Single-domain antibody targeting b7h3 protein and related biomaterials and applications thereof

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a single-domain antibody targeting a B7H3 protein and a related biomaterial and application thereof. The single-domain antibody targeting the B7H3 protein or an antigen-binding fragment thereof provided in the application comprises three complementarity determining regions named CDR1, CDR2 and CDR3 in turn, and the amino acid sequences of the CDR1, CDR2 and CDR3 are the 28th-35th, 49th-60th and 97th-103rd positions of SEQ ID NO:1, respectively. The single-domain antibody provided in the application has the property of targeting B7H3-positive tumor cells, so that the single-domain antibody can be combined with an anticancer drug or an imaging agent for targeted treatment and imaging of tumors in actual application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to single-domain antibodies targeting the B7H3 protein and related biomaterials and applications. Background Technology

[0002] The occurrence and development of malignant tumors involve multiple gene abnormalities, signaling pathway dysregulation, and the remodeling of the immune regulatory network in the tumor microenvironment. With the development of precision medicine and molecular imaging, traditional imaging methods that rely solely on changes in anatomical structures are no longer sufficient to meet the clinical needs for early tumor diagnosis, molecular subtyping, and dynamic evaluation of treatment efficacy. Molecular imaging technologies that can non-invasively, in real-time, and quantitatively reflect the expression status of tumor-related molecular targets at the in vivo level are gradually becoming an important direction for development in the field of tumor diagnosis and treatment.

[0003] B7H3 (B7 homolog 3), also known as CD276, is an important member of the B7 immune co-regulatory protein family. Belonging to the type I transmembrane glycoprotein family, it plays a complex role in the regulation of co-stimulatory and co-inhibitory signals in the immune response. Its encoding gene is located on the long arm of human chromosome 15 (15q23-24), and the protein structure includes an extracellular immunoglobulin-like domain, a transmembrane region, and an intracellular short-tail structure. The specific receptor for B7H3 has not been consistently confirmed, and its signal transduction process and biological function are significantly context-dependent, potentially exhibiting different regulatory characteristics depending on cell type, physiological state, and microenvironmental conditions. Under physiological conditions, as an immune checkpoint-related molecule, B7H3 participates in regulating T cell-mediated immune responses. However, in the tumor microenvironment, numerous studies have shown that it primarily exerts a strong immunosuppressive function, assisting tumors in achieving immune escape by inhibiting the activation and proliferation of cytotoxic T cells and promoting the function of regulatory T cells. Furthermore, B7H3 can directly promote tumor cell proliferation, invasion, metastasis, and drug resistance, making it a key molecule closely related to the malignant progression of tumors.

[0004] B7H3 expression exhibits high tissue selectivity. Under normal physiological conditions, its expression level is extremely low and its distribution is limited, mainly found in certain epithelial cells and antigen-presenting cells, participating in maintaining immune homeostasis. However, in various solid tumors, including prostate cancer, breast cancer, non-small cell lung cancer, glioblastoma, renal cell carcinoma, and neuroblastoma, B7H3 shows widespread and high-level abnormal expression. Multiple clinicopathological studies have shown that high B7H3 expression is closely associated with increased tumor invasiveness, increased risk of metastasis, and poor patient prognosis, and can serve as an important molecular marker of tumor progression.

[0005] The mechanisms by which B7H3 promotes tumor development are multifaceted. Numerous studies have shown that B7H3 exhibits abnormally elevated expression levels in various solid tumors and tumor-associated angiogenesis. This high expression is closely related to the formation of an immunosuppressive microenvironment, such as decreased T cell proliferation and activation, and limited effector function. It can also affect NK cell-mediated cytotoxicity, thereby weakening the body's overall immune clearance of tumor cells and facilitating tumor immune escape. In addition to its immunomodulatory effects, B7H3 can also directly participate in the regulation of malignant tumor cell phenotypes through non-immune-dependent mechanisms. High B7H3 expression is correlated with enhanced tumor cell proliferation, increased migration and invasion capabilities, increased metastatic potential, and the formation of resistance to radiotherapy, chemotherapy, or targeted therapy. At the molecular level, its signal transduction may involve the regulation of multiple downstream pathways, including Wnt / β-catenin, PI3K / Akt, and Jak / Stat, collectively driving the malignant phenotype of tumors. This dual nature of immunomodulation and pro-tumor effects makes B7H3 one of the hot targets in tumor research and drug development in recent years.

[0006] Given the widespread high expression of B7H3 in various tumors and its close correlation with tumor malignancy, therapeutic strategies targeting B7H3 have been continuously proposed. Currently, research targeting B7H3 mainly focuses on the therapeutic field, including monoclonal antibodies, antibody-drug conjugates (ADCs), bispecific antibodies, and CAR-T cell therapy. Some candidate drugs, such as the ADC Omburtamab, have entered clinical trials. These studies, while validating the feasibility of B7H3 as a therapeutic target, also further highlight the urgent need for accurate assessment of B7H3 expression status in clinical practice.

[0007] Single-domain antibodies are the smallest functional antigen-binding fragments composed solely of the variable region of a heavy-chain antibody. They possess unique advantages such as small molecular weight (approximately 15 kDa), high affinity, good stability, strong solubility, rapid tissue penetration, rapid blood clearance, and low immunogenicity. The screening for high-affinity B7H3-targeting single-domain antibodies provides a breakthrough direction for the development of novel diagnostic and therapeutic tools. Compared to traditional monoclonal antibodies, single-domain antibodies have inherent advantages in the field of molecular imaging: their rapid blood clearance characteristics enable high target-to-sample imaging within a short period after injection, making them highly suitable for real-time, dynamic in vivo imaging.

[0008] Nuclear medicine molecular imaging is a key technology for achieving integrated precision diagnosis and treatment. After radiolabeling specific probes with radionuclides, positron emission tomography / computed tomography (PET / CT) or single-photon emission computed tomography (SPECT) can non-invasively, quantitatively, and visually visualize the distribution of target molecules in vivo. ImmunoPET imaging combines the high sensitivity of PET / CT with the high specificity of antibodies. This design is based on a single-domain antibody targeting B7H3, which can rapidly distribute in the bloodstream and specifically accumulate at tumor sites. By radiolabeling this single-domain antibody with radionuclides, novel molecular imaging probes can be constructed to achieve early and precise localization, staging, and dynamic monitoring of treatment efficacy in B7H3-positive tumors. Simultaneously, this targeting single-domain antibody can also serve as a carrier, conjugating cytotoxic drugs or radionuclide therapeutic agents to form Nb drug conjugates (NDCs), providing a new strategy for targeted therapy of B7H3-overexpressing tumors. Summary of the Invention

[0009] The technical problem to be solved by this invention is to provide a high-affinity B7H3-targeting single-domain antibody. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: The present invention provides a single-domain antibody or antigen-binding fragment thereof, wherein the single-domain antibody or antigen-binding fragment thereof comprises three complementarity-determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is positions 28-35, 49-60 and 97-103 of SEQ ID NO:1, respectively.

[0011] In this invention, the single-domain antibody or its antigen-binding fragment binds to B7H3.

[0012] In this invention, the binding can be a specific binding.

[0013] In some specific embodiments of the present invention, the single-domain antibody or its antigen-binding fragment comprises three complementarity-determining regions CDR1, CDR2 and CDR3, wherein the amino acid sequences of CDR1 are positions 28-35, 49-60 and 97-103 of SEQ ID NO:1, respectively.

