B7h3 binding polypeptides and uses thereof

Abstract

The present application relates to the field of biological medicine. Specifically, the present application relates to a specific B7H3 binding polypeptide and uses thereof.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to a specific B7H3-binding polypeptide and its uses. Background of the Invention

[0003] B7 homologue 3 (B7H3, also known as CD276) is a type I transmembrane protein encoded by human chromosome 15. B7H3 has a short intracellular tail and lacks a known signal transduction motif. B7H3 is universally expressed across species. A soluble form of B7H3 can also be detected in human serum, produced by surface cleavage by matrix metallopeptidase (MMP) or by alternative splicing of introns.

[0004] B7H3 is induced on antigen-presenting cells and plays an important role in the suppression of T cell function. While B7H3 protein is not expressed or is expressed at very low levels in normal tissues and cells, it is highly expressed in various tumor tissues and is closely related to tumor progression, patient survival, and prognosis. For example, many studies have described the overexpression of B7H3 in human malignancies, including melanoma, leukemia, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, and other cancers. Therefore, B7H3 can serve as a novel tumor marker and a potential therapeutic target. There is still a need in the field for diagnostic and therapeutic agents for B7H3-related diseases such as cancer.

[0005] Invention Summary

[0006] This invention includes at least the following embodiments:

[0007] Implementation Scheme 1. A B7H3-binding polypeptide comprising at least one immunoglobulin single variable domain specifically binding to B7H3, said at least one immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 of any one of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 and 85.

[0008] Implementation Scheme 2. The B7H3 binding polypeptide of Implementation Scheme 1, wherein the at least one immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 selected from:

[0009] (1) CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3, and CDR3 shown in SEQ ID NO:4;

[0010] (2) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:7, and CDR3 shown in SEQ ID NO:8;

[0011] (3) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12;

[0012] (4) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO:15, and CDR3 shown in SEQ ID NO:16;

[0013] (5) CDR1 shown in SEQ ID NO:18, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:20;

[0014] (6) CDR1 shown in SEQ ID NO:22, CDR2 shown in SEQ ID NO:23, and CDR3 shown in SEQ ID NO:24;

[0015] (7) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:28;

[0016] (8) CDR1 shown in SEQ ID NO:30, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:32;

[0017] (9) CDR1 shown in SEQ ID NO:34, CDR2 shown in SEQ ID NO:35, and CDR3 shown in SEQ ID NO:36;

[0018] (10) CDR1 shown in SEQ ID NO:38, CDR2 shown in SEQ ID NO:39, and CDR3 shown in SEQ ID NO:40;

[0019] (11) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:43, and CDR3 shown in SEQ ID NO:44;

[0020] (12) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:48;

[0021] (13) CDR1 shown in SEQ ID NO:50, CDR2 shown in SEQ ID NO:51, and CDR3 shown in SEQ ID NO:52;

[0022] (14) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:55, and CDR3 shown in SEQ ID NO:56;

[0023] (15) CDR1 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:59, and CDR3 shown in SEQ ID NO:60;

[0024] (16) CDR1 shown in SEQ ID NO:62, CDR2 shown in SEQ ID NO:63, and CDR3 shown in SEQ ID NO:64;

[0025] (17) CDR1 shown in SEQ ID NO:66, CDR2 shown in SEQ ID NO:67, and CDR3 shown in SEQ ID NO:68;

[0026] (18) CDR1 shown in SEQ ID NO:70, CDR2 shown in SEQ ID NO:71, and CDR3 shown in SEQ ID NO:72;

[0027] (19) CDR1 shown in SEQ ID NO:74, CDR2 shown in SEQ ID NO:75, and CDR3 shown in SEQ ID NO:76;

[0028] (20) CDR1 shown in SEQ ID NO:78, CDR2 shown in SEQ ID NO:79, and CDR3 shown in SEQ ID NO:80;

[0029] (21) CDR1 shown in SEQ ID NO:82, CDR2 shown in SEQ ID NO:83, and CDR3 shown in SEQ ID NO:84; and

[0030] (22) CDR1 shown in SEQ ID NO:86, CDR2 shown in SEQ ID NO:87, and CDR3 shown in SEQ ID NO:88.

[0031] Implementation Scheme 3. The B7H3-binding polypeptide of Implementation Scheme 1 or 2, wherein the immunoglobulin single variable domain comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with the amino acid sequence shown in any of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, and 85.

[0032] Implementation Scheme 4. The B7H3-binding polypeptide of any one of Implementation Schemes 1-3, wherein the immunoglobulin single variable domain comprises any one of the amino acid sequences shown in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 and 85.

[0033] Implementation Scheme 5. The B7H3-binding polypeptide of any one of Implementation Schemes 1-4, wherein the immunoglobulin single variable domain is VHH.

[0034] Implementation Scheme 6. The B7H3-binding polypeptide of any one of Implementation Schemes 1-5, wherein the immunoglobulin single variable domain is humanized.

[0035] Implementation Scheme 7. Nucleic acid molecule, which encodes the B7H3-binding polypeptide of any one of Implementation Schemes 1-6.

[0036] Implementation Scheme 8. An expression vector comprising the nucleic acid molecule of Implementation Scheme 7 operatively linked to an expression regulatory element.

[0037] Implementation Scheme 9. A host cell containing the nucleic acid molecule of Implementation Scheme 7 or transformed with the expression vector of Implementation Scheme 8, and capable of expressing the B7H3 binding polypeptide.

[0038] Implementation Scheme 10. A method for generating the B7H3-binding polypeptide of any one of Implementation Schemes 1-6, comprising:

[0039] a) Culture the host cells of embodiment 9 under conditions that allow the expression of the B7H3-binding peptide;

[0040] b) Recover the B7H3-binding polypeptide expressed by the host cells from the culture obtained in step a); and

[0041] c) Optionally, further purification and / or modification of the B7H3-binding polypeptide obtained from step b) is performed.

[0042] Implementation Scheme 11. A conjugated molecule comprising a B7H3-binding polypeptide of any one of Implementation Schemes 1-6, and at least one detectable label and / or therapeutic portion conjugated to said B7H3-binding polypeptide.

[0043] Implementation Scheme 12. The conjugated molecule of Implementation Scheme 11, wherein

[0044] The detectable label is selected from radionuclides, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, and enzymes; or

[0045] The therapeutic component is selected from radionuclides, paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, epipodophyllotoxin glucoside, epipodophyllotoxin thiophene glycoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, scintillans, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, aminopyrimidine, styromycin, cephalosporin, metansine, alistatin, nitrogen mustard, chlorambucil, phenylalanine mustard, carmosin. Sine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum(II) (DDP), cisplatin, bleomycin, atrazotocin, abscisic acid, ricin A, pseudomonadine, diphtheria toxin, tumor necrosis factor, interferon-γ, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), interleukin-10 (“IL-10”), granulocyte-macrophage colony-stimulating factor (“GM-CSF”), granulocyte colony-stimulating factor (“G-CSF”) or IFN.

[0046] Implementation Scheme 13. The conjugated molecule of Implementation Scheme 12, wherein the radionuclide is selected from... 110 In、 111 In、 177 Lu、 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 68 Ge 86 Y、 90 Y、 89 Zr、 94m Tc, 99m Tc, 120 I, 123 I,124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 52m Mn, 55 Co、 72 As、 75 Br、 76 Br、 82 mRb, 83 Sr or other γ-, β-, or positron emitters, for example, the detectable marker is 68 Ga or 177 Lu.

[0047] Implementation Scheme 14. A conjugated molecule of any one of Implementation Schemes 12-13, wherein the B7H3-binding polypeptide is conjugated to the radionuclide by a chelating agent.

[0048] Implementation Scheme 15. The conjugated molecule of Implementation Scheme 14, wherein the chelating agent is selected from DTPA, EDTA, NOA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, or HEHA and their derivatives.

[0049] Implementation Scheme 16. The conjugated molecule of Implementation Scheme 15, wherein the detectable marker is 99m Tc, or the radioactive nuclide is 177 Lu and the chelating agent is DOTA.

[0050] Implementation Scheme 17. A method for detecting the presence and / or amount of B7H3 in a biological sample, comprising:

[0051] a) Under the condition that a complex can be formed between the B7H3-binding polypeptide of any one of embodiments 1-6 or the conjugate molecule of any one of embodiments 11-16 and B7H3, the biological sample and the control sample are contacted with the B7H3-binding polypeptide of any one of embodiments 1-6 or the conjugate molecule of any one of embodiments 11-16.

[0052] b) Detect the formation of the complex.

[0053] The difference in complex formation between the biological sample and the control sample indicates the presence and / or amount of B7H3 in the sample.

[0054] Implementation Scheme 18. A diagnostic agent for detecting and / or diagnosing B7H3-related diseases such as cancer, comprising a B7H3-binding polypeptide of any one of Implementation Schemes 1-6 and / or a conjugated molecule of any one of Implementation Schemes 11-15, and optionally a physiologically acceptable carrier.

[0055] Implementation Scheme 19. The diagnostic agent of Implementation Scheme 18, wherein the diagnostic agent is a contrast agent.

[0056] Implementation Scheme 20. The diagnostic agent of Implementation Scheme 19 is a contrast agent.

[0057] Implementation Scheme 21. The diagnostic agent of Implementation Scheme 20, wherein the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.

[0058] Implementation Scheme 22. Use of the B7H3-binding polypeptide of any one of Implementation Schemes 1-6 and / or the conjugate molecule of any one of Implementation Schemes 11-16 in the preparation of a diagnostic agent for detecting and / or diagnosing B7H3-related diseases such as cancer.

[0059] Implementation Scheme 23. Use of Implementation Scheme 22, wherein the diagnostic agent is a contrast agent.

[0060] Implementation Scheme 24. The purpose of Implementation Scheme 23 is as a contrast agent.

[0061] Implementation Scheme 25. Use of Implementation Scheme 24, wherein the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.

