B7-H3 is provided. In some embodiments of the present disclosure, the method comprises contacting the B7-H3 binding protein described in the first aspect of the present disclosure or the conjugate described in the sixth aspect of the present disclosure with a sample to be tested. A method for determining the presence and/or content of B7-H3 is also provided, comprising contacting the B7-H3 binding protein described in the first aspect of the present disclosure and/or the conjugate described in the sixth aspect of the present disclosure with a sample to be tested.

In the fifteenth aspect of the present disclosure, a chimeric antigen receptor is provided. In some embodiments of the present disclosure, the chimeric antigen receptor comprises an antigen recognition domain, a hinge region, a transmembrane domain, and an intracellular domain (including a co-stimulatory domain and a signal transduction domain), and the antigen recognition domain comprises the B7-H3 binding protein described in the first aspect of the present disclosure.

Further aspects and advantages will be described below, at least part of which will become apparent from the following description in conjunction with the accompanying drawings, and/or will be apparent to those of ordinary skill in the art from the embodiments described below.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of ELISA assays of VHH supernatants expressed by 33 clones with unique sequences in the initial screening using the corresponding Human B7-H3 antigen or Mouse B7-H3 antigen. Figure 1A shows the results of ELISA assays of VHH supernatants expressed by A2, A3, A9, A13, A14, A35, A75, A77, A80, A83, A84, and A94 clones using the Human B7-H3-Fc antigen. Figure 1B shows the results of ELISA assays of VHH supernatants expressed by A2, A3, A9, A13, A14, A35, A75, A77, A80, A83, A84, and A94 clones using the Mouse B7-H3-Fc antigen. Figure 1C shows the results of ELISA assays of VHH supernatants expressed by B5, B81, B90, B91, B102, B103, and B137 clones using Human B7-H3-Fc antigen. Figure 1D shows the results of ELISA assays of VHH supernatants expressed by B5, B81, B90, B91, B102, B103, and B137 clones using Mouse B7-H3-Fc antigen. Figure 1E shows the results of ELISA assays of VHH supernatants expressed by C13, C17, C23, C52, C58, C59, C80, C123, C130, C184, C217, C221, C340, and C357 clones using Human B7-H3-Fc antigen. Figure 1F shows the results of ELISA assays of VHH supernatants expressed by C13, C17, C23, C52, C58, C59, C80, C123, C130, C184, C217, C221, C340, and C357 clones using Mouse B7-H3-Fc antigen. Figure 1G shows the results of ELISA assays of VHH supernatants expressed by C157 and C190 clones using Human B7-H3-Fc antigen. Figure 1H shows the results of ELISA assays of VHH supernatants expressed by C157 and C190 clones using Mouse B7-H3-Fc antigen. Figure 1I shows the results of ELISA assays of VHH supernatants expressed by D9, D16, D22, D29, and D37 clones using Human B7-H3-Fc antigen. Figure 1J shows the results of ELISA assay of VHH supernatants expressed by D9, D16, D22, D29, and D37 clones using Mouse B7-H3-Fc antigen.

Figure 2 shows the results of ELISA detection of VHH-Fc antibodies expressed and purified from the construct using Human B7-H3-His.

Figure 3 shows the results of ELISA detection of VHH-Fc antibodies expressed and purified from the construct using Mouse B7-H3-Fc.

Figure 4 shows the results of ELISA detection of VHH-Fc antibodies expressed and purified from the construct using Cyno B7-H3-His.

Figure 5 shows the binding activity of humanized antibodies VHH21-Fc, VHH22-Fc, VHH23-Fc, VHH24-Fc, VHH25-Fc, VHH26-Fc, and VHH27-Fc to antigens. Figure 5A shows the binding activity of humanized antibodies to Human B7-H3-His, Figure 5B shows the binding activity of humanized antibodies to Mouse B7-H3 His, and Figure 5C shows the binding activity of humanized antibodies to Cyno B7-H3 His.

Figure 6 shows the in vivo antitumor efficacy of the humanized antibody VHH25-0143ADC molecule at different doses in the Calu-6 human lung cancer subcutaneous transplanted tumor mouse model. The dosage was 1 mg/kg, 3 mg/kg and 10 mg/kg, and the frequency of administration was once a week (QW), for a total of 3 times.

DETAILED DESCRIPTION OF THE INVENTION

The above features and advantages of the present invention and additional features and advantages thereof will be more clearly understood from the following detailed description of the embodiments in conjunction with the accompanying drawings.

The embodiments described herein with reference to the accompanying drawings are explanatory, illustrative, and are used for a general understanding of the present invention. The embodiments should not be construed as limiting the scope of the present invention. The same or similar elements and elements with the same or similar functions are represented by the same reference numerals in the overall description.

Unless otherwise stated or defined, all terms used have the ordinary meaning in the art known to the skilled person, for example with reference to standard manuals.

Unless otherwise indicated, the term "immunoglobulin sequence", whether it is used herein to refer to a heavy chain antibody or a conventional 4-chain antibody, is used as a general term to include full-length antibodies, single chains thereof, and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments, such as _VHH domains or _VH / _VL domains, respectively). In addition, the term "sequence" used herein (e.g., "immunoglobulin sequence", "antibody sequence", "variable domain sequence", " _VHH sequence" or "protein sequence" and other terms) should generally be understood to include the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the relevant amino acid sequence, unless the context requires a more limited interpretation.

Unless otherwise indicated, as will be clear to the skilled person, all methods, steps, techniques and operations not specifically described in detail can and have been performed in a manner known per se. For example, reference is again made to the standard manuals and general background art mentioned herein and to the further references cited therein.

For comparison of two or more nucleotide sequences, the percentage of "sequence identity" between a first sequence and a second sequence can be calculated by dividing [the number of nucleotides in the first sequence that are identical to the nucleotides at corresponding positions in the second sequence] by [the total number of nucleotides in the first sequence] and multiplying by [100%], wherein each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence is considered to be a difference of a single nucleotide (position) compared to the first nucleotide sequence.

Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment such as NCBI Blast v2.0 using standard settings.

Some other techniques, computer algorithms and settings for determining the degree of sequence identity are described, for example, in WO 04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768-A.

For comparison of two or more amino acid sequences, the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence can be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at corresponding positions in the second amino acid sequence] by [the total number of amino acids in the first amino acid sequence] multiplied by [100%], wherein the deletion, insertion, substitution or addition of each amino acid residue in the second amino acid sequence is considered to be a difference of a single amino acid residue (position) compared to the first amino acid sequence, i.e., an "amino acid difference" as defined herein.

Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms such as those described above for determining the degree of sequence identity for nucleotide sequences, again using standard settings.

Typically, to determine the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the most amino acid residues will be used as the "first" amino acid sequence and the other amino acid sequence will be used as the "second" amino acid sequence.

Furthermore, when determining the degree of sequence identity between two amino acid sequences, the skilled person may take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure, which has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example WO 04/037999, GB-A-2 357 768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferably) the type and/or combination of these substitutions may be selected according to the relevant teachings from WO 04/037999 and WO 98/49185 and the further references cited therein.

Such conservative substitutions are preferably substitutions in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and/or Phe to Val, to Ile or to Leu.

Any amino acid substitutions described herein applied to polypeptides may also be based on an analysis of amino acid variation frequencies between homologous proteins from different species developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978, an analysis of structure-forming potential developed by Chou and Fasman, Biochemistry 13:211, 1974 and Adv. Enzymol., 47:45-149, 1978, and an analysis of protein hydrophobicity patterns developed by Eisenberg et al., Proc. Nat. Acad Sci. USA 81:140-144, 1984; Kyte & Doolittle, J Mol. Biol. 157:105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15:321-353, 1986, the disclosures of which are incorporated herein by reference in their entirety.

Information on the primary, secondary and tertiary structures of nanobodies is given in the description herein and in the general background art cited above. In addition, for this purpose, the crystal structure of the VHH domain from llama is provided, for example, by Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Natural Structural Biology (1996); Vol. 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361 (1999). Further information on some amino acid residues that form the VH/VL interface in conventional _VH domains and potential camelization substitutions at these positions is provided.

Amino acid sequences and nucleic acid sequences are said to be "identical" if they have 100% sequence identity (as defined herein) over their entire length.

A nucleic acid sequence or an amino acid sequence is considered to be "in substantially isolated form", e.g., when it has been separated from at least one other component with which it is normally associated in said source or medium, such as another nucleic acid, another protein/polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component, compared to its natural biological source and/or the reaction medium or culture medium from which it is obtained. In particular, a nucleic acid sequence or an amino acid sequence is considered to be "substantially isolated" when it has been purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold and up to 1000-fold or more. A nucleic acid sequence or an amino acid sequence "in substantially isolated form" is preferably substantially homogeneous, as determined using a suitable technique, such as a suitable chromatographic technique, such as polyacrylamide-gel electrophoresis.