[0014] In this invention, the single-domain antibody or its antigen-binding fragment, in addition to the complementarity-determining region, also includes four frame regions named FR1, FR2, FR3 and FR4 in sequence; The FR1 comprises an amino acid sequence selected from the following: The amino acid sequence of A1-1 is positions 1-27 of SEQ ID NO:1; A1-2) is a sequence with more than 75% identity to A1-1); The amino acid sequence includes the following: The amino acid sequence of A2-1)FR2 is positions 36-48 of SEQ ID NO:1; A2-2) is a sequence with more than 75% identity with A2-1); The FR3 comprises an amino acid sequence selected from the following: The amino acid sequence of A3-1 is positions 61-96 of SEQ ID NO:1; A3-2) is a sequence with more than 75% identity with A3-1); The FR4 comprises an amino acid sequence selected from the following: The amino acid sequence of A4-1 is positions 104-114 of SEQ ID NO:1; A4-2) is a sequence that has more than 75% similarity to A4-1).

[0015] In this invention, the amino acid sequence of FR1 may be at least one of the above-described A1-1 or A1-2.

[0016] In this invention, the amino acid sequence of FR2 may be at least one of the above-described A2-1) or A2-2).

[0017] In this invention, the amino acid sequence of FR3 may be at least one of the above-described A3-1) or A3-2).

[0018] In this invention, the amino acid sequence of FR4 may be at least one of the above-described A4-1 or A4-2.

[0019] In one specific embodiment of the present invention, the amino acid sequences of FR1, FR2, FR3 and FR4 of the single-domain antibody or its antigen-binding fragment are positions 1-27, 36-48, 61-96 and 104-114 of SEQ ID NO:1, respectively.

[0020] In this invention, the single-domain antibody or its antigen-binding fragment consists of three CDRs and a "frame region" separated by the three CDRs. The frame region is used to align the CDRs that specifically bind to the antigen epitope. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. The single-domain antibody includes the following frame regions (FRs) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0021] In this invention, the amino acid sequence of the single-domain antibody or its antigen-binding fragment is SEQ ID NO:1 or SEQ ID NO:3 or has at least 75% similarity to SEQ ID NO:1 or SEQ ID NO:3.

[0022] In this document, "75% or more of consistency" can specifically mean at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0023] In some specific embodiments of the present invention, the amino acid sequence of the single-domain antibody or its antigen-binding fragment is SEQ ID NO:1.

[0024] To express or purify the single-domain antibody or its antigen-binding fragment provided by the present invention, tags can be added to one or both ends of the single-domain antibody or its antigen-binding fragment according to existing technology. The tags include, but are not limited to, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, HA tag protein, and Myc tag protein.

[0025] In some specific embodiments of the present invention, the C-terminus of the single-domain antibody or its antigen-binding fragment provided by the present invention is tagged with histidine.

[0026] In one specific embodiment of the present invention, the amino acid sequence of the single-domain antibody or its antigen-binding fragment provided by the present invention is SEQ ID NO:3.

[0027] In SEQ ID NO:3, positions 1-114 are the single-domain antibody PFBH01, which is the same as in SEQ ID NO:1, and positions 115-119 are histidine tags.

[0028] The present invention also provides a biomaterial, said biomaterial being any of the following: B1) A nucleic acid molecule that encodes the aforementioned single-domain antibody or its antigen-binding fragment; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

[0029] In the above-mentioned biological materials, the nucleic acid molecule mentioned in B1) can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0030] In this invention, B1) refers to nucleic acids that can be DNA molecules or RNA transcribed from said DNA molecules, as described in (F1) or (F2) below: (F1) The nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID NO:2; (F2) has more than 70% similarity to (F1) and encodes the DNA molecule of the aforementioned single-domain antibody or its antigen-binding fragment.

[0031] In this invention, the expression cassette (B2) refers to DNA capable of expressing the single-domain antibody or its antigen-binding fragment in a host cell. This DNA may include not only a promoter that initiates transcription of the gene encoding the single-domain antibody or its antigen-binding fragment, but also a terminator that terminates transcription of the gene encoding the single-domain antibody or its antigen-binding fragment.

[0032] In this invention, the vector may be a plasmid, a granule, a bacteriophage, or a viral vector.

[0033] In some embodiments of the present invention, the recombinant vector may be a recombinant vector obtained by introducing the nucleic acid molecule described in B1) into pET21b(+). Specifically, the structure of the recombinant vector may be a recombinant expression vector obtained by replacing a small fragment between the recognition sites of the restriction endonucleases NdeI and XhoI of the vector pET21b(+) with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO:2, while keeping other nucleotide sequences of the vector pET21b(+) unchanged.

[0034] In some specific embodiments of the present invention, the microorganism may be bacteria (such as Escherichia coli), yeast, algae or fungi.

[0035] In some specific embodiments of the present invention, the bacteria may be Escherichia coli.

[0036] In one specific embodiment of the present invention, the Escherichia coli is BL21(DE3).

[0037] In the above-mentioned biological materials, the cells described in B4) can be either prokaryotic cells or eukaryotic cells.

[0038] In the above-mentioned biological materials, the cells may be derived from animals, plants or microorganisms.

[0039] In some specific embodiments of the present invention, the cells derived from animals may be isolated animal cells.

[0040] In some specific embodiments of the present invention, the isolated animal cells include, but are not limited to: ExpiCHO-S™ cells, Chinese hamster ovary cells (CHO), 293F cells, 293E cells, 293-6E cells, etc.

[0041] In one specific embodiment of the present invention, the recombinant cells are recombinant Escherichia coli cells.

[0042] The present invention also provides an immunoconjugate comprising (I) and (II): (I) Antibody, said antibody comprising the aforementioned single-domain antibody or its antigen-binding fragment; (II) Coupling part: The coupling part is selected from radionuclides, drugs, toxins, nucleic acids or combinations thereof.

[0043] In one specific embodiment of the present invention, the antibody in the immunoconjugate is the aforementioned single-domain antibody or its antigen-binding fragment.

[0044] In this invention, the radionuclides include, but are not limited to, the following (i) and / or (ii): (i) Diagnostic isotopes, wherein the diagnostic isotopes are selected from the group consisting of Ga-68 (i.e., Ga-68). 68 Ga), F-18, I-123, Ga-67, Cu-64, Zr-89, C-11, Re-188, or combinations thereof; and / or, (ii) A therapeutic isotope selected from the group consisting of I-131, I-124, I-125, Lu-177, Tc-99m, In-111, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198. Ho-166, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, Lu-177, or combinations thereof.

[0045] In some embodiments of the present invention, the radionuclide is Ga-68.

[0046] In this invention, "Ga-68" and " 68 "Ga" has the same meaning.

[0047] In this invention, the radionuclide can be a detectable marker, or it can be replaced by fluorescein, enzyme, or colloidal gold.

[0048] The present invention also provides a probe molecule containing the above-described single-domain antibody or its antigen-binding fragment.

[0049] In this invention, the probe molecule may be a radioactive probe.

[0050] In this invention, the radioactive probe may include the single-domain antibody or its antigen-binding fragment and a radionuclide coupled to the single-domain antibody or its antigen-binding fragment.

[0051] In some embodiments of the present invention, the probe is 68 Ga-NOTA-PFBH01 single-domain antibody-labeled probe.

[0052] The present invention also provides a medicament comprising the aforementioned single-domain antibody or its antigen-binding fragment, the aforementioned immunoconjugate, and / or the aforementioned probe molecule.

[0053] In this invention, the pharmaceutical composition may further include a pharmaceutically acceptable carrier.

[0054] In this invention, the pharmaceutical composition also contains other drugs for treating tumors.

[0055] The drug or drug composition may also contain an agent capable of killing tumor cells.

[0056] In this invention, the preparation capable of killing tumor cells is at least one of the following: chemical drugs, biological drugs, nanomedicines, radiopharmaceuticals, photothermal therapy or photodynamic therapy drugs capable of killing tumor cells; or, at least one of the following: alkylating agents, antimetabolites, antitumor natural drugs, antitumor antibiotics, hormones, metal complexes or tumor radiotargeting markers.

[0057] In this invention, the application, use, or method is intended for the diagnosis and treatment of diseases.

[0058] In this invention, the application, use or method may not be for the purpose of diagnosing and treating diseases, but its direct purpose is only to obtain intermediate results.