[0062] Implementation Scheme 26. A method for detecting and / or diagnosing B7H3-related diseases, such as cancer or inflammatory diseases, in a subject, comprising administering to the subject a B7H3-binding polypeptide of any one of Implementation Schemes 1-6 and / or a conjugate molecule of any one of Implementation Schemes 11-16 and / or a diagnostic agent of any one of Implementation Schemes 18-21.

[0063] Implementation Scheme 27. The method of Implementation Scheme 26 further includes the step of imaging the object, for example, ECT imaging.

[0064] Implementation Scheme 28. The method of Implementation Scheme 27, wherein the ECT imaging is SPECT imaging or the ECT imaging is PET imaging.

[0065] Implementation Scheme 29. The diagnostic agent of any one of Implementation Schemes 18-21, the use of any one of Implementation Schemes 22-25, or the method of any one of Implementation Schemes 26-28, wherein the B7H3-related disease is cancer, such as the cancer selected from melanoma, leukemia, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, endometrial cancer, oral squamous cell carcinoma, cervical cancer, lung cancer such as non-small cell lung cancer, bladder cancer, clear cell renal cell carcinoma, and glioma (e.g., oligodendroglioma, anaplastic astrocytoma, glioblastoma multiforme (GBM), ependymoma, and endopontine glioma (DIPG)). Brief description of the attached diagram

[0067] Figure 1 . 99m Tc-B107 99m Tc-8H9 antibody tissue distribution results.

[0068] Figure 2 . 177 Lu-DOTA-B107 imaging and uptake results of various tissues and organs. Invention Details

[0070] definition

[0071] Unless otherwise indicated or defined, all terms used herein have their ordinary meaning as will be understood by those skilled in the art. References include, for example, standard manuals such as Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), Volumes 1–3, Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, New York (1990); and Roitt et al., “Immunology” (2nd edition), Gower Medical Publishing, London, New York (1989), and general prior art cited herein; furthermore, unless otherwise stated, all methods, steps, techniques, and operations not specifically detailed herein can and have been performed in a manner known per se as will be understood by those skilled in the art. Also refer to, for example, standard manuals, the aforementioned general prior art, and other references cited therein.

[0072] Unless otherwise stated, the interchangeable terms “antibody” or “immunoglobulin” used herein, whether referring to heavy-chain antibodies or conventional four-chain antibodies, are used generally to include full-length antibodies, their individual chains, and all their portions, domains, or fragments (including, but not limited to, antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively). Furthermore, the term “sequence” as used herein (e.g., in the terms “immunoglobulin sequence,” “antibody sequence,” “single variable domain sequence,” “VHH sequence,” or “protein sequence,” etc.) should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding said sequence, unless a more specific interpretation is required herein.

[0073] As used herein, the term (of a polypeptide or protein) “domain” refers to a folded protein structure that is able to maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of a protein and, in many cases, can be added to, removed from, or transferred to other proteins without loss of the function of the rest of the protein and / or the domain itself.

[0074] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (e.g., the chain of a conventional four-chain antibody or a heavy-chain antibody), or to a polypeptide that is essentially composed of such globular regions. Immunoglobulin domains are characterized by their ability to maintain the immunoglobulin folding characteristics of antibody molecules.

[0075] As used herein, the term "immunoglobulin variable domain" refers to an immunoglobulin domain essentially composed of four "frame regions," referred to in the art and hereinafter as "frame region 1" or "FR1," "frame region 2" or "FR2," "frame region 3" or "FR3," and "frame region 4" or "FR4," respectively, wherein these frame regions are separated by three "complementarity-determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively. Therefore, the general structure or sequence of an immunoglobulin variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain confers antibody specificity for antigens by possessing antigen-binding sites.

[0076] As used herein, the term "immunoglobulin single variable domain" refers to an immunoglobulin variable domain capable of specifically binding to an antigenic epitope without pairing with other immunoglobulin variable domains. An example of an immunoglobulin single variable domain in the context of this invention is a "domain antibody," such as the immunoglobulin single variable domains VH and VL (VH domain and VL domain). Another example of an immunoglobulin single variable domain is the "VHH domain" (or simply "VHH") of the camelid family as defined below.

[0077] The “VHH domain,” also known as heavy chain single-domain antibody, VHH, VHH domain, VHH antibody fragment, and VHH antibody, is a variable domain of antigen-binding immunoglobulin called a “heavy chain antibody” (i.e., “antibody lacking a light chain”) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)). The term “VHH domain” is used to distinguish this variable domain from the heavy chain variable domain (referred to herein as the “VH domain”) present in conventional 4-chain antibodies and the light chain variable domain (referred herein as the “VL domain”) present in conventional 4-chain antibodies. The VHH domain specifically binds to epitopes without the need for other antigen-binding domains (unlike the VH or VL domains in conventional 4-chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and highly efficient antigen-recognition unit formed by a single immunoglobulin domain.

[0078] In the context of this invention, the terms "heavy chain single-domain antibody", "VHH domain", "VHH", "VHH domain", "VHH antibody fragment", and "VHH antibody" are used interchangeably.

[0079] For example, Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999). Figure 2As shown, the amino acid residues used in the VHH domain of camelids can be numbered according to the general numbering method of VH domains given by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0080] Alternative methods for numbering amino acid residues in the VH domain are known in the art, and these alternative methods can also be similarly applied to the VHH domain. For example, the Chothia CDR refers to the position of the structural loop (Chothia and Lesk, J.Mol.Biol.196:901-917(1987)). The AbM CDR represents a compromise between the Kabat hypervariable region and the Chothia structural loop, and is used in Oxford Molecular's AbM antibody modeling software. The "Contact" CDR is based on the analysis of the available crystal structure of the complex. The residue descriptions of the CDRs from each method are as follows:

[0081] ring Kabat AbM Chothia Contact LCDR1 L24-L34 L24-L34 L26-L32 L30-L36 LCDR2 L50-L56 L50-L56 L50-L52 L46-L55 LCDR3 L89-L97 L89-L97 L91-L96 L89-L96 HCDR1 (Kabat designation) H31-H35B H26-H35B H26-H32 H30-H35B HCDR1 (Chothia designation) H31-H35 H26-H35 H26-H32 H30-H35 HCDR2 H50-H65 H50-H58 H53-H55 H47-H58 HCDR3 H95-H102 H95-H102 H96-H101 H93-H101

[0082] However, it should be noted that, as is known in the art regarding VH and VHH domains, the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, in general, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence.

[0083] For example, a CDR may include "extended CDRs", such as: 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2) and 89-97 or 89-96 (LCDR3) in VL; 26-35 (HCDR1), 50-65 or 49-65 (HCDR2) and 93-102, 94-102 or 95-102 (HCDR3) in VH.

[0084] The total number of amino acid residues in the VHH domain will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0085] The structural and functional properties of the VHH domain and its contained peptides can be summarized as follows: The VHH domain (which is naturally "designed" to functionally bind to antigens in the absence of light chain variable domains and without interaction with them) can be used as a single and relatively small functional antigen-binding structural unit, domain, or peptide. This distinguishes the VHH domain from the VH and VL domains of conventional 4-chain antibodies, which are generally not suitable on their own for practical applications as single antigen-binding proteins or single variable domains of immunoglobulins, but need to be combined in some form or another to provide a functional antigen-binding unit (e.g., in the form of conventional antibody fragments such as Fab fragments; or in the form of scFv composed of VH domains covalently linked to VL domains).

[0086] Due to these unique properties, using VHH domains—alone or as part of a larger polypeptide—offers many significant advantages over using 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 antigens with high affinity and selectivity, thus eliminating the need for two separate domains or ensuring they are in appropriate spatial conformation and configuration (e.g., scFv typically requires specially designed linkers); VHH domains can be expressed from a single gene without post-translational folding or modification; VHH domains can be easily modified into multivalent and multispecific formats (formatted); VHH domains are highly soluble and do not aggregate; VHH domains... It is highly stable to heat, pH, proteases, and other denaturing agents or conditions, and therefore can be prepared, stored, or transported without the use of refrigeration equipment, thus saving costs, time, and the environment; the VHH domain is easy to prepare and relatively inexpensive, even at the scale required for production; the VHH domain is relatively small compared to conventional 4-chain antibodies and their antigen-binding fragments (approximately 15 kDa or 1 / 10 the size of conventional IgG), thus exhibiting higher tissue permeability and allowing for higher doses compared to conventional 4-chain antibodies and their antigen-binding fragments; the VHH domain can exhibit so-called cavity-binding properties (especially due to its elongated CDR3 loop compared to conventional VH domains), thereby reaching targets and epitopes that are inaccessible to conventional 4-chain antibodies and their antigen-binding fragments.

[0087] Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185–195; Li et al., JBiol Chem., 287(2012)13713–13721; Deffar et al., African Journal of Biotechnology Vol.8(12),pp.2645-2652,17June,2009 and WO94 / 04678.

[0088] The VHH domain, derived from the Camelidae family, can be "humanized" (also referred to herein as "sequence optimization," which, in addition to humanization, can also encompass other modifications to the sequence by one or more mutations that provide improved VHH properties, such as removing potential post-translational modification sites) by replacing one or more amino acid residues in the original VHH sequence with one or more amino acid residues present at the corresponding positions in the VH domain of a conventional human 4-chain antibody. Humanized VHH domains may contain one or more fully human framework regions. Humanization can be accomplished using protein surface amino acid resurfacing and / or CDR grafting to a universal framework.

[0089] As used herein, the term “epitope” or the interchangeable term “antigenic determinant” refers to any antigenic determinant on an antigen to which the complementary site of an antibody binds. Antigenic determinants typically contain chemically active surface groups of a molecule, such as amino acid or sugar side chains, and typically possess specific three-dimensional structural features as well as specific charge features. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation, and can be a “linear” epitope or a “conformal” epitope. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996). In a linear epitope, all points of interaction between the protein and the interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. In a conformal epitope, points of interaction are separated by protein amino acid residues.

