B7h3-binding polypeptide and use thereof

Abstract

The present invention relates to the field of biomedicine. Specifically, the present invention relates to a specifically B7H3-binding polypeptide and a use thereof.

Description

B7H3 binding polypeptides and uses thereof Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a specific B7H3 binding polypeptide and its use.

[0002] Background of the Invention

[0003] B7 homolog 3 (B7H3, also known as CD276) is a type I transmembrane protein encoded by human chromosome 15. B7H3 has a short intracellular tail and no known signaling motifs. B7H3 is ubiquitously expressed across species. Soluble forms of B7H3 can also be detected in human serum, generated by surface cleavage by matrix metallopeptidases (MMPs) or alternative intronic splicing.

[0004] B7H3 is induced on antigen-presenting cells and plays an important role in suppressing T cell function. The B7H3 protein is not expressed or is expressed at very low levels in normal tissues and cells, but is highly expressed in a variety of tumor tissues and is closely associated with tumor progression, patient survival, and prognosis. For example, numerous studies have described overexpression of B7H3 in human malignancies, including melanoma, leukemia, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, and other cancers. Therefore, B7H3 may serve as a new tumor marker and potential therapeutic target. There remains a need in the art for diagnostic and therapeutic agents for B7H3-related diseases, such as cancer.

[0005] Summary of the Invention

[0006] The present invention comprises at least the following embodiments:

[0007] Embodiment 1. A B7H3-binding polypeptide comprising at least one immunoglobulin single variable domain that specifically binds to B7H3, wherein the at least one immunoglobulin single variable domain comprises the 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] Embodiment 2. The B7H3-binding polypeptide of embodiment 1, wherein the at least one immunoglobulin single variable domain comprises a CDR1, a CDR2, and a CDR3 selected from the group consisting of:

[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] Embodiment 3. The B7H3-binding polypeptide of embodiment 1 or 2, wherein the immunoglobulin single variable domain comprises an amino acid sequence that has at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the amino acid sequence shown in 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.

[0032] Embodiment 4. The B7H3-binding polypeptide of any one of embodiments 1-3, wherein the immunoglobulin single variable domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, and 85.

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

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

[0035] Embodiment 7. A nucleic acid molecule encoding the B7H3-binding polypeptide of any one of embodiments 1-6.

[0036] Embodiment 8. An expression vector comprising the nucleic acid molecule of embodiment 7 operably linked to an expression control element.

[0037] Embodiment 9. A host cell comprising the nucleic acid molecule of embodiment 7 or transformed with the expression vector of embodiment 8, and capable of expressing the B7H3-binding polypeptide.

[0038] Embodiment 10. A method of producing the B7H3-binding polypeptide of any one of embodiments 1-6, comprising:

[0039] a) culturing the host cell of embodiment 9 under conditions that allow expression of the B7H3-binding polypeptide;

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

[0041] c) optionally further purifying and / or modifying the B7H3 binding polypeptide obtained in step b).

[0042] Embodiment 11. A conjugate molecule comprising the B7H3-binding polypeptide of any one of embodiments 1-6, and at least one detectable label and / or therapeutic moiety conjugated to the B7H3-binding polypeptide.

[0043] Embodiment 12. The conjugated molecule of embodiment 11, wherein

[0044] The detectable label is selected from the group consisting of a radionuclide, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, and an enzyme; or

[0045] The therapeutic moiety is selected from the group consisting of a radionuclide, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, epipodophyllotoxin glucopyranoside, epipodophyllotoxin teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, a glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine, duocarmycin, calicheamicin, maytansine, auristatin, nitrogen mustard, chlorambucil, melphalan, camoxetine

[00135] In some embodiments, the present invention includes but is not limited to: cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin, bleomycin, anthramycin, abrin, 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.

[0046] Embodiment 13. The conjugate molecule of embodiment 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 gamma-, beta-, or positron emitters, for example, the detectable label is 68 Ga or 177 Lu.

[0047] Embodiment 14. The conjugated molecule of any one of embodiments 12-13, wherein the B7H3 binding polypeptide is conjugated to the radionuclide via a chelating agent.

[0048] Embodiment 15. The conjugate molecule of embodiment 14, wherein the chelator is selected from DTPA, EDTA, NOTA, 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 derivatives thereof.