[0059] The present invention also provides the use of the above-described single-domain antibody or its antigen-binding fragment, the immunoconjugate, the probe molecule, or the polypeptide in at least one of the following (C1) to (C7). (C1) Prepare detection reagents, detection kits, detection plates or developing agents for recognizing or assisting in the recognition of B7H3 protein; (C2) Prepare detection reagents, detection kits, detection plates or contrast agents that bind to or assist in binding to B7H3 protein; (C3) Prepare detection reagents, kits, plates or contrast agents for detecting or assisting in the detection of B7H3 expression levels in cells; (C4) Prepare diagnostic reagents, test kits, test plates or contrast agents for the diagnosis or auxiliary diagnosis of B7H3 positive diseases; (C5) Prepare diagnostic reagents, test kits, test plates or contrast agents for the diagnosis or auxiliary diagnosis of B7H3 positive disease staging; (C6) Prepare detection reagents, test kits, test plates or contrast agents for monitoring or assisting in monitoring the treatment effect of B7H3 positive diseases; (C7) Prepare a pharmaceutical composition for the treatment and / or prevention of B7H3 positive diseases.

[0060] The present invention also provides reagents or kits comprising the aforementioned single-domain antibody or its antigen-binding fragment, the aforementioned immunoconjugate, and / or the aforementioned probe molecule.

[0061] In this invention, the reagent or kit has at least one of the following uses: (K1) recognizes or assists in the recognition of B7H3 protein; (K2) binds to or helps bind to B7H3 protein; (K3) Detection or auxiliary detection of whether the sample contains B7H3 protein; (K4) Detection or auxiliary detection of B7H3 protein content in the sample to be tested; (K5) identifies or assists in the identification of B7H3 protein-positive diseases; (K6) targets the B7H3 protein; (K7) Diagnosis of diseases related to B7H3 protein.

[0062] In this invention, the reagents or kits may further include a detection-acceptable carrier.

[0063] In this invention, the detection-acceptable carrier can be a non-toxic, inert aqueous carrier medium.

[0064] In this invention, the detection-acceptable carrier is one or more reagents selected from the group consisting of: isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0065] In some specific embodiments of the present invention, the detection reagent may be a contrast agent. Furthermore, the contrast agent may also include other formulations used for contrast imaging.

[0066] In some specific embodiments of the present invention, the contrast agent is a contrast agent used for MRI (magnetic resonance imaging) or CT (computed tomography).

[0067] In some specific embodiments of the present invention, the imaging agent simultaneously chelates two or more signals, such as Ga-68 and Gd, for use in both PET / CT and MRI; or Tc-99m and a fluorescent agent, for use in both SPECT / CT and fluorescence detection.

[0068] In some specific embodiments of the present invention, the detection reagent can be used for in vivo detection.

[0069] In some specific embodiments of the present invention, the dosage form of the detection reagent is liquid or powder (such as aqueous solution, injection, lyophilized powder, tablet, lozenge, inhaler).

[0070] The present invention also provides polypeptides comprising the above-described single-domain antibody or its antigen-binding fragment.

[0071] In this invention, the polypeptide may be the above-mentioned single-domain antibody or its antigen-binding fragment.

[0072] In some specific embodiments of the present invention, the polypeptide is a recombinant single-domain antibody with a 6×His tag. The amino acid sequence of the recombinant single-domain antibody is SEQ ID NO:3.

[0073] This invention also provides any of the following applications: M1) The use of the aforementioned single-domain antibody or its antigen-binding fragment in the preparation of the aforementioned immunoconjugate, or the aforementioned probe molecule or the aforementioned drug; M2) The use of the aforementioned single-domain antibody or its antigen-binding fragment, or the aforementioned biological material, or the aforementioned immunoconjugate, or the aforementioned probe molecule, or the aforementioned drug in the preparation of products for treating diseases with positive expression of B7H3 protein; M3) The use of the aforementioned single-domain antibody or its antigen-binding fragment, or the aforementioned biological material, or the aforementioned immunoconjugate, or the aforementioned probe molecule, or the aforementioned drug in the preparation of a product for detecting the content of B7H3 protein positive expression in tumors; M4) The aforementioned single-domain antibody or its antigen-binding fragment, or the aforementioned biological material, or the aforementioned immunoconjugate, or the aforementioned probe molecule, in the preparation of a product that recognizes or assists in the recognition of the B7H3 protein; M5) The aforementioned single-domain antibody or its antigen-binding fragment, or the aforementioned biological material, or the aforementioned immunoconjugate, or the aforementioned probe molecule, in the preparation of a product that binds to or assists in binding to the B7H3 protein; M6) The aforementioned single-domain antibody or its antigen-binding fragment, or the aforementioned biological material, or the aforementioned immunoconjugate, or the aforementioned probe molecule, are used in the preparation of products for detecting or assisting in the detection of cellular B7H3 protein; M7) The aforementioned single-domain antibody or its antigen-binding fragment, or the aforementioned biological material, or the aforementioned immunoconjugate, or the aforementioned probe molecule, are used in the preparation of products for the diagnosis or auxiliary diagnosis of B7H3 protein-positive diseases; M8) The aforementioned single-domain antibody or its antigen-binding fragment, or the aforementioned biological material, or the aforementioned immunoconjugate, or the aforementioned probe molecule, are used in the preparation of products for the diagnosis or auxiliary diagnosis of B7H3 protein-positive disease staging.

[0074] The products include, but are not limited to, reagents and kits.

[0075] The amino acid sequence of BH7-HM273 is as follows:

[0076] In this invention, the application, use or method may not be for the purpose of diagnosing and treating diseases, but its direct purpose is only to obtain intermediate results.

[0077] The advantage of this invention is that the single-domain antibody provided by this invention has the characteristic of targeting B7H3 positive tumor cells. Therefore, in practical applications, the single-domain antibody of this invention can be combined with anticancer drugs or imaging agents for targeted tumor therapy and imaging. Attached Figure Description

[0078] Figure 1 Electrophoresis images of the first-round PCR amplification of the common antibody heavy chain and the heavy chain antibody VHH gene. Markers are 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp. 1 and 2 are the products obtained from PCR using different primer combinations. 1 contains a common antibody heavy chain gene amplification fragment greater than 750bp and a heavy chain antibody gene amplification fragment less than 750bp. 2 contains only a heavy chain antibody VHH gene amplification fragment of approximately 500bp.

[0079] Figure 2 The PCR product is B7H3 phage. Markers (2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp), and the PCR amplified product is approximately 400bp.

[0080] Figure 3 SDS-PAGE electrophoresis image of the protein collection solution.

[0081] Figure 4 SDS-PAGE electrophoresis images of the washing solution and eluent purified by magnetic bead method.

[0082] Figure 5 This is a curve showing the binding activity of PFBH01.

[0083] Figure 6 To determine the affinity of PFBH01 for B7H3 protein in BLI.

[0084] Figure 7 The affinity of PFBH01 for cells expressing B7H3.

[0085] Figure 8 for 68 Radiochemical purity and in vitro stability of Ga-NOTA-PFBH01 in PBS and FBS.

[0086] Figure 9 for 68 Ga-NOTA-PFBH01 in H1975 CD276 Micro PET imaging in H929 tumor-bearing mice.

[0087] Figure 10 for 68Ga-NOTA-PFBH01 in H1975 CD276 The uptake of various organs and tumors in H929 tumor-bearing mice.

[0088] Figure 11 for 68 Ga-NOTA-PFBH01 in H1975 CD276 Biodistribution of various tissues and organs and tumors in tumor-bearing mice at 2 hours. Detailed Implementation

[0089] I. Terminology in this application: Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. Generally, terms relating to cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry, and hybridization techniques described herein are well-known in the art and commonly used in the field. The methods and techniques provided herein are generally performed in accordance with standard practice.

[0090] Unless otherwise stated, methods known in the art and as described in the various general and more specific references cited and discussed herein.