[0090] Epitopes of a given antigen can be identified using many epitope mapping techniques well known in the art. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996). For instance, linear epitopes can be determined by, for example, the simultaneous synthesis of a large number of peptides on a solid support, wherein these peptides correspond to portions of a protein molecule, and by reacting these peptides with an antibody while still attached to the support. These techniques are known in the art and described, for example, in U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; and Geysen et al. (1986) Molec. Immunol. 23:709-715. Similarly, conformational epitopes can be identified by determining the spatial configuration of amino acids, such as by, for example, X-ray crystallography and 2D nuclear magnetic resonance. See, for example, Epitope Mapping Protocols (ibid.).

[0091] Antibodies can be competitively screened for binding to the same epitope using conventional techniques known to those skilled in the art. For example, competitive and cross-competitive studies can be performed to obtain antibodies that compete or cross-competitively bind to the antigen. A high-throughput method for obtaining antibodies binding to the same epitope based on their cross-competition is described in International Patent Application WO03 / 48731. Therefore, antibodies and their antigen-binding fragments that compete with the antibody molecules of the present invention for binding to the same epitope on B7H3 can be obtained using conventional techniques known to those skilled in the art.

[0092] Generally, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein (e.g., the immunoglobulin single variable domain of the present invention) can bind. Specificity can be determined based on the affinity and / or cohesion of the antigen-binding protein. Affinity, expressed as the dissociation equilibrium constant (KD) between the antigen and the antigen-binding protein, is a measure of the strength of binding between the epitope and the antigen-binding site on the antigen-binding protein: the smaller the KD value, the stronger the binding between the epitope and the antigen-binding protein (or, affinity can also be expressed as the association constant (KA), which is 1 / KD). As those skilled in the art will understand, affinity can be determined in a known manner depending on the specific antigen of interest. Affinity is a measure of the strength of binding between an antigen-binding protein (e.g., an immunoglobulin, antibody, immunoglobulin single variable domain, or polypeptide containing it) and the associated antigen. Affinity relates to both the affinity between the antigen and the antigen-binding site on the antigen-binding protein and the number of associated binding sites present on the antigen-binding protein.

[0093] As used herein, the term "B7H3-binding protein (B7H3-binding polypeptide or B7H3-binding molecule)" means any protein capable of specifically binding to the B7H3 protein. B7H3-binding proteins may include antibodies against B7H3, such as antibodies as defined herein. B7H3-binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR transplantation molecules. An exemplary amino acid sequence of B7H3 is shown in SEQ ID NO:111.

[0094] The "B7H3-binding protein" of the present invention may comprise at least one immunoglobulin single variable domain, such as VHH, that binds B7H3. In some embodiments, the "B7H3-binding molecule" of the present invention may comprise 2, 3, 4, or more immunoglobulin single variable domains, such as VHH, that bind B7H3. In addition to the immunoglobulin single variable domain that binds B7H3, the B7H3-binding protein of the present invention may also comprise a linker and / or an effector-functional portion, such as a half-life-extending portion (e.g., an immunoglobulin single variable domain that binds serum albumin), and / or a fusion coupler (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region. In some embodiments, the "B7H3-binding protein" of the present invention also encompasses bispecific antibodies containing immunoglobulin single variable domains that bind different antigens.

[0095] Typically, the B7H3 binding protein of the present invention will be measured in a preferred 10 as in a Biacore, KinExA, or Fortibio assay. -7 Up to 10 -10 mol / L (M), more preferably 10 -8 Up to 10 -10 moles per liter, or even more preferably 10 -9 Up to 10 -10 Or a lower dissociation constant (KD), and / or at least 10 7 M -1 Preferably at least 10 8 M -1 More preferably at least 10 9 M -1 More preferably at least 10 10 M -1 The association constant (KA) binds to the antigen to be bound (i.e., the B7H3 protein). Any protein with an association constant greater than 10... -4 The KD value of M is generally considered to indicate nonspecific binding. The specific binding of antigen-binding proteins to antigens or epitopes can be determined by any known suitable method, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays).

[0096] Amino acid residues will be represented according to standard three-letter or one-letter amino acid codes as known and agreed upon in the art. When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion, or substitution of a specified number of amino acid residues at a position in a reference sequence compared to the other sequence. In the case of substitution, the substitution will preferably be a conserved amino acid substitution, meaning that an amino acid residue is replaced by another amino acid residue with a similar chemical structure, and that has little or no effect on the function, activity, or other biological properties of the polypeptide. The conserved amino acid substitutions are well known in the art. For example, the conserved amino acid substitutions are preferably the substitution of one amino acid in the following groups (i)-(v) by another amino acid residue in the same group: (i) smaller aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (iii) polar positively charged residues: His, Arg and Lys; (iv) larger aliphatic nonpolar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp. The particularly preferred conserved amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gln or His; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln, or Glu; Met is substituted by Leu, Tyr, or Ile; Phe is substituted by Met, Leu, or Tyr; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is substituted by Trp or Phe; Val is substituted by Ile or Leu.

[0097] "Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence similarity" indicates the percentage of identical or conserved amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For instance, the BLAST program in the NCBI database can be used to determine identity. For determining sequence identity, see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M., Stockton Press, New York, 1991.

[0098] A polypeptide or nucleic acid molecule is considered "isolated" when it has been separated from at least one other component (e.g., another protein / peptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or trace component) that is normally associated with it in that source or medium (culture medium), compared to its natural biological source and / or the reaction medium or culture medium from which the molecule is obtained. Specifically, a polypeptide or nucleic acid molecule is considered "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. "Isolated" polypeptide or nucleic acid molecules are preferably substantially homogeneous, as determined by suitable techniques (e.g., suitable chromatographic techniques, such as polyacrylamide gel electrophoresis).

[0099] As used in this article, the term "object" refers to mammals, especially primates, and particularly humans.

[0100] The B7H3-binding polypeptide of the present invention

[0101] This invention provides a B7H3-binding polypeptide comprising at least one immunoglobulin single variable domain capable of specifically binding to B7H3. In some embodiments, the B7H3-binding polypeptide is isolated. In some embodiments, the B7H3-binding polypeptide specifically binds to B7H3.

[0102] In some embodiments, the at least one immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH shown in any of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, and 85. The CDR may be a Kabat CDR, an AbM CDR, a Chothia CDR, or a Contact CDR. In some embodiments, the CDR is a Kabat CDR.

[0103] In some embodiments, the at least one immunoglobulin single variable domain comprises a subset of CDR1, CDR2, and CDR3:

[0104] (1) CDR1 shown in SEQ ID NO:2, CDR2 shown in SEQ ID NO:3, and CDR3 shown in SEQ ID NO:4;

[0105] (2) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:7, and CDR3 shown in SEQ ID NO:8;

[0106] (3) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12;

[0107] (4) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO:15, and CDR3 shown in SEQ ID NO:16;

[0108] (5) CDR1 shown in SEQ ID NO:18, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:20;

[0109] (6) CDR1 shown in SEQ ID NO:22, CDR2 shown in SEQ ID NO:23, and CDR3 shown in SEQ ID NO:24;

[0110] (7) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:28;

[0111] (8) CDR1 shown in SEQ ID NO:30, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:32;

[0112] (9) CDR1 shown in SEQ ID NO:34, CDR2 shown in SEQ ID NO:35, and CDR3 shown in SEQ ID NO:36;

[0113] (10) CDR1 shown in SEQ ID NO:38, CDR2 shown in SEQ ID NO:39, and CDR3 shown in SEQ ID NO:40;

[0114] (11) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:43, and CDR3 shown in SEQ ID NO:44;

[0115] (12) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:48;

[0116] (13) CDR1 shown in SEQ ID NO:50, CDR2 shown in SEQ ID NO:51, and CDR3 shown in SEQ ID NO:52;

[0117] (14) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:55, and CDR3 shown in SEQ ID NO:56;

[0118] (15) CDR1 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:59, and CDR3 shown in SEQ ID NO:60;

[0119] (16) CDR1 shown in SEQ ID NO:62, CDR2 shown in SEQ ID NO:63, and CDR3 shown in SEQ ID NO:64;

[0120] (17) CDR1 shown in SEQ ID NO:66, CDR2 shown in SEQ ID NO:67, and CDR3 shown in SEQ ID NO:68;

[0121] (18) CDR1 shown in SEQ ID NO:70, CDR2 shown in SEQ ID NO:71, and CDR3 shown in SEQ ID NO:72;

[0122] (19) CDR1 shown in SEQ ID NO:74, CDR2 shown in SEQ ID NO:75, and CDR3 shown in SEQ ID NO:76;

[0123] (20) CDR1 shown in SEQ ID NO:78, CDR2 shown in SEQ ID NO:79, and CDR3 shown in SEQ ID NO:80;

[0124] (21) CDR1 shown in SEQ ID NO:82, CDR2 shown in SEQ ID NO:83, and CDR3 shown in SEQ ID NO:84; and

[0125] (22) CDR1 shown in SEQ ID NO:86, CDR2 shown in SEQ ID NO:87, and CDR3 shown in SEQ ID NO:88.

[0126] In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with the amino acid sequence shown in any of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, and 85. In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence shown in any of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, and 85.

[0127] In some embodiments, the immunoglobulin single variable domain is VHH. In some embodiments, the immunoglobulin single variable domain is humanized.

[0128] Nucleic acid, vector, host cell

[0129] In another aspect, the present invention relates to nucleic acid molecules encoding the B7H3-binding polypeptide of the present invention. The nucleic acid of the present invention may be RNA, DNA, or cDNA. Those skilled in the art can select the nucleic acid molecule encoding the B7H3-binding polypeptide of the present invention as needed or by conventional means. In some specific embodiments, the nucleic acid molecule encoding the B7H3-binding polypeptide of the present invention comprises a nucleotide sequence selected from SEQ ID NO:89-110.