[0049] Embodiment 16. The conjugate molecule of embodiment 15, wherein the detectable label is 99m Tc, or the radionuclide is 177 Lu and the chelating agent is DOTA.

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

[0051] a) contacting the biological sample and the control sample with the B7H3-binding polypeptide of any one of embodiments 1-6 or the conjugated molecule of any one of embodiments 11-16 under conditions capable of forming a complex between the B7H3-binding polypeptide of any one of embodiments 1-6 or the conjugated molecule of any one of embodiments 11-16 and B7H3;

[0052] b) detecting the formation of a complex,

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

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

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

[0056] Embodiment 20. The diagnostic agent of embodiment 19, which is a contrast agent.

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

[0058] Embodiment 22. Use of the B7H3-binding polypeptide of any one of embodiments 1-6 and / or the conjugated molecule of any one of embodiments 11-16 in the preparation of a diagnostic agent for detecting and / or diagnosing a B7H3-related disease, such as cancer.

[0059] Embodiment 23. The use of embodiment 22, wherein the diagnostic agent is a contrast agent.

[0060] Embodiment 24. The use according to embodiment 23, which is a contrast agent.

[0061] Embodiment 25. The use of embodiment 24, wherein the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.

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

[0063] Embodiment 27. The method of embodiment 26 further comprises the step of imaging the subject, such as ECT imaging.

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

[0065] Embodiment 29. The diagnostic agent of any one of embodiments 18-21, the use of any one of embodiments 22-25, or the method of any one of embodiments 26-28, wherein the B7H3-related disease is cancer, e.g., the cancer is 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 intrinsic pontine glioma (DIPG)).

[0066] BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0069] Detailed Description of the Invention

[0070] definition

[0071] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which will be understood by those skilled in the art. Reference is made, for example, to 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), as well as the general prior art cited herein; in addition, unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can and have been carried out in a manner known per se, which will be understood by those skilled in the art. Reference is also made, for example, to standard manuals, the above-mentioned general prior art and other references cited therein.

[0072] Unless otherwise indicated, the terms "antibody" or "immunoglobulin" used interchangeably herein, whether referring to heavy chain antibodies or conventional four-chain antibodies, are used as general terms to include full-length antibodies, their individual chains, and all parts, domains, or fragments thereof (including but not limited to antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively). In addition, the term "sequence" used herein (e.g., in terms such as "immunoglobulin sequence," "antibody sequence," "single variable domain sequence," "VHH sequence," or "protein sequence") is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.

[0073] As used herein, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed, or transferred to other proteins without losing the function of the rest of the protein and / or the domain.

[0074] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional 4-chain antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a globular region. An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristic of an antibody molecule.

[0075] As used herein, the term "immunoglobulin variable domain" refers to an immunoglobulin domain that essentially consists of four "framework regions," referred to in the art and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, wherein the framework 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. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity to an antibody for an antigen by having an antigen-binding site.

[0076] As used herein, the term "immunoglobulin single variable domain" refers to an immunoglobulin variable domain that is capable of specifically binding to an antigenic epitope without being paired with other immunoglobulin variable domains. One example of an immunoglobulin single variable domain within the meaning of the present 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 a "VHH domain" (or simply "VHH") of Camelidae, as defined below.

[0077] "VHH domain", also known as heavy chain single domain antibody, VHH, VHH domain, VHH antibody fragment and VHH antibody, is the variable domain of an antigen-binding immunoglobulin called a "heavy chain antibody" (i.e., an "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 the variable domain from the heavy chain variable domain present in conventional four-chain antibodies (which is referred to herein as a "VH domain") and the light chain variable domain present in conventional four-chain antibodies (which is referred to herein as a "VL domain"). The VHH domain specifically binds an epitope without the need for additional antigen-binding domains (in contrast to the VH or VL domains in conventional four-chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and efficient antigen-recognition unit formed by a single immunoglobulin domain.

[0078] In the context of the present 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, as shown in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999), the amino acid residues used for the VHH domain of Camelidae can be numbered according to the general numbering method for 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 the amino acid residues of VH domains are known in the art and can be similarly applied to VHH domains. For example, Chothia CDRs refer to the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM CDRs represent a compromise between the Kabat hypervariable regions and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. "Contact" CDRs are based on analysis of available complex crystal structures. The residues of the CDRs from each method are described below:

[0081] It should be noted, however, that, as is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

[0082] For example, the CDRs may comprise "extended CDRs", e.g., 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.