The term "specificity" refers to the number of different types of antigens or antigenic determinants that a particular antigen binding molecule or antigen binding protein (e.g., nanoantibodies or polypeptides of the present invention) molecule can bind to. The specificity of an antigen binding protein can be determined based on affinity and/or avidity. Avidity is represented by the equilibrium constant (KD) of the dissociation of antigen and antigen binding protein, and is a measure of the binding strength between an antigenic determinant and the antigen binding site of an antigen binding protein: the smaller the KD value, the stronger the binding strength between an antigenic determinant and an antigen binding molecule (or, affinity can also be expressed as an affinity constant (KA), which is 1/KD). Avidity is a measure of the binding strength between an antigen binding molecule (e.g., nanoantibodies of the present invention, antibodies, or heavy chain antibodies) and a related antigen. Avidity relates to the affinity between an antigenic determinant and the antigen binding site of an antigen binding molecule and the number of related binding sites present on the antigen binding molecule. Typically, antigen binding proteins (e.g., Nanobodies and/or heavy chain antibodies of the invention) will bind with a dissociation constant (KD) of ^10-5 to ^10-12 mol/liter or less, preferably with a dissociation constant (KD) of ^10-7 to ^10-12 mol/liter or less, more preferably with a dissociation constant (KD) of ^10-8 to ^10-12 mol/liter, and/or with a binding affinity of at least ¹⁰⁷ M ^-1 , preferably at least ¹⁰⁸ M ^-1 , more preferably at least ¹⁰⁹ M- ¹ , for example at least ¹⁰¹² M ^-1 . It is generally believed that any KD value greater than ^10-4 mol/liter represents non-specific binding. Preferably, the B7-H3 binding proteins of the invention, in particular Nanobodies, will bind to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, for example less than 500 pM. Specific binding of an antigen binding protein to an antigen or antigenic determinant can be determined by any suitable means known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competition assays and different variations known per se in the art.

The term "immunoglobulin single variable domain" as used herein refers to an immunoglobulin variable domain that is capable of specifically binding to an antigenic epitope without pairing with other immunoglobulin variable domains. An example of an immunoglobulin single variable domain of the present disclosure is a "domain antibody", such as an immunoglobulin single variable domain VH and VL (VH domain and VL domain). Another example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") of Camelidae as defined below.

The variable domain of the heavy chain of a “heavy chain antibody” (i.e., “antibody lacking a light chain”) is also called “VHH”, also known as a single domain antibody, a heavy chain single domain antibody, a VHH antibody fragment, and a VHH antibody. The term “VHH domain” is used to distinguish the variable domain from the heavy chain variable domain present in conventional 4-chain antibodies (which is referred to herein as a “VH domain”) and the light chain variable domain present in conventional 4-chain antibodies (which is referred to herein as a “VL domain”). The VHH domain specifically binds to an epitope without the need for other antigen-binding domains (this is in contrast to the VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain). The VHH domain is a small, stable, and efficient antigen recognition unit formed by a single immunoglobulin domain.

In the context of the present disclosure, the terms "heavy chain single domain antibody", "VHH domain", "VHH", "VHH antibody fragment", "VHH antibody" and "nanobody" are used interchangeably.

As further described herein, the amino acid sequence and structure of Nanobodies can be considered (but not limited to) as comprising four framework regions or "FRs", referred to in the art and herein as "Framework region 1" or "FR1"; "Framework region 2" or "FR2"; "Framework region 3" or "FR3"; and "Framework region 4" or "FR4", respectively; these framework regions are interrupted by three complementarity determining regions or "CDRs", referred to in the art as "Complementarity Determining Region 1" or "CDR1"; "Complementarity Determining Region 2" or "CDR2"; and "Complementarity Determining Region 3" or "CDR3", respectively.

The amino acid residues of Nanobodies are numbered according to the common numbering for _VH domains given by Kabat et al. ("Sequence of protein of immunological Interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to _VHH domains of camelids in Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999) (see, e.g., Figure 2 of the above reference). In this regard, it should be noted that - as is well known in the art for _VH domains and for _VHH domains - the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues represented by the Kabat numbering. That is, one or more positions numbered according to Kabat may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering. This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

Alternative methods for numbering the amino acid residues of _VH domains, which can also be applied in an analogous manner to _VHH domains and Nanobodies from Camelids, are the method described by Chothia et al. (Nature 342, 877-883 (1989), the so-called "AbM definition" and the so-called "CONTACT definition". However, in the present description, the claims and the drawings, the numbering of _VHH domains according to IMGT will be followed unless otherwise indicated.

According to the terminology used in the above references, the variable domain present in naturally occurring heavy chain antibodies will also be referred to as a “ _VHH domain” to distinguish it from the heavy chain variable domain present in conventional 4-chain antibodies (hereinafter referred to as a “ _VH domain”) and the light chain variable domain present in conventional 4-chain antibodies (hereinafter referred to as a “ _VL domain”).

As described in the prior art mentioned above, _VHH domains have many unique structural features and functional properties, which make isolated _VHH domains (and Nanobodies based on _VHH domains that share these structural features and functional properties with naturally occurring _VHH domains) and proteins containing isolated _VHH domains very advantageous for use as functional antigen-binding domains or proteins. In particular, but not limited to, _VHH domains (which have essentially been "designed" to functionally bind to antigens in the absence of light chain variable domains and without any interaction with light chain variable domains) and Nanobodies can be used as single, relatively small, functional antigen-binding structural units, domains or proteins. This distinguishes _VHH domains from the _VH and _VL domains of conventional 4-chain antibodies, which themselves are generally not suitable for practical use as single antigen-binding proteins or domains, but need to be combined in some form to provide functional antigen-binding units (e.g., in conventional antibody fragments such as Fab fragments; in ScFv fragments consisting of _VH domains covalently linked to _VL domains).

Due to these unique properties, the use of _VHH domains and Nanobodies as single antigen-binding proteins or antigen-binding domains (i.e. as part of a larger protein or polypeptide) offers a number of significant advantages over the use of conventional _VH and _VL domains, ScFv or conventional antibody fragments (e.g. Fab or F(ab')2 fragments): only a single domain is required to bind antigen with high affinity and selectivity, so there is no need for the presence of two separate domains, nor is there a need to ensure that the two domains are present in the correct spatial conformation and configuration (i.e. by using a specifically designed linker as in ScFv).

_VHH domains and nanobodies can be expressed from a single gene without the need for post-translational folding or modification.

_VHH domains and Nanobodies can be easily engineered into multivalent and multispecific formats.

_VHH domains and Nanobodies are highly soluble and have no tendency to aggregate (as described in "Mouse-derived antigen-binding domains" by Ward et al., Nature, Vol. 341, 1989, p. 544).

_VHH domains and Nanobodies are highly stable to heat, pH, proteases and other denaturing agents or conditions (see, e.g., Ewert et al., supra).

_VHH domains and nanobodies are easy and relatively cheap to prepare even on the scale expected for production. For example, _VHH domains, nanobodies and proteins/polypeptides comprising them can be produced using microbial fermentation and do not require the use of mammalian expression systems such as those used for conventional antibody fragments.

Compared to traditional 4-chain antibodies and antigen-binding fragments thereof, _VHH domains and nanobodies are relatively small (about 15 kDa, or 10 times smaller than traditional IgG) and therefore exhibit higher tissue (including but not limited to solid tumors and other dense tissues) penetration than such conventional 4-chain antibodies and antigen-binding fragments thereof.

_VHH domains and Nanobodies can display so-called cavity binding properties (particularly due to their extended CDR3 loop compared to conventional _VH domains) and can therefore also access targets and epitopes that are inaccessible to conventional 4-chain antibodies and antigen-binding fragments.

As described above, the present invention generally relates to Nanobodies against B7-H3, as well as polypeptides (eg, antibodies or heavy chain antibodies or antigen-binding fragments thereof) comprising one or more such Nanobodies, which can be used for the prophylactic, therapeutic and/or diagnostic purposes described herein.

As also further described herein, the present invention also relates to nucleic acids encoding such Nanobodies, B7-H3 binding proteins or heavy chain antibodies or antigen-binding fragments thereof, methods for preparing such Nanobodies, B7-H3 binding proteins or heavy chain antibodies or antigen-binding fragments thereof, host cells expressing or capable of expressing such Nanobodies, B7-H3 binding proteins or heavy chain antibodies or antigen-binding fragments thereof, compositions comprising such Nanobodies, B7-H3 binding proteins or heavy chain antibodies or antigen-binding fragments thereof, nucleic acids or host cells, and uses of such Nanobodies, B7-H3 binding proteins or heavy chain antibodies or antigen-binding fragments thereof, nucleic acids, host cells or compositions.