[0091] Any references cited in this article, including, for example, all patents, published patent applications and non-patent publications, are incorporated in their entirety by reference.

[0092] For ease of understanding of this disclosure, several terms and abbreviations used herein are defined as follows: In this application, "identity" refers to the similarity of amino acid or nucleotide sequences. The similarity of amino acid sequences (or nucleotide sequences) can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the similarity of a pair of amino acid sequences, the similarity value (%) can be obtained.

[0093] When used in a list of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0094] The term "single-domain antibody" refers to a variable domain antibody (VHH), which is a protein composed of the variable domain of the antibody heavy chain.

[0095] The terms "single-domain antibody," "nanobody," "VHH," or "sdAb" all have the same meaning: a single-antigen-binding polypeptide with three complementarity-determining regions (CDRs). A basic single-domain antibody has the following structure from the N-terminus to the C-terminus: FR1 CDR1FR2 CDR2 FR3 CDR3 FR4, where FR1 to FR4 refer to framework regions 1 to 4, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3.

[0096] The complementarity-determining region (CDR) is the antigen-binding site in an antibody. CDRs can be defined using various terms: (i) a complementarity-determining region (CDR) based on sequence variability; and (ii) a "hypervariant region," "HVR," or "HV" refers to a region of the antibody's variable domain that is structurally highly variable, as defined by Chothia and Lesk. The international ImMunoGeneTics (IMGT) database (http: / / www_imgt_org) provides standardized numbering and definitions of antigen-binding sites. As used herein, the terms "CDR," "CDR1," "CDR2," and "CDR3" include CDRs defined by any of the methods described above, Kabat, Chothia, or IMGT, unless otherwise explicitly stated in the specification. Frame regions (FWs) are adjacent to and located between CDRs.

[0097] As used herein, the term "fragment" refers to a polypeptide that substantially retains the same biological function or activity as the antibody of this application. The polypeptide fragment of this application may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a polypeptide having substituent groups in one or more amino acid residues; or (iii) a polypeptide formed by fusing a mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol); or (iv) a polypeptide formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6×His tag).

[0098] As used herein, the terms “specific binding,” “specific recognition,” or “specific to” refer to measurable and reproducible interactions, such as the binding between a target and a single-domain antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules.

[0099] The term "specificity" refers to the selective recognition of a specific epitope of an antigen by a single-domain antibody.

[0100] The specific binding refers to the affinity of one molecule for binding to another molecule that is significantly higher than its affinity for binding to any cross-reactive antigen or off-target antigen (collectively referred to as non-target antigen). This affinity is determined using experimental techniques such as surface plasmon resonance (SPR), fluorescence activated cell sorting (FACS) analysis, kinetic exclusion assay (KinExA), isothermal titration calorimetry (ITC), radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA).

[0101] The term "antigen-binding fragment" refers to an antigen-binding fragment comprising the single-domain antibody described in this application, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antigen-binding fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parent antibody's binding activity. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target.

[0102] In this application, "antigen-binding fragment" encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies, heteroconjugated antibodies, multispecific antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies), recombinant single-domain antibodies, bivalent antibodies, multivalent antibodies, Fab, Fab' fragments, F(ab')2 fragments, minimum recognition unit (MRU), Fv antibodies, single-chain antibodies, or heavy-chain antibodies.

[0103] Unless otherwise stated, “antigen-binding fragment” also includes full-length or complete antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA and IgD.

[0104] The terms "bivalent single-domain antibody" or "multivalent single-domain antibody" refer to a fusion polypeptide formed by linking two or more said single-domain antibodies. The single-domain antibodies in a "bivalent single-domain antibody" or "multivalent single-domain antibody" are formed by covalent or non-covalent linking of linker molecules, or by non-covalent linking of the antibody with a polymer.

[0105] The term "multispecific single-domain antibody" refers to an antibody with multispecificity formed by linking the single-domain antibody with other antigen-specific antigen-binding fragments.

[0106] The term "multispecific" means that an antigen-binding protein (the antigen-binding fragment of the single-domain antibody) has two or more different antigen-binding specificities.

[0107] The term "Fab" stands for antigen-binding fragment, which consists of a complete antibody light chain and the VH (variable region of the heavy chain) and CH1 (constant region 1) domains of the heavy chain. In other words, it is a heterodimer formed by the antibody's heavy chain Fd and the complete light chain linked by disulfide bonds.

[0108] The term "Fab' fragment" contains a portion of a complete antibody light chain and an antibody heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains. This allows interchain disulfide bonds to form between the two heavy chains of two Fab' fragments to form the F(ab')2 molecule.

[0109] The term "F(ab')2 segment" consists of two Fab' segments held together by disulfide bonds between two heavy chains.

[0110] The term "minimum recognition unit (MRU)" refers to a single CDR structure containing only the variable region, with a molecular weight of only about 1% of that of a complete antibody, which can bind to the corresponding antigen.

[0111] The term "Fv antibody" refers to a protein composed solely of the heavy chain variable region and the light chain variable region of the antibody. The heavy chain variable region and the light chain variable region are linked by non-covalent bonds.

[0112] The term "single-chain antibody" (ScFv) refers to a protein composed of short peptides that link the heavy chain variable region and the light chain variable region of an antibody.

[0113] The term "biomaterial" refers to any material that carries genetic information and is capable of self-replication or replication within a biological system, such as genes, plasmids, microorganisms, animals, and plants.

[0114] The term “protein” or “peptide” as used herein encompasses all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins, and modified proteins, including but not limited to glycoproteins and all other types of modified proteins (e.g., proteins produced by phosphorylation, acetylation, myristylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, PEGylation, biotinylation, etc.).

[0115] Unless otherwise stated, the terms “nucleic acid,” “nucleotide,” and “polynucleotide” cover both DNA and RNA.

[0116] The nucleic acid sequence encoding the single-domain antibody disclosed herein can be introduced into cells via “transfection,” “transformation,” or “transduction.” As used herein, “transfection,” “transformation,” or “transduction” refers to the introduction of one or more exogenous polynucleotides into a host cell using physical or chemical methods.

[0117] The term "transformation" refers to the introduction of one or more exogenous polynucleotides into bacterial cells that are already capable of transformation, for example, by using dimethyl sulfoxide, divalent cations (such as calcium), or polyethylene glycol. Many transformation techniques are known in the art and include heat shock and electroshock.

[0118] The term "expression" refers to the permission or inducing of the production of information from a gene or DNA sequence. For example, expression can take the form of producing a protein by activating cellular functions involved in the transcription and translation of the corresponding gene or DNA sequence. A DNA sequence is expressed in or through cells to form an "expression product" such as a protein. The expression product itself, such as the resulting protein, can also be referred to as being "expressed" by the cell. Expression products can be characterized as intracellular, extracellular, or transmembrane.

[0119] Single-domain antibodies can be prepared using recombinant methods. This typically involves cloning the single-domain antibody gene into a vector, transforming it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this application include biomolecules existing in isolated forms.

[0120] Currently, the DNA sequence encoding the protein (or a fragment thereof, or a derivative thereof) of this application can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this application through chemical synthesis.

[0121] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0122] The single-domain antibodies or peptides described in this application may be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0123] The phrase “pharmaceutically acceptable” used in conjunction with the compositions described herein refers to the molecular entities and other components of such compositions that are physiologically tolerable and typically do not produce adverse effects when administered to mammals (e.g., humans). Preferably, the term “pharmaceutically acceptable” means listed in recognized pharmacopoeias for use in mammals, and more particularly for use in humans.

[0124] For example, the term "pharmaceutically acceptable carrier" includes, but is not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.).

[0125] The terms “tumor” and “cancer” are used interchangeably in this article, including solid tumors and liquid tumors.

[0126] The terms “cancer” and “cancerous” refer to physiological diseases in mammals where cell growth is uncontrolled.

[0127] The term "tumor" refers to the growth and proliferation of all proliferative cells (whether malignant or benign) as well as all precancerous and cancerous cells and tissues. When used in this article, the terms "cancer," "cancerous," and "tumor" are not mutually exclusive.