[0083] The total number of amino acid residues in a VHH domain will generally range from 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.

[0084] Other structural characteristics and functional properties of VHH domains and polypeptides containing them can be summarized as follows: VHH domains (which have been naturally "designed" to functionally bind to antigens in the absence of, and without interacting with, light chain variable domains) can be used as single, relatively small, functional antigen-binding structural units, domains, or polypeptides. This distinguishes VHH domains from the VH and VL domains of conventional four-chain antibodies, which are generally unsuitable for practical application as single antigen-binding proteins or immunoglobulin single variable domains on their own, 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 scFvs consisting of a VH domain covalently linked to a VL domain).

[0085] Due to these unique properties, the use of VHH domains—alone or as part of a larger polypeptide—offers a number of significant advantages over the use of conventional VH and VL domains, scFvs, or conventional antibody fragments (e.g., Fab- or F(ab')2-fragments): only a single domain is required to bind antigen with high affinity and selectivity, thereby eliminating the need for the presence of two separate domains and the need to ensure that the two domains are in the proper spatial conformation and configuration (e.g., scFvs generally require the use of specially designed linkers); VHH domains can be expressed from a single gene and do not require post-translational folding or modification; VHH domains can be easily engineered into multivalent and multispecific formats (formatting); VHH domains are highly soluble and have no tendency to aggregate; VHH domains They are 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, thereby saving cost, time, and the environment; VHH domains are easy to prepare and relatively inexpensive, even at the scale required for production; VHH domains are relatively small compared to conventional four-chain antibodies and antigen-binding fragments thereof (approximately 15 kDa, or 1 / 10 the size of conventional IgG), and therefore exhibit higher tissue penetration and can be administered at higher doses than conventional four-chain antibodies and antigen-binding fragments thereof; VHH domains can exhibit so-called cavity-binding properties (particularly due to their extended CDR3 loops compared to conventional VH domains), thereby being able to access targets and epitopes that are inaccessible to conventional four-chain antibodies and antigen-binding fragments thereof.

[0086] 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., J Biol Chem., 287 (2012) 13713–13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645-2652, 17 June, 2009 and WO94 / 04678.

[0087] Camelidae-derived VHH domains can be "humanized" (also referred to herein as "sequence optimization"; in addition to humanization, "sequence optimization" may also encompass other modifications to the sequence by one or more mutations that provide improved properties of the VHH, such as removal of potential post-translational modification sites) by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in a conventional human four-chain antibody VH domain. The humanized VHH domain may contain one or more fully human framework region sequences. Humanization can be achieved using methods such as protein surface amino acid resurfacing and / or humanized universal framework CDR grafting.

[0088] As used herein, the term "epitope" or the interchangeable term "antigenic determinant" refers to any antigenic determinant on the antigen to which the paratope of an antibody is bound. Antigenic determinants typically comprise chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics and specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or non-continuous amino acids in a unique spatial conformation, which can be a "linear" epitope or a "conformational" epitope. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all interacting points between a protein and an interacting molecule (such as an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interacting points exist across separate protein amino acid residues.

[0089] Many epitope mapping techniques well known in the art can be used to identify the epitope of a given antigen. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). For example, linear epitopes can be determined by, for example, the following method: a large number of peptides are synthesized simultaneously on a solid support, wherein these peptides correspond to various parts of the protein molecule, and these peptides are reacted with antibodies while still attached to the support. These techniques are known in the art and are described in, for example, U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81: 3998-4002; 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 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols (supra).

[0090] Conventional techniques known to those skilled in the art can be used to competitively screen antibodies for binding to the same epitope. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for antigen binding. High-throughput methods for obtaining antibodies that bind to the same epitope based on their cross-competition are described in International Patent Application WO 03 / 48731. Thus, conventional techniques known to those skilled in the art can be used to obtain antibodies and antigen-binding fragments thereof that compete with the antibody molecules of the present invention for binding to the same epitope on B7H3.