In general, it should be noted that the term Nanobody, B7-H3 binding protein or heavy chain antibody or antigen-binding fragment thereof as used herein has its broadest meaning without being limited to a specific biological source or a specific method of preparation.

The humanized Nanobodies of the invention may be as defined herein, with the proviso that they have at least "one amino acid difference" (as defined herein) in at least one framework region compared to the corresponding framework region of a naturally occurring _VHH domain. More specifically, according to one non-limiting aspect of the invention, a Nanobody may be as defined herein, with the proviso that they have at least "one amino acid difference" (as defined herein) in at least one Hallmark residue compared to the corresponding framework region of a naturally occurring _VHH domain. Typically, a Nanobody has at least one such amino acid difference with a naturally occurring _VHH domain in at least one of FR2 and/or FR4, in particular at at least one Hallmark residue in FR2 and/or FR4.

Heavy chain antibodies or HCAbs consist of only two heavy chains, each of which contains only the heavy chain variable region (VHH) as well as the hinge region, CH2 and CH3. The antigen-antibody binding region of heavy chain antibodies or HCAbs consists of three complementary determining regions, which also makes it have better antigen binding ability than ordinary antibodies. On the other hand, in addition to the lack of light chains, compared with ordinary antibodies, there is no CH1 region between its heavy chain variable region and hinge region, which is also a major difference of heavy chain antibodies.

Another embodiment of the present invention is a fusion protein comprising the B7-H3 binding protein described in the present disclosure. In some embodiments, in addition to the B7-H3 binding protein, the fusion protein further comprises one or more other biologically active proteins, which can be any protein with biological, therapeutic, preventive or diagnostic significance or function, and when it is administered to a subject, mediating biological activity can prevent or alleviate a disease, disorder or condition.

Another embodiment of the invention is a nucleic acid capable of encoding a Nanobody, a B7-H3 binding protein or a heavy chain antibody as defined above.

Another embodiment of the invention is an antibody-drug conjugate comprising the nanobody, B7-H3 binding protein or heavy chain antibody or antigen binding fragment thereof, a linker and a small molecule cytotoxic drug.Antibody-drug conjugate (ADC) is a chemical connection connecting a biologically active small molecule drug to an antibody (e.g., a nanobody or antibody of the invention), which serves as a carrier to deliver the small molecule drug to target cells.

Another embodiment of the invention is a composition comprising a Nanobody, a B7-H3 binding protein, a heavy chain antibody, an antigen binding fragment, a nucleic acid, a cell and/or an antibody-drug composition as defined above. Another embodiment of the invention is a composition as defined above further comprising a pharmaceutically acceptable carrier.

Another embodiment of the invention is a B7-H3 binding protein, a heavy chain antibody, an antigen-binding fragment or a Nanobody as defined above, or a nucleic acid as defined above, or a cell as defined above, or an antibody-drug conjugate as defined above, or a composition as defined above, for use as a medicament.

Another embodiment of the present invention is a B7-H3 binding protein, a heavy chain antibody, an antigen binding fragment or a nanobody as defined above, or a nucleic acid as defined above, or a cell as defined above, or an antibody-drug conjugate as defined above, or a composition as defined above for treating, preventing and/or alleviating a B7-H3-related disease.

Another embodiment of the present invention is the use of a B7-H3 binding protein, a heavy chain antibody, an antigen binding fragment or a nanobody as defined above, or a nucleic acid as defined above, or a cell as defined above, or an antibody-drug conjugate as defined above, or a composition as defined above in the preparation of a medicament for treating, preventing and/or alleviating a B7-H3-related disease.

In some embodiments, the B7-H3-related disease or condition is a B7-H3-mediated disease or condition. In some embodiments, the B7-H3-mediated disease or condition is a tumor. In some embodiments, the B7-H3-mediated disease or condition is a solid tumor and/or a hematological tumor. In some specific embodiments, the B7-H3-mediated disease or condition is one or more tumors selected from the group consisting of lung cancer, breast cancer, prostate cancer, pancreatic cancer, colorectal cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer and kidney cancer, adrenal tumors, AIDS-related cancers, alveolar soft part sarcoma, astrocytic tumors, bone cancer, brain and spinal cord cancer, metastatic brain tumors, B cell cancer, cancer, carotid body tumors, chondrosarcoma, chordoma, benign fibrous histiocytoma of the skin, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, osteogenesis imperfecta, fibroplastic dysplasia of bone, benign, gallbladder cancer or bile duct cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, leukemia, liposarcoma/malignant lipoma, lymphoma, medulloblastoma, meningioma, multiple endocrine tumors, multiple myeloma, myelodysplastic syndrome, neuroblastoma, papillary thyroid carcinoma, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, melanocytoma, pituitary tumor, posterior uveal melanoma, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell cancer, gastric cancer, synovial sarcoma, testicular cancer, thymoma, and thyroid metastatic cancer. The cancers that can be treated by the B7-H3 binding protein or nanobody or heavy chain antibody of the present disclosure are not limited to the above-mentioned specific cancers, as long as the cancer expressing B7-H3 is suitable for treatment using the B7-H3 binding protein or nanobody or heavy chain antibody of the present disclosure.

Another embodiment of the invention is the use of a Nanobody, a B7-H3 binding protein, a heavy chain antibody, a nucleic acid, a recombinant cell, a conjugate, or a composition as defined above, wherein the medicament is administered intravenously, subcutaneously, orally, sublingually, nasally or by inhalation.

Another embodiment of the invention is a method for the prophylactic or therapeutic treatment of a B7-H3-associated disease or disorder, comprising administering to the patient an effective dose of a Nanobody, antibody, heavy chain antibody, fusion protein, nucleic acid, recombinant cell, conjugate, or composition as defined above.

Another embodiment of the invention is a method of producing a B7-H3 binding protein or a Nanobody or a heavy chain antibody as defined above, comprising:

a) cultivating a host cell comprising a nucleic acid capable of encoding a polypeptide as defined above under conditions allowing expression of the polypeptide, and,

b) recovering the produced polypeptide from the culture.

Another embodiment of the invention is the method as defined above, wherein said host cell is a bacterial, yeast or mammalian cell.

Another embodiment of the present invention is a method for diagnosing a disease or condition mediated by B7-H3, comprising the steps of:

a) contacting the sample with a B7-H3 binding protein, Nanobody, heavy chain antibody, fusion protein, or conjugate as defined above, and

b) detecting the binding of the B7-H3 binding protein, Nanobody, heavy chain antibody, fusion protein, or conjugate to the sample, and

c) wherein a binding result above the cutoff value is indicative of a B7-H3 mediated disease or disorder in the sample.

In some embodiments, the sample is a biopsy sample. In some embodiments, the disease is a tumor. In some embodiments, the disease is a solid tumor and/or a blood tumor. In some specific embodiments, the disease is one or more tumors selected from the group consisting of lung cancer, breast cancer, prostate cancer, pancreatic cancer, colorectal cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer and kidney cancer, adrenal tumors, AIDS-related cancers, alveolar soft part sarcoma, astrocytic tumors, bone cancer, brain and spinal cord cancer, metastatic brain tumors, B cell cancer, cancer, carotid body tumors, chondrosarcoma, chordoma, benign fibrous histiocytoma of the skin, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, osteogenesis imperfecta, fibrous dysplasia of bone, gallbladder carcinoma or bile duct cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposi sarcoma, leukemia, liposarcoma/malignant lipoma, lymphoma, medulloblastoma, meningioma, multiple endocrine neoplasms, multiple myeloma, myelodysplastic syndrome, neuroblastoma, papillary thyroid carcinoma, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, melanocytoma, pituitary tumor, posterior uveal melanoma, metastatic renal cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell cancer, gastric cancer, synovial sarcoma, testicular cancer, thymoma, and metastatic thyroid cancer.

Another embodiment of the present invention is a kit for diagnosing a disease or disorder associated with B7-H3. In some embodiments, the reagent can be used in the method as defined above.

Another embodiment of the invention is a B7-H3 binding protein, Nanobody, heavy chain antibody, fusion protein as defined above further comprising one or more in vivo imaging agents.

One embodiment of the present invention relates to a pharmaceutical composition comprising at least one B7-H3 binding protein, Nanobody, heavy chain antibody, fusion protein of the present invention, and at least one pharmaceutically acceptable carrier, diluent or excipient.

ELISA assays to measure the binding of B7-H3 binding proteins, Nanobodies, heavy chain antibodies, fusion proteins against B7-H3 to B7-H3 are well known.