[0128] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (e.g., a single-domain antibody) and facilitates the detection of the reagent conjugated or fused thereto. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzyme labeling, the label may catalyze a chemical change in the substrate compound or composition to make it detectable. This term is intended to cover both direct labeling of probes or antibodies by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of probes or antibodies by reacting with another directly labeled reagent. Examples of indirect labeling include using a fluorescently labeled secondary antibody to detect a primary antibody, and using biotin to end-label a DNA probe so that it can be detected using a fluorescently labeled streptavidin.

[0129] In this application, the term "B7H3 positive disease" can refer to a disease accompanied by a significant increase in B7H3 expression levels, where B7H3 can be used as a biomarker. It should be clarified that "B7H3 positive disease" also includes other diseases that may be discovered in the future that are associated with B7H3 positivity (significantly elevated expression levels).

[0130] II. Implementation Examples The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0131] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0132] The experimental results of the following examples are expressed as mean ± standard deviation. Student's t-test and one-way ANOVA were used. P < 0.05 (*) indicates that there is a significant difference.

[0133] Example 1: Construction of a single-domain antibody library targeting B7H3 antigens 1. Immunizing alpacas with B7H3 antigen Select healthy adult alpacas and inject human B7H3 antigen protein (KaiKa Bio, BH7-HM13B) subcutaneously at multiple points on the neck and back, with a total dose of 2 mg. For the first immunization, add an equal volume of Freund's complete adjuvant (manufacturer Sigma, catalog number F5881). For the second to fifth immunizations, add an equal volume of Freund's incomplete adjuvant (Sigma, F5506). Repeat the immunizations 14 days apart. After the third immunization, collect serum and determine the antigen titer using ELISA. When the titer reaches 10,000 times or more, collect 50 mL of whole blood, separate PBMC cells, add Trizol, mix thoroughly, and store at -80 °C for later use.

[0134] 2. RNA extraction Take 1 mL of isolated PBMC cells, add 0.2 mL of chloroform, and vortex to mix. Add 200 μL of chloroform, vortex vigorously for 15 s, and incubate at room temperature for 3 min; centrifuge at 12000 rpm at 4℃ for 15 min; carefully remove the centrifuge tube, aspirate the supernatant and transfer it to a new 1.5 mL EP tube, add an equal volume of isopropanol, invert thoroughly to mix, and incubate at room temperature for 10 min; centrifuge at 12000 rpm at 4℃ for 10 min; carefully discard the supernatant with a pipette tip, add 75% ethanol along the tube wall, and carefully blow away the precipitate (or gently tap the bottom of the tube); centrifuge at 7500 rpm at 4℃ for 5 min; carefully discard the supernatant, centrifuge again for 1 min, and aspirate the supernatant; open the cap and incubate at room temperature for 10-15 min until the ethanol has completely evaporated; add 30 μL of RNase-free water to dissolve the RNA. The extracted RNA is easily degraded and needs to be reverse transcribed into cDNA promptly.

[0135] 3. Obtain the VHH gene fragment (single-domain antibody gene) of the variable region of the B7H3-targeting heavy chain antibody. Following the instructions of the cDNA synthesis kit (PrimeScript™ II 1st Strand cDNA Synthesis Kit, catalog number 6210A, TAKARA), the extracted RNA was reverse transcribed into cDNA. Prepare the following reaction mixture in a centrifuge tube: 1 μL Random 6 mers (50 μM), 1 μL dNTP Mixture (10 mM each), template RNA: less than 5 μg, and RNase-free ddH2O to a final volume of 10 μL. Incubate the mixture at 65°C for 5 min, then rapidly cool on ice. Prepare the following reverse transcription reaction mixture in the same centrifuge tube, with a total volume of 20 μL. Use 10 μL of the denaturing reaction mixture, 4 μL of 5×PrimeScript II Buffer, 0.5 μL of RNase Inhibitor (40 U / μL), 1 μL of PrimeScript II RTase (200 U / μL), and RNase-free dH2O to a final volume of 20 μL. The reverse transcription reaction was carried out under the following conditions: 30℃ for 10 min, 42℃ for 30-60 min, 95℃ for 5 min, followed by cooling on ice.

[0136] Using cDNA as a template, two sets of primers were used to amplify the heavy chain VHH gene fragment by PCR. In the first PCR amplification, the fragment larger than 750 bp was the ordinary heavy chain gene fragment, and the fragment between 750 and 500 bp was the single-domain antibody gene fragment. The heavy chain antibody VHH gene fragment (single-domain antibody gene) was recovered by gel excision and used as a template to amplify the VHH target gene (~500 bp) by PCR using VHH-specific primers (YTV8 and YTV9).

[0137] Table 1. PCR primer sequences

[0138] Note: In primer YT1BN, nucleotide symbols and definitions follow the ST.26 standard. M represents A or C, and K represents G or T.

[0139] The first round of PCR amplification was divided into two groups: for the common antibody heavy chain gene, the upstream primer was YT-1 and the downstream primer was YT1BN; for the VHH gene, the upstream primer was YT-2 and the downstream primer was YT1BN. The PCR mixture consisted of 5 μL of 10×PCR buffer, 5 μL of dNTPs, 1.5 μL of forward primer YT-1, 1.5 μL of reverse primer YT-2, 3.5 μL of template, 0.5 μL of Blend Taq enzyme, and 33 μL of ddH2O. The PCR conditions were: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 1 min; 25 cycles were performed at 4℃ for 10 min.

[0140] Second round of PCR amplification: The PCR mixture consisted of 5 μL of 10×PCR buffer, 5 μL of dNTPs, 1.5 μL of forward primer YTV8, 1.5 μL of reverse primer YTV9, 3.5 μL of template (the product recovered in the first round), 0.5 μL of Blend Taq enzyme, and 33 μL of ddH2O. The PCR conditions were: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 1 min; 15 cycles were performed at 4℃ for 10 min.

[0141] Figure 1 Electrophoresis images of the first round of PCR amplification of the common antibody heavy chain and the VHH gene of the heavy chain antibody. Markers are 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp. Lanes 1 and 2 show gel electrophoresis of products obtained from PCR using different primer combinations. Lane 1 contains amplified fragments of the common antibody heavy chain gene greater than 750bp and heavy chain antibody gene amplification fragments less than 750bp. Lane 2 contains only an amplified fragment of the heavy chain antibody VHH gene of approximately 500bp.

[0142] 4. Connection Transformation PCR products and pHEN6 vector were digested with restriction endonuclease SfiI overnight at 37°C. The recovered fragments were purified using a universal recovery kit. The recovered fragments and vector were mixed at a 3:1 ratio, and T4 DNA ligase was added. After brief centrifugation, the mixture was incubated overnight at 16°C in a metal bath. The ligation product was purified using a universal recovery kit. The ligation product was electroporated into TG1 competent cells, plated, and the antibody insertion rate was verified by colony PCR. The electroporated bacterial culture was plated on LB / L / Amp plates and incubated at 37°C for 14 hours. The cells were counted and the library size was calculated the next day. Multiple electroporations were performed, and the library size was 2.61 × 10⁻⁶. 9Colonies were scraped from 2 mL agar plates of 2YT liquid medium (1L preparation: 16g tryptone, 10g yeast extract, 5g sodium chloride). All colonies were thoroughly mixed, and glycerol was added to preserve the culture as 20% glycerol culture, thus obtaining the seed bacterial bank. 1 mL agar was stored at -80℃.