[0130] The nucleic acids of the present invention may also be in vector form, and may be present in and / or part of a vector, such as plasmids, sticky-terminal plasmids, or YACs. The vectors may be, in particular, expression vectors, providing a means for expressing B7H3-binding peptides in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). The expression vector typically contains at least one nucleic acid of the present invention, operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). Selection of these elements and their sequences for expression in a particular host is common knowledge to those skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the B7H3-binding peptides of the present invention include, for example, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes.

[0131] The nucleic acids of the present invention can be prepared or obtained by known means (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on information about the amino acid sequence of the polypeptides of the present invention given herein, and / or can be isolated from suitable natural sources.

[0132] In another aspect, the present invention relates to host cells that express or are capable of expressing one or more of the B7H3-binding polypeptides of the present invention and / or contain the nucleic acids or vectors of the present invention. Preferred host cells of the present invention are bacterial cells, fungal cells, or mammalian cells.

[0133] Suitable bacterial cells include Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0134] Suitable fungal cells include cells from species of the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or cells from species of the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichiapastoris* and *Pichia methanolica*), and *Hansenula*.

[0135] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0136] However, the present invention may also use amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins.

[0137] This invention also provides a method for generating the B7H3-binding polypeptide of this invention, the method typically comprising the following steps:

[0138] - Culture the host cells of the present invention under conditions that allow expression of the B7H3 binding polypeptide of the present invention; and

[0139] -Recover the B7H3-binding polypeptide expressed by the host cells from the culture; and

[0140] -Optional further purification and / or modification of the B7H3-binding peptide of the present invention.

[0141] The B7H3 binding polypeptide of the present invention can be produced in cells as described above in an intracellular manner (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), followed by isolation from the host cell and optionally further purification; or it can be produced in an extracellular manner (e.g., in a culture medium for culturing host cells), followed by isolation from the culture medium and optionally further purification.

[0142] Methods and reagents for recombinantly generating polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, protein separation and purification techniques suitable for the methods used to produce the B7H3-binding polypeptides of the present invention are well known to those skilled in the art.

[0143] However, the B7H3 binding polypeptide of the present invention can also be obtained by other protein-generating methods known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.

[0144] Conjugated molecules

[0145] In another aspect, the present invention provides a conjugated molecule comprising the B7H3 binding polypeptide of the present invention, and at least one detectable label and / or therapeutic portion conjugated to said B7H3 binding polypeptide.

[0146] The detectable markers include, but are not limited to, radionuclides, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, and enzymes.

[0147] Fluorescent agents that can be used for conjugation include, but are not limited to, isothiocyanate fluorescein, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescein; chemiluminescent agents that can be used for conjugation include, but are not limited to, luminol, isoluminol, aromatic acridine esters, imidazole, acridine salts, and oxalates; bioluminescent agents that can be used for conjugation include, but are not limited to, fluorescein, luciferase, and jellyfish luminescent protein. Paramagnetic ions that can be used for conjugation include, but are not limited to, chromium(III), manganese(II), iron(III), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), and erbium(III), or radiation-impermeable materials such as dams, diatrizoates, ethyl iodinated oil, gallium citrate, iocarboxylic acid, iodotinic acid, iodamide, cholinesteric acid, iosaccharide, iogulum, iohexol, iopamidol, ioprosylate, iodixazoline, iosifaric acid, iodoserilic acid, iodolesulfonate meglumine, iophthalyl thiocyanate, iodotinic acid, iodotalamic acid, iodoxazoline, hydroxydiatrizoate, iopoise, meglumine, methyl diatrizoate, methyl diatrizoate salt, propiodosperidone, and thallium oxide. Enzymes that can be used for conjugation include, but are not limited to, horseradish peroxidase.

[0148] Preferably, the detectable marker is a radionuclide. Some radionuclides that can be used for conjugation are radionuclides with energies between 20 and 4000 keV, including but not limited to those listed below. 110 In、 111 In、 177 Lu、 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 68 Ge 86 Y、 90 Y、 89Zr、 94m Tc, 99m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 52m Mn, 55 Co、 72 As、 75 Br、 76 Br、 82 mRb, 83 Sr or other γ-, β-, or positron emitters. In some embodiments, the detectable marker is... 68 Ga or 177 Lu.

[0149] Methods for conjugating detectable markers and / or therapeutic moieties to peptides are well known to those skilled in the art. For example, in some embodiments, the B7H3-binding peptide may be conjugated to the detectable marker using a chelating agent.

[0150] In order to use radioactive nuclides such 68For the Ga-labeled B7H3-binding peptide of the present invention, it is necessary to react the B7H3-binding peptide of the present invention with a reagent having a long tail, the long tail having multiple integrative groups for binding ions. Such a tail can be, for example, a polymer of polylysine, a polysaccharide, or other derivative or derivatizable chains with side groups that can bind chelating groups, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid), NOA, TETA, NETA, porphyrin, polyamine, crown ether, thiourea, polyoxime, and similar groups known to be suitable for this purpose. The chelating agent is attached to the antibody using standard chemical methods. In some embodiments, the label can be detected by conjugation of the label to the B7H3-binding peptide of the present invention via the chelating agent. The chelating agents used include, but are not limited to, DTPA, EDTA, NOA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, or HEHA and their derivatives.

[0151] In some specific implementations, the detectable marker is 99m Tc. In some specific embodiments, the detectable marker is 177 Lu, and the chelating agent is DOTA.

[0152] On the other hand, the present invention provides a method for preparing the radionuclide of the present invention, such as... 99m Tc or 177 A method for Lu-labeled conjugated molecules, comprising: 1) conjugating the B7H3-binding polypeptide of the present invention with a chelating agent to generate a conjugate of the B7H3-binding polypeptide and the chelating agent; and 2) reacting the product of step 1) with a radionuclide such as 99m Tc or 177 Lu contact, thereby radioactive nuclides such as 99m Tc or 177 Lu labels the B7H3-binding polypeptide of the present invention by chelation of a chelating agent. In some embodiments, the chelating agent is NOA, and in step 1), the B7H3-binding polypeptide is reacted with p-SCN-Bn-NOTA or p-NH2-Bn-NOTA to generate a conjugate of the B7H3-binding polypeptide and NOA. In some embodiments, the chelating agent is DOTA, and in step 1), the B7H3-binding polypeptide is reacted with SCN-Bn-DOTA to generate a conjugate of the B7H3-binding polypeptide and DOTA.

[0153] In some embodiments, the therapeutic component includes, but is not limited to, paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, epipodophyllotoxin glucoside, epipodophyllotoxin thiophene glycoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraxetine dione, mitoxantrone, scintillans, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, aminopyrimidine, styromycin, cephalosporin, metansine, aristostatin, nitrogen mustard, chlorambucil, phenylalanine mustard Carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum(II) (DDP), cisplatin, bleomycin, atrazotocin, abscisicin, ricin A, Pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor, interferon-γ, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), interleukin-10 (“IL-10”), granulocyte-macrophage colony-stimulating factor (“GM-CSF”), granulocyte colony-stimulating factor (“G-CSF”), or IFN. In some embodiments, the therapeutic component may be the radionuclides described above.

[0154] Detection / Diagnostic Uses

[0155] In another aspect, the present invention provides a method for detecting the presence and / or amount of B7H3 in a biological sample, comprising:

[0156] a) Under conditions where a complex can be formed between the B7H3-binding polypeptide of the present invention or the conjugate molecule of the present invention and B7H3, the biological sample and the control sample are contacted with the B7H3-binding polypeptide of the present invention or the conjugate molecule of the present invention.

[0157] b) Detect the formation of the complex.

[0158] The difference in complex formation between the biological sample and the control sample indicates the presence and / or amount of B7H3 in the sample. In some embodiments, the biological sample is an ex vivo sample.

[0159] In another aspect, the present invention provides a composition comprising the B7H3-binding polypeptide of the present invention and / or the conjugated molecule of the present invention, and optionally a physiologically acceptable carrier. The composition can be used as a detection or diagnostic agent, for example, a diagnostic agent for detecting and / or diagnosing B7H3-related diseases.

[0160] In another aspect, the present invention provides a diagnostic agent for detecting and / or diagnosing B7H3-related diseases such as cancer, comprising the B7H3-binding polypeptide of the present invention and / or the conjugated molecule of the present invention, and optionally a physiologically acceptable carrier. In some embodiments, the diagnostic agent is a contrast agent.

[0161] The B7H3-binding peptides and / or conjugate molecules of the present invention are particularly suitable for in vivo imaging, such as for emission computed tomography (ECT). For example, the B7H3-binding peptides and / or conjugate molecules of the present invention can be applied to single-photon emission computed tomography (SPECT) and positron emission tomography (PET) depending on the labeling. In tumor diagnosis, they can provide high-resolution tumor imaging and allow for quantitative analysis of the images. The SPECT imaging may also include SPECT / CT imaging, and the PET imaging may also include PET / CT imaging, which can provide even better imaging results.

[0162] Therefore, in some embodiments, the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.

[0163] In another aspect, the present invention provides the use of the B7H3-binding peptide and / or the conjugate molecule of the present invention in the preparation of diagnostic agents for detecting and / or diagnosing B7H3-related diseases such as cancer. In some embodiments, the diagnostic agent is a contrast agent. In some embodiments, the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.

[0164] In another aspect, the present invention provides a method for detecting and / or diagnosing B7H3-related diseases such as cancer in a subject, comprising administering the subject a B7H3-binding polypeptide of the present invention and / or a conjugate molecule of the present invention and / or a diagnostic agent of the present invention.

[0165] In some embodiments, the method further includes the step of imaging the object, such as ECT imaging. In some embodiments, the ECT imaging is SPECT imaging. In some embodiments, the ECT imaging is PET imaging. Imaging techniques and apparatus for SPECT or PET scans are well known in the art, and any such known ECT imaging techniques and apparatus can be used.