One aspect of the invention is an anti-B7-H3 polypeptide comprising at least one anti-B7-H3 heavy chain antibody, in particular a nanobody derived therefrom. One aspect of the invention is that such a polypeptide may comprise other components. Such other components may be polypeptide sequences, such as one or more anti-B7-H3 nanobodies or one or more anti-serum albumin nanobodies. Other fusion proteins are also within the scope of the invention and may include, for example, fusions with carrier polypeptides, signaling molecules, tags, and enzymes. Other components may include, for example, radioactive labels, organic dyes, fluorescent compounds.

According to one aspect of the present invention, the anti-B7-H3 polypeptide of the present invention may comprise at least two identical or different anti-B7-H3 nanobody sequences. The anti-B7-H3 polypeptide may comprise at least two of the above sequences that do not have the same affinity for B7-H3, thereby forming an anti-B7-H3 polypeptide that combines weak affinity and high affinity binding sequences.

Methods for constructing bivalent polypeptides are known in the art (eg US 2003/0088074) and are also described below.

It may be desirable to modify the B7-H3 binding proteins of the invention in terms of effector function to enhance their therapeutic efficacy. For example, nanobody fusions fused to certain Fc domains, especially to Fc domains of human origin, may be advantageous.

In sequential administration, the polypeptide may be administered once or any number of times before and/or after administration of the agent and in various doses. Sequential administration may be combined with simultaneous or sequential administration.

Another embodiment of the present invention is a B7-H3 binding protein as described herein, comprising one or more immunoglobulin single variable domains, wherein one or more of said immunoglobulin single variable domains are humanized.

Humanization refers to mutations that result in little or no potential immunogenicity when administered to human patients. According to the present invention, humanization of a polypeptide may include the following steps: replacing one or more non-human immunoglobulin amino acids with human counterparts present in a human consensus sequence or human germline gene sequence without causing the polypeptide to lose its typical characteristics, i.e., humanization will not significantly affect the antigen binding ability of the resulting polypeptide.

According to one aspect of the invention, humanized Nanobodies are defined as Nanobodies that have at least 50% homology (e.g. 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%) with human framework regions.

One embodiment of the invention relates to polypeptides comprising at least one Nanobody, in which one or more amino acid residues have been substituted without substantially altering the antigen binding capacity.

The skilled artisan will recognize that the B7-H3 binding proteins of the invention may be modified, and such modifications are within the scope of the invention. For example, the polypeptide may be used as a drug carrier, in which case it may be fused to a therapeutic activator, or its solubility properties may be altered by fusion to an ionic/bipolar group, or it may be used for imaging by fusion to a suitable imaging marker, or it may contain modified amino acids, etc. The polypeptide may also be prepared as a salt. Such modifications that substantially retain binding to B7-H3 are within the scope of the invention.

It is clear from the disclosure herein that the use of natural or synthetic analogs, mutants, variants, alleles, homologues and orthologues of the Nanobodies of the invention as defined herein (collectively referred to herein as "analogues"), in particular analogues of the Nanobodies of SEQ ID NO: 1-20, is also within the scope of the invention. Thus, according to one embodiment of the invention, the term "Nanobody of the invention" in its broadest sense also covers such analogues.

Typically, in such analogs, one or more amino acid residues may have been replaced, deleted and/or added compared to the Nanobodies of the invention as defined herein. Such substitutions, insertions or deletions may be made in one or more framework regions and/or in one or more CDRs. When such substitutions, insertions or deletions are made in one or more framework regions, they may be made at one or more other positions in one or more Hallmark residues and/or framework residues, but substitutions, insertions or deletions of Hallmark residues are generally less preferred (unless these are suitable humanizing substitutions as described herein).

Yet another modification may comprise the introduction of one or more detectable labels or other signal generating groups or moieties, depending on the intended use of the labelled Nanobody. Suitable labels and techniques for attaching, using and detecting Nanobodies will be clear to the skilled person and include, for example, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine and fluorescent metals such as ¹⁵² Eu or other lanthanide metals), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, thermoacridinium esters, imidazoles, acridinium salts, oxalates, dioxetanes or GFP and its analogs), radioactive isotopes (such as ³ H, ¹²⁵ I, ³² P, ³⁵ S, ¹⁴ C, ⁵¹ Cr, ³⁶ Cl, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, and ⁷⁵ Se), metals, metal chelates or metal cations (such as metal cations such as ^99m Tc, ¹²³ I, ¹¹¹ In, ¹³¹ I, ⁹⁷ Ru, ⁶⁷ Cu, ⁶⁷ Ga, and ⁶⁸ Ga or other metals or metal cations particularly suitable for in vivo, in vitro or in situ diagnosis and imaging, such as ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr, and ⁵⁶ Fe), as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, biotin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase). Other suitable labels will be clear to the skilled person, for example including moieties that can be detected using NMR or ESR spectroscopy.

Depending on the choice of the specific label, such labeled Nanobodies and polypeptides of the invention can, for example, be used in in vitro, in vivo or in situ assays (including immunoassays known per se, such as ELISA, RIA, EIA and other "sandwich assays", etc.) as well as for in vivo diagnostic and imaging purposes.

As will be clear to the skilled person, another modification may involve the introduction of a chelating group which, for example, chelates one of the above metals or metal cations. Suitable chelating groups include, for example, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may include the introduction of a functional group that is part of a specific binding pair, such as a biotin-(streptavidin) avidin binding pair. Such a functional group can be used to connect the nanobodies of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair (i.e., by forming a binding pair). For example, the nanobodies of the invention can be conjugated to biotin and connected to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such conjugated nanobodies can be used as reporter genes, such as in a diagnostic system in which a detectable signal-generating agent is conjugated to avidin or streptavidin. For example, such binding pairs can also be used to bind the nanobodies of the invention to a carrier including a carrier suitable for pharmaceutical purposes. A non-limiting example is the case of liposome preparations described in Cao and Suresh, Journal of Drug Targeting, 8, 4, 257 (2000). Such binding pairs can also be used to connect therapeutic activators to the nanobodies of the invention.

Other potential chemical and enzymatic modifications will be clear to the skilled person. Such modifications may also be introduced for research purposes (e.g., to study function-activity relationships). See, for example, Lundblad and Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).

As mentioned above, the invention also relates to proteins or polypeptides that essentially consist of at least one Nanobody of the invention. "Essentially consisting of" means that the amino acid sequence of the polypeptide of the invention is identical to the amino acid sequence of a Nanobody of the invention or corresponds to the amino acid sequence of a Nanobody of the invention with a limited number of amino acid residues, such as 1-20 amino acid residues, such as 1-10 amino acid residues, preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, added to the amino-terminus, the carboxyl-terminus or both the amino-terminus and the carboxyl-terminus of the amino acid sequence of the Nanobody.

Said amino acid residues may or may not change, alter or otherwise affect the (biological) properties of the Nanobody, and may or may not add further functionality to the Nanobody.

According to another embodiment, the polypeptide of the invention comprises a Nanobody of the invention, which is fused at its amino terminus, at its carboxyl terminus, or at its amino terminus and at its carboxyl terminus to at least one further amino acid sequence, i.e. to provide a fusion protein comprising said Nanobody of the invention and one or more further amino acid sequences. Such fusions will also be referred to herein as "Nanobody fusions".

The one or more additional amino acid sequences may be any suitable and/or desired amino acid sequences. The additional amino acid sequences may or may not change, alter or otherwise affect the (biological) properties of the Nanobody, and may or may not add further functionality to the Nanobody or polypeptide of the invention. Preferably, the additional amino acid sequence is such that it confers one or more desired properties or functionalities to the Nanobody or polypeptide of the invention.

The nucleic acid of the present invention can be in the form of single-stranded or double-stranded DNA or RNA, preferably in the form of double-stranded DNA. For example, the nucleotide sequence of the present invention can be genomic DNA, cDNA or synthetic DNA (e.g., DNA with codon usage that has been specifically adapted for expression in the intended host cell or host organism).

According to one embodiment of the invention, the nucleic acid of the invention is in essentially isolated form as defined herein.

The nucleic acid of the invention may also be in the form of, present in and/or be part of a vector, such as a plasmid, cosmid or YAC, which may also be in essentially isolated form.

Nucleic acid of the present invention can also be the form of genetic construct, be present in genetic construct and/or as a part of genetic construct, which is clear to those skilled in the art.This type of genetic construct usually comprises at least one nucleic acid of the present invention, and this nucleic acid is optionally connected to one or more elements of genetic construct known per se, for example one or more suitable regulatory elements (for example suitable promoter, enhancer, terminator etc.) and other elements of genetic construct mentioned herein.This type of genetic construct comprising at least one nucleic acid of the present invention will also be referred to as " genetic construct of the present invention " in this article.