[0091] In general, 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., an immunoglobulin single variable domain of the present invention) can bind to. Specificity can be determined based on the affinity and / or avidity of an antigen binding protein. Avidity, represented by the dissociation equilibrium constant (KD) of an antigen and an antigen binding protein, is a measure of the binding strength between an epitope and an antigen binding site on an antigen binding protein: the smaller the KD value, the stronger the binding strength between the epitope and the antigen binding protein (or, affinity can also be expressed as an association constant (KA), which is 1 / KD). As will be appreciated by those skilled in the art, affinity can be measured in a known manner depending on the specific antigen of interest. Avidity is a measure of the binding strength between an antigen binding protein (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing the same) and a related antigen. Avidity is related to both the affinity between the antigen binding site on its antigen binding protein and the number of related binding sites present on the antigen binding protein.

[0092] As used herein, the term "B7H3 binding protein (B7H3 binding polypeptide or B7H3 binding molecule)" means any protein that can specifically bind to a B7H3 protein. B7H3 binding proteins can include antibodies against B7H3, such as antibodies as defined herein. B7H3 binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules. An exemplary amino acid sequence of B7H3 is shown in SEQ ID NO: 111.

[0093] The "B7H3 binding proteins" of the present invention may comprise at least one immunoglobulin single variable domain, such as a VHH, that binds to B7H3. In some embodiments, the "B7H3 binding molecules" of the present invention may comprise 2, 3, 4, or more immunoglobulin single variable domains, such as a VHH, that bind to B7H3. The B7H3 binding proteins of the present invention may also comprise, in addition to the immunoglobulin single variable domain that binds to B7H3, a linker and / or a portion having effector function, such as a half-life extending portion (such as an immunoglobulin single variable domain that binds to serum albumin), and / or a fusion partner (such as serum albumin), and / or a conjugated polymer (such as PEG), and / or an Fc region. In some embodiments, the "B7H3 binding proteins" of the present invention also encompass bispecific antibodies, which contain immunoglobulin single variable domains that bind to different antigens.

[0094] Typically, the B7H3 binding proteins of the invention will be expressed as preferably 10 -7 to 10 -10 Mole / liter (M), more preferably 10 -8 to 10 -10 mol / L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (KD), and / or with a dissociation constant of 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) of the protein binds to the antigen (i.e., B7H3 protein). -4 KD values ​​of M are generally considered to indicate nonspecific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be determined in any suitable manner known in the art, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays.

[0095] Amino acid residues will be represented according to the standard three-letter or one-letter amino acid code as is well 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 another sequence. In the case of substitutions, the substitution will preferably be a conservative amino acid substitution, which refers to the replacement of an amino acid residue with another amino acid residue of similar chemical structure and which has little or substantially no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art. For example, conservative amino acid substitutions are preferably substitutions of an amino acid within the following groups (i) to (v) by another amino acid residue within the same group: (i) smaller aliphatic non-polar 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 non-polar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative 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.

[0096] "Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative 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 example, the BLAST program from the NCBI database can be used to determine identity. For the determination of 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.

[0097] A polypeptide or nucleic acid molecule is considered "isolated" when it has been separated from at least one other component with which it is normally associated in that source or medium (e.g., another protein / polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or trace component) compared to its natural biological source and / or the reaction medium or culture medium from which it was obtained. In particular, 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. An "isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous as determined by a suitable technique (e.g., a suitable chromatographic technique, such as polyacrylamide gel electrophoresis).

[0098] As used herein, the term "subject" means a mammal, particularly a primate, especially a human.

[0099] B7H3-binding polypeptides of the present invention

[0100] The present invention provides a B7H3-binding polypeptide comprising at least one immunoglobulin single variable domain that specifically binds to B7H3. In some embodiments, the B7H3-binding polypeptide is isolated. In some embodiments, the B7H3-binding polypeptide specifically binds to B7H3.

[0101] In some embodiments, the at least one immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of a VHH as set forth in any one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, and 85. The CDRs may be Kabat CDRs, AbM CDRs, Chothia CDRs, or Contact CDRs. In some embodiments, the CDRs are Kabat CDRs.