[0143] 5. Preparation of B7H3-targeting VHH phage library (single-domain antibody library) Add 1 mL of the seed bacterial bank to 200 mL of 2YT medium (containing a final concentration of 50 μg / mL Amp + 1% glucose), and incubate at 37ºC until OD. 600nm After adjusting the culture medium to 0.8-1, add M13KO7 helper phage at a ratio of 1:20 for infection. After culturing for 1 hour, replace with fresh 2YT medium (containing a final concentration of 100 μg / mL Amp + 50 μg / mL Kana) and incubate overnight at 37ºC with shaking. Collect the bacterial culture into sterile centrifuge tubes, centrifuge to collect the supernatant, add 1 / 5 volume of 20% PEG-2.5M NaCl, mix well, incubate on ice for 1 hour, centrifuge at 12000 rpm for 30 min, discard the supernatant, dissolve the precipitate in 3 mL of sterile 15% glycerol-PBS solution, and store the phage library at -80℃.

[0144] Example 2: Screening, expression, and purification of B7H3-targeting single-domain antibodies 1. Screening for B7H3-specific single-domain antibodies Human B7H3 antigen protein (KaiKa Biotechnology, BH7-HM13B) was coated onto immunoassay tubes using coating buffer for the first round of screening. The coating buffer was discarded, and the tubes were completely blocked with blocking buffer. After 1 h at room temperature, phage solution was added, and the tubes were washed 20 times with PBST, followed by 15 washes with PBS. Elution was performed with 500 μL of elution buffer, followed by neutralization with neutralization buffer. 2 μL of the eluted phage was used for titer determination, and the remaining phage solution was used for scale-up culture.

[0145] Determining the titer facilitates the next round of screening. The screening results are detailed in the table below: Table 2. Screening of B7H3-targeting specific single-domain antibodies

[0146] Using B7H3 protein as the target, a three-round screening method was used to screen the total phage antibody library. The titer of the phage eluted in each round was detected. As shown in the table above, as the number of screening rounds increased, the coating concentration decreased step by step, but the titer of the eluted phage increased, indicating that B7H3-specific phages were enriched.

[0147] 2. Screening of B7H3-targeting single-domain antibody phage monoclonals Human B7H3 antigen protein (KaiKa Biotechnology, BH7-HM13B) was coated for ELISA assay. The coating concentration of B7H3 antigen protein was 1 μg / mL, 100 μL / well, and incubated overnight at 4 ℃. After 2-3 rounds of solid-phase phage selection, single clones were randomly selected and cultured in deep-well plates. Helper phages were added for infection, followed by overnight culture. The supernatant from the overnight culture was used for ELISA detection, with Anti-M13-HRP as the secondary antibody, and incubated at 37 ℃ for 1 h. After washing, TMB was added for color development, and the reaction was terminated by adding 2 M H2SO4. The OD value of the sample was measured at 450 nm using a microplate reader. B7H3-targeting positive wells were selected, and DNA sequencing was performed to identify the gene sequences of B7H3-targeting single-domain antibody clones, obtaining a series of single-domain antibody gene sequences. Through analysis and genotyping of the sequence-encoded amino acids, single-domain antibodies of various genotypes were obtained for further expression and screening of highly specific and highly active single-domain antibodies.

[0148] 3. Construction of B7H3-targeting single-domain antibody expression plasmid Using 5-nano-NdeI (5'-CATATGCAGGTTCAGCTGGTTGA-3') and 3-nano-XhoI (5'-CTCGAGTGAGGAGACGGTGACCTGGGT-3') as primers, and phage-selected positive monoclonal products as templates, a specific B7H3-targeting single-domain antibody gene was obtained by PCR amplification. The PCR product and the pET-21b(+) vector (UBO Biotechnology, VT1198) were treated with restriction endonucleases NdeI and XhoI, respectively. The fragments were ligated with T4 DNA ligase and transformed into BL21(DE3) competent cells to obtain a plasmid that can efficiently express single-domain antibodies in E. coli, named pET-21b(+)-B7H3.

[0149] Figure 2 The PCR product is B7H3 phage. Markers (2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp), and the PCR amplified product is approximately 400bp.

[0150] 4. Expression and purification of B7H3-targeting single-domain antibody The bacterial strain containing the target gene (i.e., *E. coli* containing the aforementioned single-domain antibody expression plasmid pET-21b(+)-B7H3) was streaked into a culture plate containing ampicillin sodium and incubated overnight at 37°C. Single clones were selected and inoculated into 3 mL of 2YT medium containing ampicillin sodium and incubated overnight. The clones were then transferred to 100 mL of 2YT medium containing ampicillin sodium and cultured on a shaker at 37°C and 220 rpm / min until the OD (Organic Oxygen Demand) was reached. 600nmWhen the pH reaches 0.6–1.0, add 0.5–1 M IPTG and incubate overnight at 20°C with shaking at 200 rpm / min. Centrifuge at 6500 rpm / min for 20 min to collect the bacteria. Wash the cells twice with 0.05 M Tris buffer, add 20 mL of 0.05 M Tris buffer, place in an ultrasonic homogenizer, sonicate on ice for 15 min, and centrifuge to collect the supernatant. Separate the single-domain antibody protein with a purity of over 90% using Ni+ ion affinity chromatography magnetic beads (BeaverBeads™ His-tag Protein Purification, Suzhou Beaver Biomedical Engineering Co., Ltd.), and collect the protein for dialysis replacement buffer. Results are shown in SDS-PAGE electrophoresis. Figure 3 , Figure 4 (Approximately 15 kDa), the resulting single-domain antibody was named single-domain antibody PFBH01 (or simply single-domain antibody PFBH01).

[0151] The amino acid sequence of the PFBH01 single-domain antibody is shown in SEQ ID NO:1, comprising a frame region (named FR, including FR1, FR2, FR3, and FR4) and a complementarity-determining region (named CDR, including CDR1, CDR2, and CDR3). Amino acids 1-27 of SEQ ID NO:1 are FR1, amino acids 36-48 are FR1, amino acids 61-96 are FR1, amino acids 104-114 are FR1, amino acids 28-35 are CDR1, amino acids 49-60 are CDR2, and amino acids 97-103 are CDR3. The coding gene for the above single-domain antibody PFBH01 has the nucleotide sequence shown in SEQ ID NO:2, and the amino acid sequence of the corresponding His-tagged recombinant single-domain antibody is shown in SEQ ID NO:3.

[0152] The aforementioned plasmid pET-21b(+)-B7H3, which expresses a single-domain antibody, is a recombinant expression plasmid obtained by replacing a small fragment between the restriction endonucleases NdeI and XhoI of the pET-21b(+) vector with a DNA fragment whose nucleotide sequence is SEQ ID NO:2, while keeping the other nucleotide sequences of pET-21b(+) unchanged. The protein expressed by this plasmid has the amino acid sequence of SEQ ID NO:3 (where positions 1-114 are the single-domain antibody PFBH01, and positions 115-119 are histidine tags).

[0153] BL21(DE3), containing the plasmid pET-21b(+)-B7H3, is a recombinant *E. coli* strain capable of expressing a single-domain antibody. This recombinant *E. coli* strain can express a histidine-tagged single-domain antibody with the amino acid sequence SEQ ID NO:3.

[0154] SEQ ID NO:1 QVQLVDSGGGLVQPGGSLRLSCAISGTRFSTKSMGWYRQAPGKQREFVAVIFGDASTNYKSSVAGRFTISKNNANNNTLYLQMNNLKPEDTAVYYCNLVSPLDYWGQGTQVTVSS.

[0155] SEQ ID NO:2 5'-CAGGTGCAGCTGGTGGATAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGATTAGCGGCACCCCGCTTTAGCACCAAAAGCATGGGCTGGTATCGCCAGGCGCCGGGCAAACAGCGCGAATTTGTGGCGGTGATTTTTGGCGATGCGAGCACC AACTATAAAAGCAGCGTGGCGGGCCGCTTTACCATTAGCAAAAACAACGCGAACAACACCCTGTATCTGCAGATGAACAACCTGAAACCGGAAGATACCGCGGTGTATTATTGCAACCTGGTGAGCCCGCTGGATTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC-3'.

[0156] The sequence of the complementarity determination region (CDR) is defined according to the Kabat numbering system.