Genetic construct of the present invention can be DNA or RNA, and is preferably double-stranded DNA.Genetic construct of the present invention can also be the form suitable for transforming desired host cell or host organism, the form suitable for being integrated into the genomic DNA of desired host cell, or the form suitable for independently replicating, maintaining and hereditary in desired host organism.For example, genetic construct of the present invention can be the form of the carrier of for example plasmid, cosmid, YAC, viral vector or transposon.Especially, carrier can be expression vector, i.e. can provide the carrier of external and/or internal expression (for example in suitable host cell, host organism and/or expression system).

In a preferred, but non-limiting embodiment, the genetic construct of the invention comprises: a) at least one nucleic acid of the invention, operably linked to b) one or more regulatory elements, such as a promoter and optionally a suitable terminator; and optionally c) one or more other elements of a genetic construct known per se; wherein the terms "regulatory element", "promoter", "terminator" and "operably linked" have their usual meaning in the art (as further described herein); and wherein the "other elements" present in the genetic construct may be, for example, 3'-UTR or 5'-UTR sequences, leader sequences, selection markers, expression markers/reporter genes, and/or elements that may facilitate or increase transformation or integration (efficiency). These and other suitable elements for such genetic constructs are clear to the skilled person and may, for example, depend on the type of construct used, the desired host cell or host organism; the manner in which the nucleotide sequence of interest of the invention is expressed (e.g., by constitutive, transient or inducible expression); and/or the transformation technique to be used. For example, regulatory sequences, promoters and terminators known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments) may be used in an essentially analogous manner.

Preferably, in the genetic construct of the present invention, the at least one nucleic acid of the present invention and the regulatory element, and optionally the one or more other elements, are "operably connected" to each other, which generally means that there is a functional relationship between them. For example, if the promoter is capable of initiating or otherwise controlling/regulating the transcription and/or expression of a coding sequence (wherein the coding sequence is understood to be "under the control of the promoter"), the promoter is considered to be "operably connected" to the coding sequence. Generally, when two nucleotide sequences are operably connected, they will be in the same orientation and generally in the same reading frame. It is also generally substantially continuous, although this may not be necessary.

Preferably, the regulatory elements and other elements of the genetic construct of the invention are such that it is able to provide its desired biological function in the desired host cell or host organism.

For example, a promoter, enhancer or terminator should be "operable" in the desired host cell or host organism, which means that (for example) the promoter should be able to initiate or otherwise control/regulate the transcription and/or expression of a nucleotide sequence, such as a coding sequence, to which it is operably linked (as defined herein).

Some particularly preferred promoters include, but are not limited to, promoters known per se for expression in the host cells mentioned herein; and in particular promoters for expression in bacterial cells, such as those mentioned herein and/or used in the Examples.

Selectable markers should be those which allow - i.e. under appropriate selection conditions - host cells and/or host organisms which have been (successfully) transformed with the nucleotide sequence of the invention to be distinguished from host cells/organisms which have not been (successfully) transformed. Some preferred, but non-limiting examples of such markers are genes which provide resistance to antibiotics (e.g. kanamycin or ampicillin), genes which provide temperature resistance, or genes which allow the host cell or host organism to be maintained in the absence of certain factors, compounds and/or (food) components in the culture medium which are essential for the survival of non-transformed cells or organisms.

The leader sequence should be one that allows the post-translational modification to be performed in the desired host cell or host organism and/or allows the mRNA to be directed to the desired part or organelle of the cell. The leader sequence can also allow the expression product to be secreted from the cell. Therefore, the leader sequence can be any prosequence, presequence or preprosequence that is operable in the host cell or host organism. Expression in bacterial cells may not require a leader sequence. For example, the leader sequence known per se for expressing and producing antibodies and antibody fragments (including but not limited to single domain antibodies and ScFv fragments) can be used in a substantially similar manner.

The expression marker or reporter gene should be one that allows detection of the expression of the genetic construct (the gene or nucleotide sequence present on the genetic construct) in a host cell or host organism. The expression marker can also optionally allow localization of the expression product, such as in a specific part or organelle of a cell and/or in a specific cell, tissue, organ or part of a multicellular organism. Such reporter genes can also be expressed as proteins fused to the amino acid sequence of the present invention. Some preferred but non-limiting examples include fluorescent proteins such as GFP.

Some preferred but non-limiting examples of suitable promoters, terminators and other elements include those useful for expression in the host cells mentioned herein; and in particular those suitable for expression in bacterial cells, such as those mentioned herein and/or used in the examples below. For some (other) non-limiting examples of promoters, selection markers, leader sequences, expression markers and other elements that may be present/used in the genetic constructs of the invention - such as terminators, transcription and/or translation enhancers and/or integration factors - reference is made to the general handbooks of Sambrook et al. and Ausubel et al., for example, mentioned above, and to the examples given in WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, US-A-6,207,410, US-A-5,693,492 and EP 1 085 089. Other examples will be clear to the skilled person. Reference is also made to the general background technology cited above and to the further references cited herein.

The genetic constructs of the invention may generally be provided by appropriately ligating a nucleotide sequence of the invention to one or more of the other elements described above, for example using the techniques described in the general manuals of Sambrook et al. and Ausubel et al. mentioned above.

Typically, the genetic construct of the invention will be obtained by inserting the nucleotide sequence of the invention into a suitable (expression) vector known per se. Some preferred, but non-limiting examples of suitable expression vectors are those used in the following examples and those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of the invention can be used to transform host cells or host organisms, i.e. for expression and/or production of Nanobodies or polypeptides of the invention. Suitable hosts or host cells will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism.

In general, for the prevention and/or treatment of the diseases and disorders mentioned herein, the Nanobodies and polypeptides of the invention are typically administered in an amount of 1 gram to 0.01 microgram per kg body weight per day, preferably 0.1 gram to 0.1 microgram per kg body weight per day, for example about 1, 10, 100 or 1000 micrograms per kg body weight per day, continuously (e.g. by infusion) as a single daily dose or as multiple divided doses throughout the day, depending on the specific disease or disorder to be treated, the potency of the specific Nanobodies and polypeptides of the invention to be used, the specific route of administration and the specific pharmaceutical formulation or composition used. Based on the factors mentioned herein, the clinician will generally be able to determine a suitable daily dose. It is also clear that in specific circumstances the clinician may choose to deviate from these amounts, for example based on the above factors and their professional judgment. In general, some guidance on the amount of administration can be obtained from the usual administration amounts of comparable conventional antibodies or antibody fragments against the same target administered by essentially the same route, but differences in affinity/avidity, efficacy, biodistribution, half-life and similar factors well known to the skilled person need to be taken into account.

It should also be noted that when the Nanobodies of the invention contain one or more other CDR sequences that are different from the preferred CDR sequences described above, these CDR sequences can be obtained in any manner known per se, for example from Nanobodies (preferred), _VH domains from conventional antibodies (in particular from human antibodies), heavy chain antibodies, conventional 4-chain antibodies (e.g. conventional human 4-chain antibodies) or other immunoglobulin sequences against A-β. Such immunoglobulin sequences against A-β can be generated in any manner known per se, as will be clear to the skilled person, i.e. by immunization with A-β or by screening a library of suitable immunoglobulin sequences with A-β or any suitable combination. Optionally, this may be followed by techniques such as random or site-directed mutagenesis and/or other affinity maturation techniques known per se. Suitable techniques for generating such immunoglobulin sequences are clear to the skilled person and include, for example, the screening techniques reviewed by Hoogenboom, Nature Biotechnology, 23, 9, 1105-1116 (2005). Other techniques for producing immunoglobulins against specific targets include, for example, nanocloning technology (e.g. described in non-prepublished US provisional patent application 60/648,922), the so-called SLAM technology (e.g. described in European patent application 0 542 810), the use of transgenic mice expressing human immunoglobulins or the well-known hybridoma technology (see, for example, Larrick et al., Biotechnology, Vol. 7, 1989, p. 934). All of these techniques can be used to produce immunoglobulins against A-beta, and the CDRs of such immunoglobulins can be used in the Nanobodies of the invention, i.e. as described above. For example, the sequences of such CDRs can be determined, synthesized and/or isolated, and inserted into the sequences of the Nanobodies of the invention (e.g., to replace the corresponding natural CDRs), all using techniques known per se such as those described herein, or Nanobodies of the invention comprising such CDRs (or nucleic acids encoding them) can be synthesized de novo, again using the techniques mentioned herein.

The invention will now be further described by way of the following non-limiting examples and figures.

Example

Example 1 Construction of alpaca immune library

1.1 Alpaca Immunity

The antigens used for immunization, Human B7-H3-Fc (ACRO B73-H5253), Human B7-H3-His (ACRO B73-H52E2), and Mouse B7-H3-Fc (ACRO B73-M5255), were all purchased commercially. Immunization was performed by subcutaneous injection, and two alpacas, NM018 and NM019, were immunized in total. NM018 was immunized alternately with human antigens Human B7-H3-Fc and Human B7-H3-His; NM019 was immunized alternately with Human B7-H3-Fc and Mouse B7-H3-Fc human and mouse antigens. The single immunization dose was 500μg. Freund's incomplete adjuvant was used for the first immunization, and Freund's complete adjuvant was used for subsequent immunizations. Immunization was performed once every two weeks, for a total of 5 times.