[0102] In some embodiments, the at least one immunoglobulin single variable domain comprises a CDR1, CDR2, and CDR3 selected from:

[0103] (1) CDR1 shown in SEQ ID NO: 2, CDR2 shown in SEQ ID NO: 3, and CDR3 shown in SEQ ID NO: 4;

[0104] (2) CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 8;

[0105] (3) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12;

[0106] (4) CDR1 shown in SEQ ID NO: 14, CDR2 shown in SEQ ID NO: 15, and CDR3 shown in SEQ ID NO: 16;

[0107] (5) CDR1 shown in SEQ ID NO: 18, CDR2 shown in SEQ ID NO: 19, and CDR3 shown in SEQ ID NO: 20;

[0108] (6) CDR1 shown in SEQ ID NO: 22, CDR2 shown in SEQ ID NO: 23, and CDR3 shown in SEQ ID NO: 24;

[0109] (7) CDR1 shown in SEQ ID NO: 26, CDR2 shown in SEQ ID NO: 27, and CDR3 shown in SEQ ID NO: 28;

[0110] (8) CDR1 shown in SEQ ID NO: 30, CDR2 shown in SEQ ID NO: 31, and CDR3 shown in SEQ ID NO: 32;

[0111] (9) CDR1 shown in SEQ ID NO: 34, CDR2 shown in SEQ ID NO: 35, and CDR3 shown in SEQ ID NO: 36;

[0112] (10) CDR1 shown in SEQ ID NO: 38, CDR2 shown in SEQ ID NO: 39, and CDR3 shown in SEQ ID NO: 40;

[0113] (11) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:43, and CDR3 shown in SEQ ID NO:44;

[0114] (12) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:48;

[0115] (13) CDR1 shown in SEQ ID NO: 50, CDR2 shown in SEQ ID NO: 51, and CDR3 shown in SEQ ID NO: 52;

[0116] (14) CDR1 shown in SEQ ID NO: 54, CDR2 shown in SEQ ID NO: 55, and CDR3 shown in SEQ ID NO: 56;

[0117] (15) CDR1 shown in SEQ ID NO: 58, CDR2 shown in SEQ ID NO: 59, and CDR3 shown in SEQ ID NO: 60;

[0118] (16) CDR1 shown in SEQ ID NO: 62, CDR2 shown in SEQ ID NO: 63, and CDR3 shown in SEQ ID NO: 64;

[0119] (17) CDR1 shown in SEQ ID NO: 66, CDR2 shown in SEQ ID NO: 67, and CDR3 shown in SEQ ID NO: 68;

[0120] (18) CDR1 shown in SEQ ID NO: 70, CDR2 shown in SEQ ID NO: 71, and CDR3 shown in SEQ ID NO: 72;

[0121] (19) CDR1 shown in SEQ ID NO: 74, CDR2 shown in SEQ ID NO: 75, and CDR3 shown in SEQ ID NO: 76;

[0122] (20) CDR1 shown in SEQ ID NO: 78, CDR2 shown in SEQ ID NO: 79, and CDR3 shown in SEQ ID NO: 80;

[0123] (21) CDR1 shown in SEQ ID NO: 82, CDR2 shown in SEQ ID NO: 83, and CDR3 shown in SEQ ID NO: 84; and

[0124] (22) CDR1 shown in SEQ ID NO:86, CDR2 shown in SEQ ID NO:87, and CDR3 shown in SEQ ID NO:88.

[0125] In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence that has at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 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 that has at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, and 85.

[0126] In some embodiments, the immunoglobulin single variable domain is a VHH. In some embodiments, the immunoglobulin single variable domain is humanized.

[0127] Nucleic acids, vectors, host cells

[0128] In another aspect, the present invention relates to nucleic acid molecules encoding the B7H3-binding polypeptides of the present invention. The nucleic acids of the present invention may be RNA, DNA, or cDNA. A person skilled in the art can select a nucleic acid molecule encoding the B7H3-binding polypeptide of the present invention as needed or by conventional means. In some embodiments, the nucleic acid molecule encoding the B7H3-binding polypeptide of the present invention comprises a nucleotide sequence selected from SEQ ID NOs: 89-110.

[0129] The nucleic acids of the present invention may also be in the form of, present in, and / or be part of, a vector, such as a plasmid, cosmid, or YAC. The vector may particularly be an expression vector, i.e., a vector that provides for expression of a B7H3-binding polypeptide in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). The expression vector typically comprises at least one nucleic acid of the present invention, operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the skill of the art. Specific examples of regulatory elements and other elements useful or necessary for expression of a B7H3-binding polypeptide of the present invention include, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.

[0130] The nucleic acids of the invention can be prepared or obtained by known means (e.g. by automated DNA synthesis and / or recombinant DNA technology) based on the information on the amino acid sequences of the polypeptides of the invention given herein, and / or can be isolated from suitable natural sources.