[0166] Diseases that can be detected and / or diagnosed by the B7H3 binding peptide of the present invention and / or the conjugated molecules of the present invention and / or the diagnostic agents of the present invention include diseases that cause abnormally elevated B7H3 expression in cells, tissues or organs, such as cancer.

[0167] In this article, B7H3-related diseases include, but are not limited to, cancers, such as melanoma, leukemia, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, endometrial cancer, oral squamous cell carcinoma, cervical cancer, lung cancer such as non-small cell lung cancer, bladder cancer, clear cell renal cell carcinoma, and gliomas (such as oligodendroglioma, anaplastic astrocytoma, glioblastoma multiforme (GBM), ependymoma, and endophytic pontine glioma (DIPG)).

[0168] Pharmaceutical Compositions and Therapeutic Uses

[0169] On the other hand, the present invention provides a pharmaceutical composition comprising the B7H3-binding polypeptide or conjugate molecule of the present invention, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is used to treat B7H3-related diseases. The B7H3-related diseases, as described above, are preferably cancers.

[0170] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion).

[0171] In another aspect, the present invention provides the use of the B7H3-binding polypeptide or conjugate molecule of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating B7H3-related diseases. The B7H3-related diseases, as described above, are preferably cancers.

[0172] In another aspect, the present invention provides a method for treating a B7H3-related disease in a subject, comprising administering to the subject a therapeutically effective amount of the B7H3-binding polypeptide or conjugate molecule of the present invention or a pharmaceutical composition of the present invention. The B7H3-related disease, as described above, is preferably cancer.

[0173] Reagent test kit

[0174] In another aspect, the present invention provides a kit comprising the B7H3-binding polypeptide of the present invention, or the conjugated molecule of the present invention, or the diagnostic agent of the present invention. The kit is used to carry out the methods of the present invention. The kit generally includes a label indicating the intended use of the kit contents. The term "label" includes any written or recorded material provided on or with the kit or otherwise accompanied by the kit. Example

[0175] Example 1: Antibody Screening and Construction

[0176] 1.1 Construction of the Library

[0177] The B7H3-cHis fusion protein used for immunization was expressed in 293F cells and purified by nickel affinity chromatography. One healthy alpaca was selected for immunization. After immunization, peripheral blood was collected from the alpaca, and PBMCs were isolated according to the instructions for using the lymphocyte separation medium. RNA was extracted using RNAiso Plus reagent and processed using PrimeScript. TM The II 1st Strand cDNA Synthesis Kit (Takara, catalog number: 6210A) reverse transcribes extracted RNA into cDNA. Nested PCR is then used to amplify the nucleic acid fragment encoding the variable region of the heavy chain antibody.

[0178] The VHH fragment was purified using a DNA product purification kit. Both the vector and the fragment were digested with the restriction endonuclease SfiI overnight at 50°C. The digested fragment was then recovered via gel extraction and cloned into the phage display vector pComb3XSS. The product was subsequently electroporated into *E. coli* electroporated competent cells TG1 to construct a phage display library of a heavy chain single-domain antibody against B7H3, and the library was validated. The library size was calculated to be 2.36 × 10⁻⁶ cells after serial dilution plating. 13 CFU. To test the insertion rate of the library, 48 clones were randomly selected for identification. The results showed that the insertion rate reached 100% and the size was correct.

[0179] 1.2 Panning of heavy chain single-domain antibodies against B7H3

[0180] Dilute the B7H3-cHis fusion protein to a final concentration of 5 μg / mL with carbonate buffer at pH 9.6, and add 100 μL / well to each microplate. Incubate overnight at 4°C. The next day, block with 300 μL / well of 3% BSA-PBS blocking buffer at 37°C for 1 hour. After washing the plate three times with PBS, add 100 μL of phage (2 × 10⁻⁶). 11CFU (from the heavy chain single-domain antibody phage display library constructed in section 1.1) was incubated at 37°C for 1 hour. Afterwards, the cells were washed 6 times with PBST (PBS containing 0.05% Tween 20) and then 2 times with PBS solution to remove unbound phages. Next, 100 μL of Gly-HCl (pH = 2.5) was added to each well and incubated at 37°C for 8 min to dissociate the phages specifically bound to B7H3 and transfer them to a sterile centrifuge tube. 10 μL of Tris-HCl (pH = 9.0) neutralization buffer was quickly added. 10 μL of the solution was serially diluted, the titer was determined, and the panning recovery rate was calculated. The neutralized phages were then used to infect E. coli TG1 bacteria in the logarithmic growth phase to generate and purify phages for the next round of screening. This screening process was repeated several times, with different panning conditions for each round. Thus, positive clones were enriched, achieving the goal of screening B7H3-specific antibodies from the antibody library using phage display technology. Table 1 shows the affinity panning conditions, and Table 2 shows the recovery rate and enrichment degree of the two rounds of screening.

[0181] Table 1 Affinity Selection Criteria

[0182]

[0183] Table 2. Recovery rates after one round of acid elution screening targeting B7H3 antigen.

[0184]

[0185] 1.3. Screening for specific single positive clones using phage enzyme-linked immunosorbent assay (ELISA).

[0186] After two rounds of panning, 192 clones were randomly selected from the panned plates for identification. The 192 randomly selected single colonies were cultured separately to produce and purify bacteriophages. The B7H3-cHis fusion protein was diluted to a final concentration of 2 μg / mL with carbonate buffer at pH 9.6, and 100 μL was added to each well of the ELISA plate. The plates were incubated overnight at 4°C. Blocking was performed with 5% skim milk and incubated at 37°C for 1 hour. Then, 50 μL of phage culture supernatant and 50 μL of 5% skim milk were added to each well, and the plates were incubated at 37°C for 1 hour. After washing with PBST, horseradish peroxide-labeled anti-M13 secondary antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., diluted 1:10000 with PBS) was added, and the plates were incubated at 37°C for 1 hour. After washing, TMB chromogenic buffer was added, and the absorbance was read at 450 nm. According to the ELISA results, 316 positive clones were found out of 372 clones, and these were sequenced for analysis. Thirteen of these clones were non-monoclonal.

[0187] Based on the results of phage ELISA with the B7H3-cHis antigen, clones that showed good binding to the antigen protein were selected. Simultaneously, the protein sequences of each clone were analyzed using the DNAMAN sequence alignment software. Clones with identical CDR1, CDR2, and CDR3 sequences were considered the same antibody strain, while clones with different CDR sequences were considered different antibody strains. Finally, 22 candidate antibodies were selected for subsequent experiments. The specific antibody names and amino acid sequences are shown in Table 3 below.

[0188] 1.4 Preparation of B7H3 antibody protein using mammalian cells

[0189] Primers were designed based on the nucleotide sequences of the screened B7H3 single-domain antibodies. Using plasmids as templates, the nucleotide sequences (including signal peptides and His tags) of each antibody were amplified by PCR, subcloned into the expression vector PSNA008 (pCDNA4 (Invitrogen, Cat V86220)), and transfected into HEK293 cells for antibody expression. The recombinant expression plasmid was diluted with Freestyle293 medium and PEI (Polyethylenimine) solution was added for transformation. The plasmid / PEI mixture was added to HEK293 cell suspension and cultured at 37°C, 10% CO2, and 90 rpm. After four hours, EX293 medium and 2 mM glutamine were added, and the cells were cultured at 135 rpm. After 24 hours, 3.8 mM VPA was added. After 6–7 days of culture, the transient expression culture supernatant was collected and purified using a nickel column. The final antibody protein had a purity of over 90%.

[0190] Table 3. B7H3 antibodies obtained through screening and their amino acid sequences.

[0191]

[0192]

[0193]

[0194]

[0195]

[0196] The nucleotide sequence of the B115-cHis antibody (SEQ ID NO:89):

[0197] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGC CTCTGGACGCACTTTCAGTAATCGTAATCTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCAG CTATATGGTGGACTAGTGGTACCATATACTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTGCAAATAGACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCTGGGCGTGG GAGTTACTACTCGGAGCATCGGGGATATGACTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0198] Nucleotide sequence of B149-cHis antibody (SEQ ID NO:90):

[0199] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCCGGGGGAGGCTCGGTGCAACCTGGGGGGTCTCTGAGGCTCACCTGTGCAGC CTCTGGACGCACTTTCAGTAATCGTAACATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCAA CTATATGGTGGACTCCTGGTACCGTATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTCGAAATAGACAGACTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCTGGGCGTGG GAGTGCCTACTCGGAGCATCGGGGATATGACTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0200] The nucleotide sequence of the B65-cHis antibody (SEQ ID NO:91):

[0201] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCCGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTACAGC CTCTGAACGCACCTCCAGTGATCGTAACGTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAG CTATATGGTGGACTAGTGGTACCATATACTATGTAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTGCAAATAGACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCTGGGCGTGG GAGTAACTACTCGGAGCGACGGGGATATGTCTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0202] Nucleotide sequence of B143-cHis antibody (SEQ ID NO:92):

[0203] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCCGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAAC CTCTGGACGCACCTCCAGTGATCGTAACGTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAG CTATATGGTGGACTAGTGGTACCATATACTATGTAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTGCAAATAGACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCTGGGCGTGG GAGTAACTACTCGGAGCGACGGGGATATGTCTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0204] Nucleotide sequence of B179-cHis antibody (SEQ ID NO:93):

[0205] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCGGGGGGAGGATTAGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGC CTCTGGCAGCACCTCCAGTAACTATCGCGTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGATTGTCGCAG CTATTGGGGGGTGGCCTAGTAGTACGATATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGAC GCCAAGAATACGGTCTATCTGCAAATGAACAGCCTGAAACCCGAAGACACGGCCGTTTATTACTGTGCAGCAAATCC CCGACGTTGGGTGGTGTCTTTTCACAGCGATGACTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0206] Nucleotide sequence of B75-cHis antibody (SEQ ID NO:94):