1.2 Serum titer detection

After the 4th and 5th immunizations, blood samples were collected to test the titer of the target antigen Human B7-H3-Fc antibody in the serum. The specific detection method is as follows:

The antigen Human B7-H3-Fc was diluted with PBS to a final concentration of 2μg/mL, and 30μL of the dilution was added to the first ELISA plate and coated overnight at 4℃. On the day of the immunopotency assay, the plate was rinsed three times with PBST, then blocked with PBSM containing 5% skim milk at room temperature for two hours, and then rinsed three times with PBST. On another plate, the negative serum without immunization and the serum after the fourth or fifth immunization were diluted with PBS, with a 2000-fold dilution in the first well, and then a 2-fold gradient dilution was used for the subsequent 7 wells. The diluted serum was added to the first ELISA plate and incubated at room temperature for 1 hour. After washing the plate three times with PBST, the secondary antibody Goat anti-Llama IgG (H+L) Secondary Antibody [HRP] (purchased from NOVUS Cat. NBP1-75088) was added at 1:5000 and incubated at room temperature for 0.5 hours. After incubation, the plate was washed six times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to terminate the reaction and the plate was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

The results are shown in Table 1. After 5 immunizations, the antibody titer of the targeting antigen Human B7-H3-Fc in the sera of the two alpacas reached a dilution ratio of 1:32K or above.

Table 1. Detection of IgG titer in serum of B7-H3 immunized alpaca

Example 2 Construction of phage display library and screening of nanoantibodies targeting B7-H3

Using phage display technology, the antibody genes of alpaca peripheral blood cells (PBMC) immunized with B7-H3 antigen protein were cloned into phage display vectors to construct an antibody library. The library was screened using Human B7-H3-Fc, Human B7-H3-His, and Mouse B7-H3-Fc as screening antigens, respectively, and multiple nanoantibodies that specifically bind to B7-H3 protein were obtained.

2.1 Construction of phage display library of camel-derived nanobodies

100 mL of blood was collected from alpacas to separate PBMC cells, and RNA was extracted from PBMC cells by chloroform method and reverse transcribed into cDNA. Degenerate primers were designed based on the situation of VHH antibody germline genes, and the DNA fragment encoding VHH-CH2 was obtained by PCR amplification and recovery of PCR products by agarose gel electrophoresis. Then, by the method of secondary PCR, the DNA fragment encoding the variable domain (VHH) was amplified using the DNA fragment encoding VHH-CH2 as a template. Then, the DNA fragment encoding VHH was digested and purified and constructed into a phage display vector. Finally, the vector expressing VHH was transformed into competent Escherichia coli by an electrotransformer, and 10 μl was taken to coat a monoclonal plate for sequencing. The storage capacity of the antibody library and the correct insertion rate of the antibody gene were determined by dilution point plate method and monoclonal sequencing analysis, respectively. The storage capacity of the single domain antibody library was determined to be 10 ⁸ level, and sequence analysis showed that the correct insertion rate of the antibody gene was greater than 80%.

2.2 Screening of antibody gene phage display library

Immunotubes and magnetic bead screening were used to screen the phage display library, and three rounds of screening were performed using Human B7-H3-Fc, Human B7-H3-His, and Mouse B7-H3-Fc.

2.2.1 Screening of antibody gene phage display library by immunotube method

The principle of immunotube screening is to coat the antigen protein Human B7-H3-Fc or Human B7-H3-His on the surface of an immunotube with high adsorption capacity, and then enrich the specific monoclonal antibodies against the antigen by adding the phage display antibody library into the immunotube and incubating, washing and eluting the antigen protein adsorbed on the surface of the immunotube.

The specific method is as follows. Add 1mL 300μg/mL Human B7-H3-Fc or Human B7-H3-His to different groups of immunotubes and coat at 4℃. Incubate the phage suspension with the protein-coated immunotubes, and combine and wash according to the immunotube screening system method. Then elute the phage with Trypsin, mix the eluted phage solution with the logarithmic phase Escherichia coli SS320 cells, incubate at 37℃ for 30min, spread on 2YT medium, and culture in a 37℃ incubator overnight for the next round of screening. At the same time, the eluted phage solution was diluted 10 times with logarithmic phase SS320 cells, incubated at 37℃ for 30min, mixed well, and 2μL was dropped on the plate, and cultured in a 37℃ incubator overnight for detection.

2.2.2 Screening of antibody gene phage display library by magnetic bead method (Kingfisher method)

Human B7-H3-Fc and Human B7-H3-His were labeled with biotin and then bound to Dynabeads magnetic beads. The antigen-bound magnetic beads and the antibody gene phage display library were incubated, washed, and eluted for panning, thereby enriching a large number of monoclonal antibodies specific for the antigen.

The specific method is as follows. The prepared alpaca immune library phage suspension was diluted and blocked with 5% BSA, incubated with Dynabeads, and the phages after negative screening incubation were collected. According to the Kingfisher magnetic bead screening system method, the magnetic beads were bound and washed with Human B7-H3-Fc or Human B7-H3-His, and 5% BSA was incubated with the magnetic beads. The phage suspension collected after negative screening was incubated with Dynabeads coated and blocked with biotin-labeled antigen, and bound and washed according to the Kingfisher magnetic bead screening system method, and the phages were eluted with Trypsin. After elution, the phage solution was fully mixed with the logarithmic phase Escherichia coli SS320 cells and incubated at 37°C for 30 minutes, coated in 2YT culture medium, and cultured overnight in a 37°C incubator for the next round of screening. At the same time, the eluted phage solution was diluted 10-fold with logarithmic phase SS320 cells, incubated at 37°C for 30 min, mixed and dropped 2 μL on the plate, and cultured in a 37°C incubator overnight for detection.

2.2.3 Selection of single clones

The phage pools obtained from each round of elution by immunotube screening and magnetic bead screening were diluted 5 times with 5% PBSM and screened by ELISA.

Add 2μg/mL Human B7-H3-Fc or Mouse B7-H3-Fc to a 96-well plate, 30μL per well, and coat overnight at 4℃; block with 5% PBSM at room temperature for 1h; add 30μL/well of the gradient diluted VHH or Phage expression supernatant obtained in 2.2.1 and 2.2.2 and incubate at room temperature for 1h; add 30μL/well of the secondary antibody Anti-Flag-HRP (Sigma Cat H7425-1VL) diluted 1:5000 to the VHH expression supernatant, and add 30μL/well of the secondary antibody Anti-M13-HRP (SinoBiological Cat 11973-MM05T) diluted 1:20000 to the Phage expression supernatant and incubate at room temperature for 1h; add TMB for color development for 5min to 20min, then add stop solution to terminate the reaction, and read the data by OD450 on an enzyme reader.

A large number of monoclones were selected for ELISA screening. After sequencing analysis combined with ELISA, 33 nanoantibodies with sequence diversity that bind to the B7-H3 antigen were obtained. 33 sequences were selected for ELISA affinity retest. The results are shown in Figure 1.

Finally, 13 monoclones were selected based on affinity and mouse cross-reactivity for the construction of full-length sequence (VHH and Fc fusion protein) expression vectors, as shown in Table 2.

Table 2. Amino acid sequences of VHH and VHH-Fc antibodies

Table 3. CDR sequences of VHHs defined according to the AbM numbering system

Example 3 Construction, expression and purification of antibodies

The 13 nanobodies obtained in Example 2 were constructed as heavy chain antibodies of human IgG1 subtype, i.e., VHH-Fc.

3.1 Plasmid construction

The VHH-encoding DNA fragment was constructed into the eukaryotic expression vector plasmid pcDNA3.4-IgG1, wherein the plasmid was obtained by connecting the human IgG1 Fc fragment (SEQ ID NO: 69) to the backbone pCDNA3.4 (purchased from Invitrogen, A14697), and finally a protein expression plasmid containing the complete VHH-Fc full-length gene was obtained.

3.2 Antibody expression and purification

The constructed plasmid was transfected into Expi CHO cells (Thermo Fisher, A29133) for transient expression. Seven days after transfection, the cell culture supernatant expressing the target protein was centrifuged at 15,000 g for 10 min. The supernatant was affinity purified using Protein A (purchased from cytiva (GE Life)), and the resulting protein was stored in PBS buffer.