[0131] In another aspect, the present invention relates to a host cell that expresses or is capable of expressing one or more B7H3-binding polypeptides of the invention and / or contains a nucleic acid or vector of the invention. Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.

[0132] Suitable bacterial cells include cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0133] Suitable fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0134] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0135] However, the present invention can also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0136] The present invention also provides methods for producing the B7H3-binding polypeptides of the present invention, which generally comprise the following steps:

[0137] - culturing the host cell of the invention under conditions that allow expression of the B7H3-binding polypeptide of the invention; and

[0138] - recovering the B7H3-binding polypeptide expressed by the host cell from the culture; and

[0139] - Optionally further purifying and / or modifying the B7H3-binding polypeptides of the invention.

[0140] The B7H3-binding polypeptides of the present invention can be produced intracellularly in the cells described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified.

[0141] Methods and reagents for recombinant production of polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc., are known in the art. Similarly, protein isolation and purification techniques suitable for use in methods for producing the B7H3-binding polypeptides of the present invention are well known to those skilled in the art.

[0142] However, the B7H3-binding polypeptides of the present invention can also be obtained by other methods known in the art for producing proteins, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0143] Conjugated molecules

[0144] In another aspect, the present invention provides a conjugate molecule comprising a B7H3-binding polypeptide of the present invention and at least one detectable label and / or therapeutic moiety conjugated to the B7H3-binding polypeptide.

[0145] Such detectable labels include, but are not limited to, radionuclides, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, and enzymes.

[0146] Fluorescent agents that can be used for conjugation include, but are not limited to, fluorescein isothionine, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine; chemiluminescent agents that can be used for conjugation include, but are not limited to, luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters; bioluminescent agents that can be used for conjugation include, but are not limited to, luciferin, luciferase, and aequorin. Paramagnetic ions that can be used for conjugation include, but are not limited to, chromium (III), manganese (II), iron (III), iron (II), 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 radiopaque materials such as dam, diatrizoate, ethiodized oil, gallium citrate, iocaproic acid, iodamide, cholinesterase, iodoxic acid, iodine, iohexol, iopamidol, iopamidol, iopamidol, iodopamine, ioproxil, iodoxic acid, iodine, iodinesulfonamide, iothalamic acid, iotral acid, iotral acid, iodoxic acid, iodine, hydroxydiatrizoate, iopate, meglumine, meglumine, meglumine, meglumine, propiodine and thallium oxide. Enzymes that can be used for conjugation include, but are not limited to, horseradish peroxidase and the like.

[0147] Preferably, the detectable label is a radionuclide. Some radionuclides that can be used for conjugation are radionuclides with energies between 20-4000 KeV, including but not limited to 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 gamma-, beta-, or positron emitters. In some embodiments, the detectable label is 68 Ga or 177 Lu.

[0148] Methods for conjugating detectable labels and / or therapeutic moieties to polypeptides are well known to those skilled in the art. For example, in some embodiments, the B7H3-binding polypeptide can be conjugated to the detectable label via a chelating agent.

[0149] In order to use radionuclides such as 68To label a B7H3-binding polypeptide of the invention with Ga, it is necessary to react the B7H3-binding polypeptide of the invention with a reagent having a long tail to which are attached multiple incorporating groups for binding ions. Such a tail can be, for example, polylysine, a polysaccharide, or other polymer having a derivatized or derivatizable chain with pendant groups that can bind to chelating groups such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid), NOTA, TETA, NETA, porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and similar groups known for this purpose. The chelating agent is attached to the antibody using standard chemical methods. In some embodiments, the detectable label is conjugated to the B7H3-binding polypeptide of the invention via the chelating agent. The chelating agents used include, but are not limited to, DTPA, EDTA, NOTA, 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.

[0150] In some embodiments, wherein the detectable label is 99m In some embodiments, wherein the detectable label is 177 Lu, and the chelating agent is DOTA.