[0157] SEQ ID NO:3 QVQLVDSGGGLVQPGGSLRLSCAISGTRFSTKSMGWYRQAPGKQREFVAVIFGDASTNYKSSVAGRFTISKNNANNNTLYLQMNNLKPEDTAVYYCNLVSPLDYWGQGTQVTVSSHHHHHH.

[0158] Example 3: Detection of the affinity between single-domain antibody PFBH01 and human B7H3 protein by enzyme-linked immunosorbent assay (ELISA). Dilute human B7H3 antigen protein (Kaikai Biotechnology, BH7-HM13B) to 1 µg / mL with 0.05 M NaHCO3 (pH 9.6) coating buffer, 100 µL / well, and coat overnight at 4°C. Block 96-well plates with 2% BSA at 37°C for 1 hour. Add single-domain antibody PFBH01 at different dilutions (concentrations shown in Table 3), and incubate at 37°C for 1 hour. Wash the plate three times with 0.05% PBST. Add 1:5000 diluted Anti-VHH-antibody-HRP secondary antibody at 100 µL / well, and incubate at 37°C for 1 hour. Wash the plate three times with 0.05% PBST. Add 100 μL of TMB, and incubate at room temperature in the dark for 10 minutes for color development. Stop the reaction by adding 50 μL of 2 M H2SO4. Measure the OD value of the samples at 450 nm using a microplate reader. The results are shown in Table 3 and [Table data missing]. Figure 5 It is evident that the obtained single-domain antibody PFBH01 has excellent specificity and biological activity in detecting human B7H3 antigen protein.

[0159] Table 3. Specificity and bioactivity assays of single-domain antibody PFBH01 and B7H3 protein

[0160] Example 4: Identification of the affinity between single-domain antibody PFBH01 and B7H3 protein using Bio-Layer Interferometry (BLI). The affinity of the single-domain antibody PFBH01 for human B7H3 antigen protein (Kaikai Biotechnology, BH7-HM273) was determined using an Octet RED96 (Sartorius) biomembrane interferometer. The HIS1K biosensor was immersed in PBS solution for 10 min to capture the single-domain antibody. The sensor was then immersed in wells containing 100, 50, 25, 12.5, 6.25, 3.125, and 1.563 nM human B7H3 antigen protein for 2 min, followed by dissociation in PBS solution. Data were then analyzed using Octet Analysis Studio 13.0 software. Results are shown below. Figure 6 The single-domain antibody PFBH01 has an affinity of 8.197 nM (K) for the B7H3 antigen. D ).

[0161] Example 5: Flow cytometry analysis of the affinity between B7H3-targeting single-domain antibody and B7H3-expressing cells. Human non-small cell lung cancer H1975 CD276The cells are B7H3 positive cells, and the cell line is disclosed in the non-patent literature "Lei Xia, Yan Wu, Yanan Ren, Zhen Wang, Nina Zhou, Wenyuan Zhou, Lixin Zhou, Ling Jia, Chengxue He, Xiangxi Meng, Hua Zhu, Zhi Yang. A whole-body imaging technique for tumor-specific diagnostics and screening of B7H3-targetedtherapies. J Clin Invest. 2025 Jan 23;135(6):e186388. doi: 10.1172 / JCI186388.", which is available to the public from the applicant. The biological material is only for repeating the relevant experiments of this invention and cannot be used for other purposes.

[0162] H1975 was cultured in RPMI 1640 medium (manufacturer: GIBCO, catalog number: C11875500BT) containing 10% FBS and 1% penicillin and streptomycin. CD276 Cells; negative cells were the human multiple myeloma cell line H929 (ATCC, CRL-3580), cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin. 50 µg / mL of single-domain antibody PFBH01 was added to H1975. CD276 Cells and H929 cells were incubated on ice for 1 h, washed three times with PBS, then incubated on ice for 1 h with secondary antibody (A0027, GenScript), washed three times with PBS, and resuspended in 500 µL PBS. Fluorescence intensity was measured using a FACS Calibur analyzer (BD Biosciences) and FlowJo software (Tree Star).

[0163] The results are as follows Figure 7 As shown, single-domain antibodies PFBH01 and H1975 CD276 The cells showed significant displacement, unlike H929 cells which showed almost no displacement, indicating that the single-domain antibody PFBH01 was compatible with H1975 cells. CD276 The cells showed good binding affinity, but no binding affinity to H929 cells, indicating that the single-domain antibody PFBH01 is specific and only recognizes B7H3 positive cells.

[0164] Example 6: PET imaging and biodistribution analysis of the single-domain antibody PFBH01 probe in a tumor-bearing mouse model. All animal experiments were conducted in accordance with the protocols approved by the Animal Management and Use Committee of Peking University First Hospital. BALB / c nude mice (4-5 weeks old, female) lacking T cells were selected for the construction of a B7H3-positive tumor model. BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (RRID:IMSR_CRL:194). NCG-severe immunodeficient mice (4-5 weeks old, female) lacking T cells, B cells, and NK cells were selected for the construction of a B7H3-negative tumor model. NCG immunodeficient mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. (strain number: T001475).

[0165] Positive tumor-bearing mouse group: BALB / c nude mice were subcutaneously injected with a suspension in the right axilla. The suspension consisted of 1×10⁻⁶ mmol / L. 7 H1975 in Example 5 CD276 Cells were obtained by mixing with 100 μL Matrigel (Corning, USA). The health status and tumor volume of mice were monitored the following day. When the tumor volume reached 1 cm... 3 It can be used for in vivo imaging experiments, with 4 mice in each group.

[0166] Negative tumor-bearing mouse group: H929 cells were used instead of H1975 cells. CD276 Cells were seeded subcutaneously in the right axilla of NCG mice, and the remaining procedures were the same as those in the positive tumor-bearing mouse group.

[0167] Rinse with 0.1 M high-purity hydrochloric acid 68 Ge- 68 Ga germanium-gallium generator (ITM Isotope Technologies, Germany) 68 GaCl3 solution, take 2 mL 68Add 120 μL of sodium acetate (1 M) to the GaCl3 solution, cap, mix well, and adjust the pH of the solution to 4-4.5 (measured as 4-4.5 using 0-6 precision pH paper). Add the single-domain antibody PFBH01 prepared in Example 2 (already conjugated with a Nota chelating agent, purchased from Macrocyclics, catalog number B-605) to the adjusted solution to obtain a mixed solution containing 200 μg of single-domain antibody PFBH01. Heat the mixed solution at 37°C for 15 min, then elute. After cooling the reaction solution, add it to an activated PD-10 column (activated with 15 mL of PBS buffer, purchased from Cytiva, catalog number 17085101), and add 380 μL of PBS to bring the total volume to 2.5 mL. Prepare five 1.5 mL EP tubes, labeled 1, 2, 3, 4, and 5. Take 500 μL of liquid from each EP tube from the PD-10 column, measure the activity, and set aside for later use. 68 Ga-NOTA-PFBH01 single-domain antibody-labeled probe.

[0168] 68 The radiochemical purity and in vitro stability of the Ga-NOTA-PFBH01 single-domain antibody-labeled probe in PBS and FBS were determined by thin-layer chromatography (TLC). The specific measurement procedure is as follows: First, lightly draw a horizontal line 1 cm from the end of the chromatography paper with a pencil. Then, use a capillary tube to gently spot the sample solution (probe incubated in PBS or FBS solution) on the horizontal line. Next, develop the spotted chromatography paper in the developing solvent (EDTA: sodium citrate - 90:10). After drying, place the chromatography paper on a MINI-SCAN (Eckert & Ziegler) thin-layer chromatograph, aligning the dial of the spotted point with the 0 mark of the thin-layer chromatograph. The MiniGita TCL software was used to detect the radioactivity distribution on the chromatography paper. The scan time was 1 minute, and the energy range was 450-750 keV. GINA software was used to analyze the Rf value and peak area of ​​the spectrum. The software automatically calculated the radiochemical purity of the probe. The results showed that the probe had a radiochemical purity >92% and a stability >95% in both systems. Figure 8 As shown.