[0207] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTCGGTGCAACCTGGGGGGTCTCTGAGACTCTCCTGTGCA GCCTCTAGACGCACCTTCAGTACGTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTTG CAACTATTAACTGGAGTGGTGGTACCACGGACTATGCAGACTCCATGAAGGGCCGGTTCACCATCTCCAGAGACAA CGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGGGGACACGGCCGTTTATTACTGTGCA GCCGG CGGCGCCTGCTCAGGCTACGGGTGTCGGTTGAGTTCCGCCTATTACTACTGGGGCCAGGGGACCCAGGTCACCGTCT CCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0208] Nucleotide sequence of B107-cHis antibody (SEQ ID NO:95):

[0209] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGC CTCTAGACGCACCTTCAGTACCTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTTGCAA CTATTAACTGGAGTGGTGGTACCACGGACTATGCAGACTCCATGAAGGGCCGGTTCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTGCAAATGAACCGCCTGAAACCTGGGGACACGGCCGTTTATTACTGTGCAGCCGGCGGCGC CTGCTCAGGCTATGGGTGTCGGTTGAGTTCTGCCTATTACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0210] Nucleotide sequence of B108-cHis antibody (SEQ ID NO:96):

[0211] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCTGGTGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGCCTCTCCTGTGCAGC CTCTAGCCGCACCTTCAGTACCTATCTCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAG CTGCTAACTGGAGTGGCGGTAGAACGCATTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTGGAAATGAACAGACTGAAACCTGAGGACACGGCCCTTTATTACTGTGCAGCAGGGGGAGC AAGTGCTTCGGTCGGAGGCGACTATGATTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0212] Nucleotide sequence of B106-cHis antibody (SEQ ID NO:97):

[0213] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCGGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGCCTCTCCTGTGCAGC CTCTAGCCGCACCTTCAGTACCTATCTCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAG CTGCTACCTGGAGTGGCGGTAGAACATATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTGCAAATGAACAGACTGAAACCTGAGGACACGGCCCTTTATTACTGTGCAGCAGGGGGTGC AAGTGCTTCTGTCGGAGGCGACTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0214] Nucleotide sequence of B134-cHis antibody (SEQ ID NO:98):

[0215] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCGGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGC CTCTCCACGCACCTTCAGTACATATGTCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTCGTAGCCA GTATTAACTGGAGTGGTGGTGAGAAATACCATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC GAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTACAGCAGGGGGCCC ACCCTTCGGAGAGGTATTTAGTACCTCGCGACAGGTGGCATACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0216] The nucleotide sequence of the B154-cHis antibody (SEQ ID NO:99):

[0217] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCGGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGCCTCTCCTGTGCAGC CTCTAGCCGCACCTTCAGTACCTACGTCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAG CTGCTACCTGGAGTGGCGGTAGAACATTTTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTGCGTATGAACAGACTGAAACCTGAGGACACGGCCCTTTATTACTGTGCAGCAGGGGGTGC AAGTGCTTCTGTCGGAGGCGACTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0218] Nucleotide sequence of B85-cHis antibody (SEQ ID NO:100):

[0219] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGCCTCTCCTGTGCAGC CTCTGGAATCATCTTTAGTAAAAATGACATGGGCTGGTTCCGCCAGGCTCCAGGGAAGCAGCGCGAGTGGGTCGCAA CTGTTACTACTGGTGGTAGGGCGTACTACTTCGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAG AACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATAAAATTCCCGCGTT TGGTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0220] Nucleotide sequence of B22-cHis antibody (SEQ ID NO:101):

[0221] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCAGGGGGAGGATTGGCGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGC CTCTGGACGCACCCTCAGTGTGTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAA CTATTAGCAAGAATGGTAATGGATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAG ATTACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGCTTATCACTGTGCAGCCTCCTCTCGGGT GGCAGTGGCTACCCAGGGTCCTTTGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0222] Nucleotide sequence of B2-41-cHis antibody (SEQ ID NO:102):

[0223] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCAAGGTGCAGCCTGGGGACTCTCTGAGACTCTCCTGTGCA GCCTCTAGACGCACCTTCAGTACCTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGT T GCAACTATTAACTGGAGTGGTGGTACCACGGACTATGCAGACTCCATGAAGGGCCGGTTCACCATCTCCAGAGACAA CGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGGGGACACGGCCGTTTATTACTGTGCAGCCGGCG GCGCGTGCTCAGGCTATGGGTGTCGGTTGAGTTCTGCCTATTACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCC TCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0224] Nucleotide sequence of B2-39-cHis antibody (SEQ ID NO:103):

[0225] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGTTGCAGCTCGTGGAGTCCGGTGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGC CTCTGGACGCACTTTCAGTAATCGTAACATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCAA CTATATGGTGGACTCCTGGTACCGTATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTCGAAATAGACAGACTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCTGGGCGTGG GAGTGCCTACTCGGAGCATCGGGGATATGACTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0226] Nucleotide sequence of B2-22-cHis antibody (SEQ ID NO:104):

[0227] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCCGGTGGAGGCTCGGTGCAACCTGGGGGGTCTCTGAGGCTCTCCTGTGCAGC CTCTGGACGCACTTTCAGTAATCGTAATCTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGCAG CTATATGGTGGACTAGTGGTACCATATACTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGGTGTATCTGCAAATAGACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCTGGGCGTGG GAGTTACTACTCGGAGCATCGGGGATATGACTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0228] Nucleotide sequence of B4-165-cHis (SEQ ID NO:105):

[0229] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCTGGTGGTGGATTGGTCCACATTGGGGGCTCTCTGAGGCTCTCCTGTGCAGT CTCTGGACGCACCTCCAGTAACTATCGCGTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGATTGTCGCAG CTATTGGGGGGTGGCCTAGTAGTACGATATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGAC GCCAAGAATACGGTCTATCTGCAAATGAACAGCCTGAAACCCGAAGACACGGCCGTTTATTACTGCGCAGCAAATCC CCGACGTTGGGTGGTGTCTTTTCACAGCGATGACTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0230] Nucleotide sequence of B4-148-cHis (SEQ ID NO:106):

[0231] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCCGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCA GCCTCTGGACGCACCTCCAGTAACTATCGCGTGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGATTGTCG CAGCTATTGGGGGGTGGCCTAGTAGTACGATATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGA CGACGCCAAGAATACGGTCTATCTGCAAATGAACAGCCTGAAACCCGAAGACACGGCCGTTTATTACTGTGCAGCA AATCCCCGACGTTGGGTGGTGTCTTTTCACAGCGATGACTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCT CCTCA Nucleotide sequence of B2-124-cHis antibody (SEQ ID NO:107):

[0232] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCCGGGGGAGGATTGGCGCGGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGC CTCTGAACGCACCCTCAGTGTGTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAA CTATTAGCAAGAATGGTTATGGATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAGAACGCCAAG ATTACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTATTGTGCAGCCTCCTCTCGGGT GGCAGTGGCTACCCAAACTCCTTTGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0233] Nucleotide sequence of B6-cHis antibody (SEQ ID NO:108):

[0234] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCTGGAGGAGGATTGGTCCAGGCTGGGGGCTCTCTGAGACTCTCCTGTTCAGC CTCAGGACTCACCTTAAGTAATTATGGAATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAACTTGTAGCGG ATATTACTAGGAGTTCTGGTACGACACGCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACACGGCTTATCTCCAAATGAACAGCCTGATACCTGATGACACGGCCGTTTATTACTGTGCAGCCCGGCTGCC AGGGACGTGGAATACGAGGAAGTATGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0235] Nucleotide sequence of B38-cHis antibody (SEQ ID NO:109):

[0236] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGT CAGGTGCAGCTCGTGGAGTCCGGGGGAGGCTCGGTGCAACCTGGGGGGTCTCTGAGGCTCACCTGTGCAGC CGCTGGATTTCCCGTGGAGGCTGAAGCCATAGGCTGGTTCCGCCAGGTCCCAGGGAAGGAGCGTGAGGGGGTCTCAT GCATCACTAGAAGTAGTAGACGCACAACCTATTCAGACTCCGTGAAGGGCCGATTCGCCGTCTCCAGAGGGGACAAC GACAACACTGTGTATCTGCAGATGAACAGCCTGAAACCTGAGGACACAGGGCGTTATTACTGCGCAGTCATGAAGGA CGAGTACTGTTCAGACTGGATTCCCACTGGCTATATGGGCCGGGGGACGCAGGTCACCGTCTCCTCA GGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0237] Nucleotide sequence of B133-cHis antibody (SEQ ID NO:110):

[0238] AAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCAC CGGT CAGTTGCAGCTCGTGGAGTCGGGGGGAGGCTTGGTGCAGGCTGGGGGGTCCCTGAGACTCTCCTGTGC AGCCTCTGGACGCACCAGCAGTACAGGTGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTCG CGAATATTCGCTGAGTGGTGGCAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAC GCCAAGAATATGGTATATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCATCTATTACTGTAATGCCCGCTG GTTCATTAGATCCTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCAGGCAGCATGGATCCTGGAGGATCTCATCATCACCACCACCATCATCACTAAGAATTC

[0239] Note: The underlined portion is the nucleotide sequence of the target antibody.

[0240] Example 2: In vitro verification of B7H3 antibody function

[0241] The B7H3-cHis fusion protein was constructed for detection. The B7H3-cHis fusion protein was obtained by transient expression in HEK293 cells and affinity purification using nickel-based filler.

[0242] The amino acid sequence of the B7H3-cHis fusion protein is (SEQ ID NO:111):

[0243] MLRRRGSPGMGVHVGAALGALWFCLTGA LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSL QVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPT GAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAV SLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMT GSMDPGGSHHHHHHHH

[0244] Note: Underlined lines represent signal peptides and His tags.

[0245] 2.1. Antibody Expression Levels

[0246] Of the 22 antibodies, 5 showed no expression, and 17 showed expression. Expression levels were calculated based on the total amount of the target protein obtained after one-step affinity chromatography purification.