[0151] In another aspect, the present invention provides a method for preparing a radionuclide of the present invention such as 99m Tc or 177 The method for producing a Lu-labeled conjugate molecule comprises 1) conjugating a B7H3-binding polypeptide of the present invention to a chelating agent to generate a conjugate of the B7H3-binding polypeptide and the chelating agent; and 2) conjugating the product of step 1) to a radionuclide such as 99m Tc or 177 Lu contact, whereby radionuclides such as 99m Tc or 177 Lu labels the B7H3-binding polypeptide of the present invention through chelation with a chelating agent. In some embodiments, the chelating agent is NOTA, and in step 1), a conjugate of the B7H3-binding polypeptide and NOTA is generated by reacting the B7H3-binding polypeptide with p-SCN-Bn-NOTA or p-NH2-Bn-NOTA. In some embodiments, the chelating agent is DOTA, and in step 1), a conjugate of the B7H3-binding polypeptide and DOTA is generated by reacting the B7H3-binding polypeptide with SCN-Bn-DOTA.

[0152] In some embodiments, the therapeutic moiety includes but is not limited to paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, epipodophyllotoxin glucopyranoside, epipodophyllotoxin teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine, duocarmycin, calicheamicin, maytansine, auristatin, nitrogen mustard, chlorambucil, melphalan , carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin, bleomycin, anthramycin, abrin, ricin A, Pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor, interferon-γ, lymphokine, 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 moiety can be a radionuclide as described above.

[0153] Detection / diagnostic use

[0154] In another aspect, the present invention provides a method for detecting the presence and / or amount of B7H3 in a biological sample, comprising:

[0155] a) contacting the biological sample and the control sample with a B7H3-binding polypeptide of the invention or a conjugated molecule of the invention under conditions allowing formation of a complex between the B7H3-binding polypeptide of the invention or the conjugated molecule of the invention and B7H3;

[0156] b) detecting the formation of a complex,

[0157] wherein 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.

[0158] In another aspect, the present invention provides a composition comprising a B7H3-binding polypeptide of the present invention and / or a conjugated molecule of the present invention, and optionally a physiologically acceptable carrier. The composition can be used as a detection agent or diagnostic agent, for example, a diagnostic agent for detecting and / or diagnosing a B7H3-related disease.

[0159] In another aspect, the present invention provides a diagnostic agent for detecting and / or diagnosing a B7H3-related disease, such as cancer, comprising a B7H3-binding polypeptide of the present invention and / or a conjugated molecule of the present invention, and optionally a physiologically acceptable carrier. In some embodiments, the diagnostic agent is a contrast agent.

[0160] The B7H3-binding polypeptides and / or conjugate molecules of the present invention are particularly suitable for in vivo imaging, for example, for emission computed tomography (ECT). For example, the B7H3-binding polypeptides and / or conjugate molecules of the present invention can be used in single-photon emission computed tomography (SPECT) and positron emission tomography (PET) depending on the labeling. High-resolution tumor imaging can be provided in tumor diagnosis, and quantitative analysis can be performed using the images. The SPECT imaging can also include SPECT / CT imaging, and the PET imaging can also include PET / CT imaging, which can provide even better imaging results.

[0161] Thus, in some embodiments, the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.

[0162] In another aspect, the present invention provides a use of a B7H3-binding polypeptide of the present invention and / or a conjugate molecule of the present invention in the preparation of a diagnostic agent for detecting and / or diagnosing a B7H3-related disease, 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.

[0163] In another aspect, the present invention provides a method for detecting and / or diagnosing a B7H3-associated disease, such as cancer, in a subject, comprising administering to the subject a B7H3-binding polypeptide of the invention and / or a conjugate molecule of the invention and / or a diagnostic agent of the invention.

[0164] In some embodiments, the method further comprises the step of imaging the subject, 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 devices for scanning by SPECT or PET are well known in the art, and any such known ECT imaging techniques and devices may be used.

[0165] Diseases that can be detected and / or diagnosed by the B7H3-binding polypeptides of the present invention and / or the conjugated molecules of the present invention and / or the diagnostic agents of the present invention include diseases in which B7H3 expression in cells, tissues or organs is abnormally elevated, such as cancer.

[0166] Herein, B7H3-related diseases include, but are not limited to, cancer, for example, the cancer can be 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 intrinsic pontine glioma (DIPG)).

[0167] Pharmaceutical compositions and therapeutic uses

[0168] In another aspect, the present invention provides a pharmaceutical composition comprising a 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 a B7H3-associated disease. The B7H3-associated disease is as described above, preferably cancer.

[0169] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0170] In another aspect, the present invention provides use of a B7H3-binding polypeptide or conjugate molecule of the present invention or a pharmaceutical composition of the present invention in the preparation of a medicament for treating a B7H3-related disease, wherein the B7H3-related disease is as described above, and is preferably cancer.