Example 4 Identification of the physicochemical properties of antibodies

4.1 VHH-Fc antibody SDS-PAGE identification

Sample preparation: 1 μg of VHH-Fc antibody was added to 4×LDS loading buffer (containing iodoacetamide at a final concentration of 40 mM), heated in a 75°C dry bath for 10 min, cooled to room temperature and centrifuged at 12,000 rpm for 5 min, and the supernatant was used as a non-reduced SDS-PAGE sample; 2 μg of VHH-Fc antibody was added to 4×LDS loading buffer (containing DTT at a final concentration of 5 mM), heated in a 100°C dry bath for 10 min, cooled to room temperature and centrifuged at 12,000 rpm for 5 min, and the supernatant was used as a reduced SDS-PAGE sample. The quality control product IPI (Ipilimumab) has a non-reduced band molecular weight of about 150 kDa and a purity of more than 90%, a reduced heavy chain band molecular weight of about 55 kDa, a light chain molecular weight of about 25 kDa, and a heavy chain plus light chain purity of more than 90%. The samples were added to precast gel for gel electrophoresis and stained with Coomassie Brilliant Blue. After decolorization, they were scanned with EPSON V550 color scanner. The purity of the reduced and non-reduced bands was calculated using Image J according to the peak area normalization method.

The results are shown in Table 3: the band of VHH-Fc antibody in non-reducing gel is about 80 kDa, and the band of reducing gel is about 40 kDa, which is consistent with the expected size. Except for C184-Fc, the purity of other antibodies is greater than 90%.

4.2 SEC-HPLC Monomer Purity Identification of VHH-Fc Antibodies

Material preparation: 1. Mobile phase preparation: 150mmol/L PB+NaCl, pH adjusted to 6.0; 2. Sample treatment: dilute the sample concentration to 0.5mg/mL; 3. Agilent HPLC 1100 column (XBridge BEH SEC 3.5μm, 7.8mm×300mm, Waters) flow rate set to 0.8mL/min, injection volume 20μL, detector wavelength 280nm. According to the area normalization method, the percentage of high molecular weight polymers, monomers and low molecular weight polymers in the sample was calculated. The results are shown in Table 4. The purity of VHH-Fc antibody monomers except C184-Fc is greater than 95%.

Table 4. Results of physical and chemical properties test of VHH-Fc antibodies

Example 5 VHH-Fc Antibody Affinity (Elisa-Binding) Detection

Antigens Human B7-H3-His, Mouse B7-H3-Fc and Cyno B7-H3-His were diluted with 1×PBS to a concentration of 2μg/mL, and coated on 96-well plates at 30μL/well at 4°C overnight. The next day, the 96-well plates were washed 3 times with PBST and blocked with 5% skim milk for 2h. After washing 3 times with PBST, the test antibodies (ipilimumab (IPI) as a negative control and DX008-BMG-MGC (self-made sample, the amino acid sequence of the heavy chain is shown in SEQ ID NO:21, and the amino acid sequence of the light chain is shown in SEQ ID NO:22) as a positive control) diluted with 1% PBSM were added and incubated for 1h. After washing 3 times with PBST, secondary antibodies were added: for Human B7-H3-His and Cyno B7-H3-Fc, the secondary antibodies were added. o B7-H3-His, use Anti-human-Fc-HRP (abcam; ab97225); for Mouse B7-H3-Fc, use Anti-VHH1+VHH2-HRP (Genescript, A01861-200, CP0001); for ipilimumab and DX008-BMG-MGC, use Anti-human κ+λ-HRP (Millipore; AP502P; AP506P), and incubate for 1 hour; after incubation, wash the plate six times with PBST and add TMB for color development; according to the color development results, add 2M HCl to terminate the reaction, and read the plate at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

The results are shown in Figures 2, 3 and 4. The results showed that except for C184, C23 and A13, the remaining antibodies had high affinity activity with Human B7-H3-His antigen. A13, A2, A3, A77, A83, B91, C357, C59 and D16 had human-mouse cross-activity. Nine antibodies were selected for human-monkey cross-activity detection, and the results showed that A2, A3, A77, A83, B91, C59, C357 and D16 had human-monkey cross-activity.

DX008-BM-MGC positive control antibody VH (SEQ ID NO: 21)

DX008-BM-MGC positive control antibody VL (SEQ ID NO: 22)

Example 6 Kinetic detection of VHH-Fc antibody affinity

The Kientics experimental mode based on GATOR detects the affinity kinetics of VHH antibodies to the antigens Human B7-H3-His and Mouse B7-H3-His. DX008-BM-MGC is used as a positive control. The antibody to be tested is diluted to 30nM in a buffer containing PBS (10mM PH7.4), 0.02% Tween 20 and 0.2% BSA, and then carried out according to the preset program, with a binding time of 120s and a dissociation time of 120s. The antigen is diluted 2 times from 2400nM to 4.69nM. Finally, KD (affinity kinetic constant), Kon (binding constant) and Koff (dissociation constant) are obtained by fitting the binding and dissociation data of different concentrations of antigens. Kon can be written as Ka and Koff can be written as Kd.

The test results are shown in Tables 5 and 6. The results showed that the affinities of antibodies A2, A77, B91, and C59 were higher than those of the control antibody, and the affinities of antibodies A3, A83, B102, C357, and D16 were similar to or slightly lower than those of the control antibody, among which A2, A77, B91, C357, and C59 also had affinity for mouse B7-H3 antigen.

Table 5. Kinetic test results of affinity of VHH-Fc antibody to antigen Human B7-H3-His

Table 6. Kinetic test results of affinity between VHH-Fc antibody and antigen Mouse B7-H3-His

Example 7 Humanized transformation of nanobodies

In order to reduce the immunogenicity that may be caused by camel-derived nanobodies, the framework region of the nanobody was humanized and reversely mutated to obtain a heavy chain antibody with a higher degree of humanization while maintaining the affinity of the humanized antibody for the antigen.

7.1 Nanobody Humanization Process

VHH antibody A2 was selected for humanization. The antibody sequence was compared with the human antibody germline gene database to find the human germline with high homology to the VHH sequence. After defining the CDR and framework regions, humanized sequences of different degrees were designed according to the differential sites in the framework region. After humanization of the A2 sequence, 7 humanized antibodies were obtained: VHH21 to VHH27, and their amino acid sequences are shown in Table 7.

The expression vectors of humanized antibodies VHH21 to VHH27 were constructed as described in Example 3.1. The humanized VHH-Fc antibodies respectively comprised the VHH amino acid sequence described above and the Fc fragment of human IgG1 (SEQ ID NO: 69). The expression and purification of the humanized VHH-Fc antibodies are described in Example 3.2. The sequences of the humanized VHH-Fc antibodies are shown in Table 7.

Table 7. Sequences of humanized VHHs and humanized VHH-Fc of A2 VHH

Table 8. CDR sequences of humanized VHHs of A2 VHH

7.2 Affinity activity test of humanized antibodies (ELISA-Binding test)

The affinity of humanized VHH-Fc antibodies (VHH21 to VHH27) to the antigens Human B7-H3-His, Mouse B7-H3-His and Cyno B7-H3-His was determined by ELISA and compared with the parental antibody A2-VHH-Fc.

The specific method is as follows: dilute the antigen Human B7-H3-His, Mouse B7-H3 His or Cyno B7-H3-His with 1×PBS to a concentration of 2μg/mL, and coat 96-well plates with 30μL/well at 4°C overnight. The next day, wash with PBST three times and block with 5% PBS-Milk for 2h; wash the plate, add the gradient dilution of the VHH-Fc antibody to be tested (VHH21 to VHH27; Ipilimumab (IPI) as a negative control, DX008-BMG-MGC as a positive control) and incubate for 1h; then, wash the plate three times with PBST, add the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab97225), and incubate at room temperature for 60min. Wash the plate six times with PBST, add TMB for color development; according to the color development results, add 2M HCl to terminate the reaction and detect OD450 at the same time.

The results are shown in Figure 5. The results showed that VHH21-Fc to VHH27-Fc all maintained affinity for human and cynomolgus monkey B7-H3 antigens, and VHH21-Fc, VHH22-Fc, VHH23-Fc and VHH25-Fc retained the parent's ability to bind to mouse antigens.

Example 8 Humanized Antibody Affinity Kinetics Evaluation

The GATOR-based Kientics experimental model was used to detect the affinity kinetics of humanized VHH-Fc antibodies to the antigens Human B7-H3-His, Mouse B7-H3-His and Cyno B7-H3-His. The parent antibody A2 VHH-Fc was used as a positive control. The detection method is shown in Example 6.

The test results are shown in Table 9. The results showed that the KD values of VHH21 and VHH25 were comparable to the parent KD value, and the KD values of the other molecules were greater than the KD value of the parent A2 VHH-Fc antibody.

Table 9. Kinetic test results of the affinity of humanized VHH-Fc antibodies to antigens Human B7-H3-His, Mouse B7-H3-His and Cyno B7-H3-His

Note: N/A means no binding at 1200nm, NA means no dissociation.