[0247] Table 4.17 Antibody Expression Levels

[0248] name Expression level (mg / L) name Expression level (mg / L) name Expression level (mg / L) B149 59.6 B143 75.8 B179 78 B75 28.6 B107 45.8 B108 55.2 B106 53 B22 13.6 B133 54 B38 56.3 B6 91 B2-124 85 B2-22 69 B2-39 95 B2-41 79 B4-148 45.7 B4-165 78

[0249] The expression level detection results show that most antibodies have an expression level of around 30 mg / L after one-step affinity purification, while some antibodies can reach an expression level of around 90 mg / L. The corresponding protein can be provided for subsequent detection by instantaneous transfection.

[0250] 2.2 Detection of the affinity of B7H3 heavy chain single-domain antibody for human B7H3 protein

[0251] B7H3-cHis fusion protein was coated onto plates at 5 μg / mL, 100 μL per well, and incubated overnight at 4°C. After washing, 3% BSA was added for blocking, and the plates were incubated at 37°C for 1 hour. The test antibody (the heavy chain single-domain antibody obtained in Example 1.4) was serially diluted 4-fold at a starting concentration of 10 μg / mL, resulting in 10 different concentrations. 100 μL of each diluted antibody was added to each well, and the plates were incubated at 37°C for 1 hour. After washing, anti-His (HRP) secondary antibody (1:5000 LOT#GR3248851-4) was added, and the plates were incubated at 37°C for 1 hour. After washing, TMB chromogenic buffer was added, and the absorbance was read at 450 nm. Data processing and plotting were performed using SotfMaxPro v5.4 software. The antibody-B7H3 binding curve and EC50 were obtained through four-parameter fitting. 50 Value. Among the selected 17 series, B107-cHis was used as a control to compare the relative activities of different B7H3 heavy chain single-domain antibodies to reflect the affinity of candidate antibodies for B7H3.

[0252] Table 5. ELISA results of 17 antibodies binding to B7H3

[0253] name EC50 (ng / mL) Relative activity (%) name EC50 (ng / mL) Relative activity (%) B107 11.23 100 B75 23.07 48.7 B108 41.23 27.2 B179 67.71 16.6 B22 100.9 11.1 B143 150.3 7.5 B149 355.1 3.2 B106 1161 1.0 B133 1997 0.2 B2-124 3.356 145 B2-22 293.8 16.5 B2-39 160.8 3.0 B2-41 6.573 73.9 B38 1.192 407.3 B4-148 12.05 40.3 B4-165 32.62 14.9 B6 1134 0.4

[0254] The test results in Table 5 show that some antibodies bind well to the B7H3 antigen. Among the 17 antibodies, B107, B2-124, B2-41, and B38 have the best affinity for the B7H3 antigen. Further cell assays such as FACS will be used to verify the binding of the candidate antibodies to B7H3.

[0255] 2.3 Alpaca secondary antibody was used for ELISA activity screening.

[0256] The B7H3-cHis fusion protein was coated onto plates at a concentration of 5 μg / mL, 100 μL per well, and incubated overnight at 4°C. After washing, 3% BSA was added for blocking, and the plates were incubated at 37°C for 1 hour. 5 μg / mL of the test antibody (the heavy chain single-domain antibody obtained in Example 1.4) was added to each well, 100 μL, and the plates were incubated at 37°C for 1 hour. After washing, anti-camile (HRP) secondary antibody (1:10000 LOT#18E001484) was added, and the plates were incubated at 37°C for 1 hour. After washing, TMB chromogenic buffer was added, and the absorbance was read at 450 nm. The detection results are shown in Table 6.

[0257] Table 6. ELISA activity screening results of 17 alpaca secondary antibodies

[0258] name <![CDATA[OD 450nm ]]> name <![CDATA[OD 450nm ]]> name <![CDATA[OD 450nm ]]> B22 2.644 B75 2.732 B106 2.471 B107 2.709 B108 2.683 B143 2.699 B149 2.693 B179 2.665 B133 1.976 B2-124 0.24 B2-22 1.526 B2-39 1.541 B2-41 1.859 B38 1.854 B4-148 1.716 B4-165 1.743 B6 0.785

[0259] As can be seen from the test results in Table 6, 16 of the 17 antibodies can bind to alpaca secondary antibody, while 1 antibody binds poorly to alpaca secondary antibody.

[0260] 2.4 The binding of B7H3 heavy chain single-domain antibody to B7H3 on the cell surface was investigated by FACS.

[0261] Both A-204 (human rhabdomyosarcoma) and NCI-H322 (human non-small cell lung cancer) cells express the B7H3 antigen. Cells were cultured and collected, and their density was adjusted to 1 × 10⁻⁶. 6 The concentration was determined to be cells / mL. A 100 μL aliquot of cells was mixed with 5 μg of primary antibody and incubated on ice for 30 minutes. After centrifugation and washing, the cells were resuspended, and 2 μL of the Anti-His-PE conjugate was added. The cells were then incubated on ice in the dark for 30 minutes. The cells were washed twice with 300 μL of 1×PBS + 0.5% BSA and resuspended for flow cytometry analysis.

[0262] Eight antibodies with good activity were screened from the above antibodies and their binding ability to NCI-H322 cells was tested, with 8H9 as a positive control. The binding of B107, B38, and B2-124 was tested again using A-204 cells.

[0263] The FACS results are shown in Tables 7 and 8. Among the eight antibodies, only B22 and B38 showed poor binding ability to cells, while the positive rates of the remaining antibodies were all above 99%. The positive control antibody 8H9 showed a 100% binding rate on A-204 and NCI-H322 cells.

[0264] Table 7. Binding of candidate antibodies to NCI-H322 cells

[0265] name Positive rate (%) name Positive rate (%) name Positive rate (%) 8H9 100 B108 100 B107 100 B143 99.9 B2-124 100 B179 100 B2-41 100 B22 0 B38 0.6

[0266] Table 8. Binding of candidate antibodies to A204 cells

[0267] name Positive rate (%) name Positive rate (%) 8H9 100 B107 100 B38 0.6 B2-124 99.9

[0268] 2.5 The binding of the B7H3 heavy chain single-domain antibody to B7H3 was investigated using the ForteBio method.

[0269] B107, B108, B75, and B179 were selected as detection antibodies. A candidate single-domain antibody against B7H3—biotin—was immobilized on the SA biosensor. Then, B7H3-cHis at concentrations of 6.25–100 nM was bound to the nanobodies, followed by dissociation. Octet Data Analysis version 9.0 was used to evaluate and determine the binding kinetic variables for the four antibodies (B107, B108, B75, and B179), including Kon, Koff, and Kd.

[0270] Table 9. Results of KD affinity assay for four antibodies

[0271] Loading Sample ID KD(M) kon(1 / Ms) kdis(1 / s) Full R^2 B107 8.96E-10 1.84E+05 1.65E-04 0.9584 B75 4.56E-10 1.90E+05 8.65E-05 0.9598 B108 8.84E-10 3.10E+05 2.74E-04 0.9665 B179 5.61E-10 5.31E+05 2.98E-04 0.9744

[0272] As shown in Table 9, the KD values ​​of the four candidate antibodies with B7H3 are all less than 1 nM, indicating that they all have good affinity for the B7H3 antigen.

[0273] Example 3: In vivo verification of B7H3 antibody function

[0274] 3.1 99m Imaging of Tc-labeled B7H3 single-domain antibody on MC38-B7H3 tumors

[0275] Based on the analysis of the in vitro validation results, the B107 antibody was ultimately selected as the candidate antibody for in vivo validation. Simultaneously, the positive antibody 8H9 (positive antibody 8H9 is a protein obtained by self-expression based on the antibody sequence in patent US20200197546A1; the construction and expression process is similar to that in Example 2, obtained through transient expression in HEK293 cells and affinity purification with nickel packing material) was also labeled and subjected to in vivo imaging.

[0276] Take 200 μL 99m Add TcO4 (pH ~ 5.0) to the ISOLINK KIT kit (21.7 mCi), add 200 μL of physiological saline, and incubate at 100 °C for 30 min; remove 100 μL (4.05 mCi). 99m Tc(H2O)3(CO)3 (pH ~ 12.5) was mixed with 400 μg (150 μL) of 8H9 protein and incubated at 50 °C for 90 min. 100 μL (4.04 mCi) was then collected. 99mTc(H2O)3(CO)3 (pH ~12.5) was mixed with 300 μg (164 μL) of 8H9 protein and incubated at 50 °C for 90 min. After labeling, the protein was purified using a PD10 column, and the purified protein was measured by TLC using PBS as the developing solvent. 99m The labeling efficiency of Tc(H2O)3(CO)3 for B107 and 8H9 was measured. The labeling efficiency for 8H9 was 93%, and the labeling efficiency for B107 was greater than 99%.

[0277] Six female BALBc-Nude nude mice, 6–8 weeks old, were housed in an SPF-barrier environment with free access to food and water. A standard 12-hour day-night light cycle was maintained. 100 μL of MC38-B7H3 / PBS cells were subcutaneously injected into the right axilla of each mouse. The cell seeding density was approximately 5–6 × 10⁶ cells / mL. 5 Cells / mouse. Tumor volume becomes usable after 3-4 weeks, approximately 100-300 mm. 3 .

[0278] Will 99m Tc-B107 / 8H9 test products were administered via tail vein injection to MC38-B7H3 tumor-bearing nude mice, with 3 mice in each group. The dosage of B107 was 802 μCi / 59.5 μg, and the dosage of 8H9 was 786 μCi / 77.6 μg. SPECT / CT imaging was then performed, and the ROI was used to delineate the uptake of different tissues.