[0169] The tumor volume of the tumor-bearing mouse is about 1 cm. 3 At that time, 6.7 MBq was injected via the tail vein. 68 Ga-NOTA-PFBH01.

[0170] Static PET scans were performed for 10 minutes each 1 h and 2 h post-injection using a micro-PET / CT scanner (Super Nova®, China). Regions of interest (ROIs) were plotted using Avatar 1.0 software and quantitative analysis was performed to obtain the radioactivity concentration in tumors, cardiac blood pools (blood), liver, spleen, kidneys, and muscles at different time points in vivo. The content of the radioactive probe was expressed as the percentage of radioactivity count per gram of tissue relative to the total injected radioactivity count (%ID / g), representing the radioactive uptake.

[0171] The results are as follows Figure 9 As shown, the maximum intensity projection (MIP) results indicate that H1975 cells with high B7H3 expression exhibit... CD276 In tumor models, 68 Significant radioactive accumulation was observed in tumors 1 h and 2 h after Ga-NOTA-PFBH01 injection, while no significant radioactive accumulation was observed in the H929 tumor model that was negative for B7H3 expression. Regions of interest (ROIs) were plotted using the corresponding software, and quantitative analysis was performed to obtain the radioactive accumulation of tumors at different time points in vivo.

[0172] The results are as follows Figure 10 As shown, 68 Ga-NOTA-PFBH01 probe in H1975 CD276 Tumor uptake (1.50 ± 0.31 %ID / g at 1h, 1.46 ± 0.29 %ID / g at 2h) was higher than that of H929 tumor (0.69 ± 0.01 %ID / g at 1h, P < 0.01; 0.60 ± 0.02 %ID / g at 2h, P < 0.01). Furthermore, at the 2h time point... 68 Ga-NOTA-PFBH01 in H1975 CD276 It continues to remain within the tumor. 68 Two hours after Ga-NOTA-PFBH01 injection, tumors, blood, heart, liver, spleen, lungs, kidneys, stomach, small intestine, bladder, muscles, long bones of the lower leg, brain, and tail were dissected. The samples were weighed, and the radioactivity of the samples was counted using an automatic gamma counter. The uptake of the radiotracer in different organs / tissues was calculated, and the biodistribution was calculated and expressed as %ID / g (mean ± SD).

[0173] Biological distribution results as follows Figure 11 As shown, 68Ga-NOTA-PFBH01 in H1975 CD276 Significant uptake and retention were observed in the tumor (1.71 ± 0.53 %ID / g), consistent with PET imaging results.

[0174] The above results demonstrate that the single-domain antibody probe of the present invention has rapid targeting of B7H3 and good tumor penetration ability, enabling high-sensitivity in vivo imaging of small tumors.

[0175] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A single-domain antibody or antigen-binding fragment thereof that binds to B7H3, characterized in that, The single-domain antibody or its antigen-binding fragment contains three complementarity-determining regions named CDR1, CDR2, and CDR3, with the amino acid sequences of CDR1, CDR2, and CDR3 being positions 28-35, 49-60, and 97-103 of SEQ ID NO:1, respectively.

2. The single-domain antibody or its antigen-binding fragment according to claim 1, characterized in that, The single-domain antibody or its antigen-binding fragment also includes four frame regions named FR1, FR2, FR3 and FR4; The FR1 comprises an amino acid sequence selected from the following: The amino acid sequence of A1-1 is positions 1-27 of SEQ ID NO:1; A1-2) is a sequence with more than 75% identity to A1-1); The amino acid sequence includes the following: The amino acid sequence of A2-1)FR2 is positions 36-48 of SEQ ID NO:1; A2-2) is a sequence with more than 75% identity with A2-1); The FR3 comprises an amino acid sequence selected from the following: The amino acid sequence of A3-1 is positions 61-96 of SEQ ID NO:1; A3-2) is a sequence with more than 75% identity with A3-1); The FR4 comprises an amino acid sequence selected from the following: The amino acid sequence of A4-1 is positions 104-114 of SEQ ID NO:1; A4-2) is a sequence that has more than 75% similarity to A4-1).

3. The single-domain antibody or its antigen-binding fragment according to claim 2, characterized in that, The amino acid sequence of the single-domain antibody is SEQ ID NO:1 or SEQ ID NO:

3.

4. A biomaterial, characterized in that, The biomaterial is any one of the following: B1) A nucleic acid molecule, wherein the nucleic acid molecule encodes a single-domain antibody or an antigen-binding fragment thereof as described in any one of claims 1-3; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) Recombinant cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

5. The biomaterial according to claim 4, characterized in that, The cells are derived from animals or microorganisms.

6. An immunoconjugate, characterized in that, The immunoconjugates include (I) and (II): (I) An antibody, said antibody comprising a single-domain antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 3; (II) Coupling part: The coupling part is selected from radionuclides, drugs, toxins, nucleic acids or combinations thereof.

7. A probe molecule, characterized in that, The probe molecule contains the single-domain antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

8. A drug, characterized in that, The drug contains a single-domain antibody or its antigen-binding fragment as described in any one of claims 1 to 3, an immunoconjugate as described in claim 6, or / and a probe molecule as described in claim 7.

9. A reagent or kit, characterized in that, It includes the single-domain antibody or its antigen-binding fragment as described in any one of claims 1 to 3, the immunoconjugate as described in claim 6, or / and the probe molecule as described in claim 7.

10. Application, characterized in that, The application is any one of the following: M1) The use of the single-domain antibody or its antigen-binding fragment according to any one of claims 1-3 in the preparation of the immunoconjugate according to claim 6, or the probe molecule according to claim 7, or the medicament according to claim 8; M2) The use of the single-domain antibody or antigen-binding fragment thereof as described in any one of claims 1-3, or the biomaterial as described in claim 4 or 5, or the immunoconjugate as described in claim 6, or the probe molecule as described in claim 7, or the medicament as described in claim 8 in the preparation of a product for treating diseases with positive expression of B7H3 protein; M3) The use of the single-domain antibody or antigen-binding fragment thereof as described in any one of claims 1-3, or the biomaterial as described in claim 4 or 5, or the immunoconjugate as described in claim 6, or the probe molecule as described in claim 7, or the drug as described in claim 8 in the preparation of a product for detecting the content of B7H3 protein positively expressed in tumors; M4) The single-domain antibody or its antigen-binding fragment as described in any one of claims 1-3, or the biomaterial as described in claim 4 or 5, or the immunoconjugate as described in claim 6, or the probe molecule as described in claim 7, in the preparation of a product that recognizes or assists in the recognition of the B7H3 protein; M5) The single-domain antibody or antigen-binding fragment thereof as described in any one of claims 1-3, or the biomaterial as described in claim 4 or 5, or the immunoconjugate as described in claim 6, or the probe molecule as described in claim 7, in the preparation of a product that binds to or assists in binding to the B7H3 protein; M6) The single-domain antibody or its antigen-binding fragment as described in any one of claims 1-3, or the biomaterial as described in claim 4 or 5, or the immunoconjugate as described in claim 6, or the probe molecule as described in claim 7, in the preparation of products for detecting or assisting in the detection of cellular B7H3 protein; M7) The single-domain antibody or its antigen-binding fragment as described in any one of claims 1-3, or the biomaterial as described in claim 4 or 5, or the immunoconjugate as described in claim 6, or the probe molecule as described in claim 7, in the preparation of products for the diagnosis or auxiliary diagnosis of B7H3 protein-positive diseases; M8) The single-domain antibody or its antigen-binding fragment as described in any one of claims 1-3, or the biomaterial as described in claim 4 or 5, or the immunoconjugate as described in claim 6, or the probe molecule as described in claim 7, in the preparation of products for the diagnosis or auxiliary diagnosis of B7H3 protein positive disease staging.

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