Example 9 In vivo efficacy experiment of humanized candidate ADC molecules

By chemical coupling, the topoisomerase toxin 0143 (obtained from MCE, catalog number: HY-114233), which is formed by connecting the DNA topoisomerase I inhibitor DX8951 and the protease cleavable linker MC-GGFG, is coupled to the humanized antibody molecule VHH25-Fc of the present application to prepare the VHH25-0143 ADC molecule with a DAR value of about 4. The structure of the topoisomerase toxin 0143 is as follows:

Calu-6 cells were subcutaneously inoculated on the right back of Balb/c nude mice to establish a subcutaneous xenograft mouse model of human lung cancer Calu-6 cell line. When the tumor grew to an average volume of 100 mm3, the mice were randomly divided into four groups according to tumor size and mouse weight: vehicle control group, VHH25-0143 ADC low-dose group (1 mg/kg), VHH25-0143 ADC medium-dose group (3 mg/kg) and VHH25-0143 ADC high-dose group (10 mg/kg), with 5 mice in each group. The day of grouping was recorded as day 1, and medication was started on the day of grouping, once a week (QW), for a total of 3 times. After medication, the tumor size was measured twice a week, and the results are shown in Figure 6.

Figure 6 shows that the VHH25-0143 ADC molecule can inhibit tumor growth at three doses, indicating that it has effective anti-tumor activity in vivo.

Claims (27)

A B7-H3 binding protein, wherein the binding protein comprises an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 contained in the VHH shown in any one of SEQ ID NO:1-13. The B7-H3 binding protein of claim 1, wherein the CDR1, CDR2 and CDR3 are encoded according to Kabat, AbM, Chothia or IMGT. The B7-H3 binding protein of claim 2, wherein the CDR1, CDR2 and CDR3 are encoded according to AbM, and: (1) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:29, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:30, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:31; (2) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:23, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:24, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:25; (3) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:26, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:27, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:28; (4) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:32, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:33, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:34; (5) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:35, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:36, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:37; (6) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:38, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:39, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:40; (7) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:41, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:42, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:43; (8) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:44, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:45, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:46; (9) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:47, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:48, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:49; (10) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:50, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:51, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:52; (11) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:53, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:54, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence of SEQ ID NO:55; (12) the CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:56, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:57, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence set forth in SEQ ID NO:58; or (13) The CDR1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:59, the CDR2 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:60, and the CDR3 comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in SEQ ID NO:61. The B7-H3 binding protein of any one of claims 1-3, wherein the immunoglobulin single variable domain comprises an amino acid sequence that is at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1-13, or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1-13. The B7-H3 binding protein of any one of claims 1 to 4, wherein the B7-H3 binding protein is monovalent, divalent or multivalent. The B7-H3 binding protein of any one of claims 1 to 5, wherein the B7-H3 binding protein is monospecific, bispecific or multispecific. The B7-H3 binding protein of any one of claims 1 to 3 and 5 to 6, wherein the immunoglobulin single variable domain comprises a heavy chain framework region, and at least a portion of the heavy chain framework region is derived from at least one of a mouse antibody, a human antibody, a primate antibody, and a mutant thereof. The B7-H3 binding protein of claim 7, wherein at least a portion of the heavy chain framework region is derived from a human antibody, preferably, the immunoglobulin single variable domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 14-20, more preferably, the immunoglobulin single variable domain comprises or consists of an amino acid sequence shown in any one of SEQ ID NOs: 14-20. The B7-H3 binding protein of any one of claims 1-8, wherein the B7-H3 binding protein is a heavy chain antibody, preferably, the heavy chain antibody further comprises human IgG Fc, more preferably, the heavy chain antibody further comprises human IgG1 Fc, more preferably, the human IgG1 Fc comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% identity with the amino acid sequence shown in SEQ ID NO:69, more preferably, the human IgG1 Fc comprises or consists of the amino acid sequence shown in SEQ ID NO:69. The B7-H3 binding protein of any one of claims 1 to 9, wherein the B7-H3 binding protein is a nanobody. The B7-H3 binding protein of claim 10, wherein the nanoantibody comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1-13, preferably, the nanoantibody comprises or consists of an amino acid sequence shown in any one of SEQ ID NOs: 1-13. The B7-H3 binding protein of claim 10, wherein the nanoantibody comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 14-20. Preferably, the nanoantibody comprises or consists of an amino acid sequence shown in any one of SEQ ID NOs: 14-20. A fusion protein comprising the B7-H3 binding protein according to any one of claims 1 to 12. A nucleic acid molecule encoding the B7-H3 binding protein of any one of claims 1 to 12 or the fusion protein of claim 13, optionally wherein the nucleic acid molecule is DNA. An expression vector comprising the nucleic acid molecule of claim 14, optionally wherein the expression vector is a prokaryotic expression vector or a eukaryotic expression vector. A cell comprising the nucleic acid molecule of claim 14 or the expression vector of claim 15, optionally wherein the cell is a prokaryotic cell or a eukaryotic cell (eg, a mammalian cell). A conjugate comprising the B7-H3 binding protein of any one of claims 1 to 12 or the fusion protein of claim 13 conjugated to a therapeutic agent, a diagnostic agent or an imaging agent; optionally, wherein the therapeutic agent is a small molecule cytotoxic drug. The conjugate of claim 17, wherein the therapeutic agent is a topoisomerase inhibitor, preferably exotecan. The conjugate of claim 17 or 18, wherein the B7-H3 binding protein or the fusion protein is conjugated to the therapeutic agent, diagnostic agent or imaging agent via a linker, preferably, the linker is MC-GGFG. A composition comprising the B7-H3 binding protein of any one of claims 1 to 12, the fusion protein of claim 13, the nucleic acid molecule of claim 14, the expression vector of claim 15, the cell of claim 16, or the conjugate of any one of claims 17 to 19, optionally, the composition is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or diluent. Use of the B7-H3 binding protein of any one of claims 1 to 12, the fusion protein of claim 13, the nucleic acid molecule of claim 14, the expression vector of claim 15, or the cell of claim 16, or the conjugate of any one of claims 17 to 19 in the preparation of a medicament for preventing, treating or alleviating a B7-H3-related disease. Use of the B7-H3 binding protein of any one of claims 1 to 12, the fusion protein of claim 13, the nucleic acid molecule of claim 14, the expression vector of claim 15, or the cell of claim 16, or the conjugate of any one of claims 17 to 19 in combination with another agent in the preparation of a medicament for preventing, treating or alleviating a B7-H3-related disease, wherein the other agent is preferably an immunotherapeutic agent or a chemotherapeutic agent. The use according to claim 21 or 22, wherein the B7-H3-related disease is a tumor, preferably a solid tumor and/or a blood tumor, and further preferably: lung cancer, breast cancer, prostate cancer, pancreatic cancer, colorectal cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer and kidney cancer, adrenal tumors, AIDS-related cancers, alveolar soft part sarcoma, astrocytic tumors, bone cancer, brain and spinal cord cancer, metastatic brain tumors, B cell cancer, cancer, carotid body tumors, chondrosarcoma, chordoma, benign fibrous histiocytoma of the skin, desmoplastic small round cell tumor, ependymoma, Ewing's tumor, extraskeletal myxoid chondrosarcoma, osteogenesis imperfecta, fibroblastic fibrosis, osteoporosis ... fibroblastic dysplasia, gallbladder or bile duct cancer, gestational trophoblastic disease, germ cell tumors, head and neck cancer, hepatocellular carcinoma, islet cell tumors, Kaposi sarcoma, leukemia, liposarcoma/malignant lipoma, lymphoma, medulloblastoma, meningioma, multiple endocrine neoplasms, multiple myeloma, myelodysplastic syndrome, neuroblastoma, papillary thyroid cancer, parathyroid tumor, childhood cancer, peripheral nerve sheath tumors, melanocytoma, pituitary tumors, posterior uveal melanoma, metastatic kidney cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell cancer, gastric cancer, synovial sarcoma, testicular cancer, thymoma, and metastatic thyroid cancer. The use according to claim 21 or 22, wherein the B7-H3 related disease is lung cancer. A kit for detecting B7-H3 or cells containing B7-H3, comprising the B7-H3 binding protein of any one of claims 1 to 12, or the conjugate of any one of claims 17 to 19. Use of the B7-H3 binding protein of any one of claims 1 to 12 or the conjugate of any one of claims 17 to 19 in the preparation of a kit for detecting B7-H3 or cells containing B7-H3. A chimeric antigen receptor comprising an antigen recognition domain, a hinge region, a transmembrane domain, and an intracellular domain (including a co-stimulatory domain and a signal transduction domain), wherein the antigen recognition domain comprises the B7-H3 binding protein according to any one of claims 1 to 12.