[0279] Figure 1 show 99m The uptake of Tc-labeled B7H3 single-domain antibody and positive antibody 8H9 in various organs was analyzed. Both B107 and 8H9 antibodies showed high radioactive concentrations in tumors, with B107 showing higher tumor uptake than 8H9, demonstrating that the selected candidate B7H3 single-domain antibody B107 has high affinity for the B7H3 antigen in vivo. In addition to high tumor uptake, B107 single-domain antibody also showed high radioactive concentrations in the kidneys, indicating... 99m Tc-labeled B7H3 single-domain antibodies are metabolized out of the body through the kidneys, maintaining the typical distribution characteristics of single-domain antibodies.

[0280] 3.2 177 Imaging of Lu-labeled B7H3 single-domain antibody on MC38-B7H3 tumors

[0281] The B107 antibody was replaced with NaHCO3-Na2CO3 buffer (pH 9.5) using a PD-10 column. The SCN-Bn-DOTA solution was added to the B107 antibody at 5 times the molar number of lysine residues on the B107 antibody and mixed well. The reaction was carried out at 37°C for 21 h. After the reaction was completed, the solution was replaced with ultrafiltration and the concentration was determined by measuring the absorbance at 280 nm using a spectrophotometer.

[0282] 250 μg of DOTA-B107 nanobody conjugate was mixed with 100 μL of 0.1 M HCl and 500 μL of 0.25 M sodium acetate, and finally 1.91 mCi was added. 177 LuCl3 solution. React at 45℃ for 1 hour. If the reaction volume is too small, 0.25M sodium acetate can be added to increase the volume of the reaction mixture.

[0283] After 1 hour of reaction, samples were spotted and analyzed by TLC using citric acid as the developing solvent. If the TLC labeling rate was >95%, the sample was released for use. If the labeling rate was <95%, purification was performed using a PD-10 column, saturated with 1% BSA, and eluted with PBS. Radioactivity was measured in 500 μL samples. The radioactive eluates were combined and analyzed by TLC again. If the radiochemical purity was >95%, the sample was released for use. Testing confirmed that the labeling rate of B107 was greater than 99%.

[0284] Three female BALBc-Nude nude mice, 6–8 weeks old, were housed in an SPF-barrier environment with free access to food and water. A standard 12-hour day-night light cycle was maintained. 100 μL of MC38-B7H3 / PBS cells were subcutaneously injected into the right axilla of each mouse. The cell seeding density was approximately 5–6 × 10⁶ cells / mL. 5 Cells / mouse. Tumor volume becomes usable after 3-4 weeks, approximately 100-300 mm. 3 .

[0285] Will 177 Lu-B107 was administered via tail vein injection to MC38-B7H3 tumor-bearing nude mice (n=3 per group). The dose of B107 was 270 μCi / 51.3 μg, and 8 mg of lysine was administered concurrently. SPECT / CT imaging was then performed, and the ROI was used to delineate the uptake in different tissues.

[0286] Figure 2 The images show the imaging results of B107 antibody after 2 hours and its uptake in various tissues and organs. B107 antibody showed significant enrichment in tumors, enhancing its in vivo specific targeting of the B7H3 antigen. High renal uptake suggests... 177 Lu-DOTA-B7H3 nanobody is primarily metabolized by the kidneys.

Claims

1. A B7H3-binding polypeptide comprising at least one immunoglobulin single variable domain specifically binding to B7H3, wherein the at least one immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 in any VHH of any of SEQ ID NO: 5, 13, 17, 21, 25, 29, 33, 49, 53, 57, 61, 65, 69, 73, 77, 81, and 85; The single variable domain of the immunoglobulin mentioned above is VHH; The CDR mentioned therein is a Kabat CDR, AbM CDR, Chothia CDR, or Contact CDR.

2. The B7H3-binding polypeptide of claim 1, wherein the at least one immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 selected from the following: (1) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:7, and CDR3 shown in SEQ ID NO:8; (2) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO:15, and CDR3 shown in SEQ ID NO:16; (3) CDR1 shown in SEQ ID NO:18, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:20; (4) CDR1 shown in SEQ ID NO:22, CDR2 shown in SEQ ID NO:23, and CDR3 shown in SEQ ID NO:24; (5) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:28; (6) CDR1 shown in SEQ ID NO:30, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:32; (7) CDR1 shown in SEQ ID NO:34, CDR2 shown in SEQ ID NO:35, and CDR3 shown in SEQ ID NO:36; (8) CDR1 shown in SEQ ID NO:50, CDR2 shown in SEQ ID NO:51, and CDR3 shown in SEQ ID NO:52; (9) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:55, and CDR3 shown in SEQ ID NO:56; (10) CDR1 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:59, and CDR3 shown in SEQ ID NO:60; (11) CDR1 shown in SEQ ID NO:62, CDR2 shown in SEQ ID NO:63, and CDR3 shown in SEQ ID NO:64; (12) CDR1 shown in SEQ ID NO:66, CDR2 shown in SEQ ID NO:67, and CDR3 shown in SEQ ID NO:68; (13) CDR1 shown in SEQ ID NO:70, CDR2 shown in SEQ ID NO:71, and CDR3 shown in SEQ ID NO:72; (14) CDR1 shown in SEQ ID NO:74, CDR2 shown in SEQ ID NO:75, and CDR3 shown in SEQ ID NO:76; (15) CDR1 shown in SEQ ID NO:78, CDR2 shown in SEQ ID NO:79, and CDR3 shown in SEQ ID NO:80; (16) CDR1 shown in SEQ ID NO:82, CDR2 shown in SEQ ID NO:83, and CDR3 shown in SEQ ID NO:84; and (17) CDR1 shown in SEQ ID NO:86, CDR2 shown in SEQ ID NO:87, and CDR3 shown in SEQ ID NO:

88.

3. The B7H3-binding polypeptide of claim 1, wherein the sequence of the single variable domain of the immunoglobulin is as shown in any one of the amino acid sequences represented by SEQ ID NO: 5, 13, 17, 21, 25, 29, 33, 49, 53, 57, 61, 65, 69, 73, 77, 81 and 85.

4. The B7H3-binding polypeptide of any one of claims 1-3, wherein the immunoglobulin single variable domain is humanized.

5. A nucleic acid molecule encoding the B7H3-binding polypeptide of any one of claims 1-4.

6. An expression vector comprising the nucleic acid molecule of claim 5 operatively linked to an expression regulatory element.

7. A host cell comprising the nucleic acid molecule of claim 5 or transformed with the expression vector of claim 6, and capable of expressing the B7H3 binding polypeptide.

8. A method for generating the B7H3-binding polypeptide of any one of claims 1-4, comprising: a) Culture the host cells of claim 7 under conditions that allow the expression of the B7H3-binding polypeptide; b) Recover the B7H3-binding polypeptide expressed by the host cells from the culture obtained in step a); and c) Further purification and / or modification of the B7H3-binding polypeptide obtained from step b).

9. A conjugated molecule comprising the B7H3-binding polypeptide of any one of claims 1-4, and at least one detectable label conjugated to said B7H3-binding polypeptide.

10. The conjugated molecule of claim 9, wherein The detectable markers are selected from radionuclides, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, and enzymes.

11. The conjugated molecule of claim 10, wherein the radionuclide is selected from... 110 In、 111 In、 177 Lu、 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 68 Ge 86 Y、 90 Y、 89 Zr、 94m Tc, 99m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 32 P, 11 C 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 52m Mn, 55 Co、 72 As、 75 Br、 76 Br、 82 mRb, 83 Sr or other γ-, β-, or positron emitters.

12. The conjugated molecule of claim 10, wherein the detectable marker is 68 Ga or 177 Lu.

13. The conjugated molecule of any one of claims 10-12, wherein the B7H3-binding polypeptide is conjugated to a radionuclide by a chelating agent.

14. The conjugated molecule of claim 13, wherein the chelating agent is selected from DTPA, EDTA, NOA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, or HEHA and their derivatives.

15. The conjugated molecule of claim 14, wherein the detectable marker is 99m Tc, or the radioactive nuclide is 177 Lu and the chelating agent is DOTA.

16. A non-diagnostic method for detecting the presence and / or amount of B7H3 in a biological sample, comprising: a) Under the condition that a complex can be formed between the B7H3-binding polypeptide of any one of claims 1-4 or the conjugate molecule of any one of claims 9-15 and B7H3, the biological sample and the control sample are contacted with the B7H3-binding polypeptide of any one of claims 1-4 or the conjugate molecule of any one of claims 9-15. b) Detect the formation of the complex. The difference in complex formation between the biological sample and the control sample indicates the presence and / or amount of B7H3 in the sample.

17. A diagnostic agent for detecting and / or diagnosing B7H3-related diseases, comprising a B7H3-binding polypeptide of any one of claims 1-4 and / or a conjugated molecule of any one of claims 9-15, and a physiologically acceptable carrier.

18. The diagnostic agent of claim 17, wherein the diagnostic agent is a contrast agent.

19. The diagnostic agent of claim 18, wherein the contrast agent is an ECT contrast agent.

20. The diagnostic agent of claim 19, wherein the ECT contrast agent is a SPECT contrast agent or a PET contrast agent.

21. The diagnostic agent of any one of claims 17-20, wherein the B7H3-related disease is cancer, said cancer being selected from melanoma, leukemia, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, endometrial cancer, oral squamous cell carcinoma, cervical cancer, lung cancer, bladder cancer, clear cell renal cell carcinoma, and glioma.

22. Use of the B7H3-binding polypeptide of any one of claims 1-4 and / or the conjugated molecule of any one of claims 9-15 in the preparation of a diagnostic agent for detecting and / or diagnosing B7H3-related diseases, wherein the B7H3-related diseases are cancers selected from melanoma, leukemia, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, endometrial cancer, oral squamous cell carcinoma, cervical cancer, lung cancer, bladder cancer, clear cell renal cell carcinoma, and glioma.

23. The use of claim 22, wherein the diagnostic agent is a contrast agent.

24. The use of claim 23, wherein the contrast agent is an ECT contrast agent.

25. The use of claim 24, wherein the ECT contrast agent is a SPECT contrast agent or a PET contrast agent.

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