[0171] In another aspect, the present invention provides a method for treating a B7H3-associated disease in a subject, comprising administering to the subject a therapeutically effective amount of a B7H3-binding polypeptide or conjugate molecule of the present invention or a pharmaceutical composition of the present invention. The B7H3-associated disease is as described above, preferably cancer.

[0172] Reagent test kit

[0173] In another aspect, the present invention provides a kit comprising a B7H3-binding polypeptide of the present invention, a conjugated molecule of the present invention, or a diagnostic agent of the present invention. The kit is used to perform 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 associated with the kit. Example

[0174] Example 1. Antibody screening and construction

[0175] 1.1 Library Construction

[0176] The B7H3-cHis fusion protein used for immunization was expressed in 293F cells and purified by nickel affinity chromatography. A healthy alpaca was selected for immunization. After immunization, peripheral blood was collected from the alpaca, PBMCs were isolated according to the instructions of the lymphocyte separation solution, and RNA was extracted using RNAiso Plus reagent. PrimeScript TM The extracted RNA was reverse transcribed into cDNA using II 1st Strand cDNA Synthesis Kit (Takara, Cat. No. 6210A), and the nucleic acid fragment encoding the variable region of the heavy chain antibody was amplified by nested PCR.

[0177] The VHH fragment was purified using a DNA product purification kit. The vector and fragment were digested with the restriction endonuclease SfiI at 50°C overnight. The digested fragments were recovered by gel excision and cloned into the phage display vector pComb3XSS. The product was then electroporated into electrocompetent E. coli TG1 cells to construct and assay a phage display library of heavy chain single-domain antibodies targeting B7H3. The library was then assayed by serial dilution plating, and the library size was calculated to be 2.36×10 13 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.

[0178] 1.2 Selection of heavy chain single domain antibodies targeting B7H3

[0179] The B7H3-cHis fusion protein was diluted with carbonate buffer at pH 9.6 to a final concentration of 5 μg / mL, and 100 μL / well was added to the ELISA plate and placed at 4°C overnight. The next day, 300 μL / well of 3% BSA-PBS blocking solution was used to block the plate at 37°C for 1 hour. After washing the plate three times with PBS, 100 μL of phage (2×10 11cfu, from the heavy chain single domain antibody phage display library constructed in 1.1), incubated at 37°C for 1 hour. Then wash 6 times with PBST (PBS containing 0.05% Tween 20) and then wash twice with PBS solution to wash off unbound phages. Then add 100 μL of Gly-Hcl (PH = 2.5) to each well and act at 37°C for 8 minutes to dissociate the phages that specifically bind to B7H3 and transfer them to a sterile centrifuge tube, to which 10 μL of Tris-Hcl (PH = 9.0) solution is quickly added to neutralize the buffer. Take 10 μL for gradient dilution, determine the titer and calculate the panning recovery rate, and take the neutralized phage to infect Escherichia coli TG1 in the logarithmic phase of growth to produce and purify phages for the next round of screening. The same screening process is repeated for several rounds, and the panning conditions need to be changed for each round of panning. 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 conditions for affinity panning, and Table 2 shows the recovery rate and enrichment of the two rounds of screening.

[0180] Table 1 Affinity panning conditions

[0181] Table 2 Recovery of one round of acid elution screening using B7H3 antigen as target molecule

[0182] 1.3. Screening of specific single positive clones using phage enzyme-linked immunosorbent assay (ELISA)

[0183] After two rounds of panning, 192 colonies were randomly selected from the panning plates for identification. Each of the 192 randomly selected colonies was cultured, and phage was produced and purified. The B7H3-cHis fusion protein was diluted to a final concentration of 2 μg / mL in carbonate buffer (pH 9.6). 100 μL / well was added to an ELISA plate and coated overnight at 4°C. The plate was blocked with 5% skim milk and incubated at 37°C for 1 hour. Subsequently, 50 μL of phage culture supernatant and 50 μL of 5% skim milk were added to each well and incubated at 37°C for 1 hour. After washing with PBST, horseradish peroxide-conjugated anti-M13 secondary antibody (purchased from Beijing Sino-Biotech Co., Ltd., diluted 1:10,000 in PBS) was added and incubated at 37°C for 1 hour. After washing, TMB colorimetric solution was added, and absorbance was read at 450 nm. Based on the ELISA results, 316 of the 372 colonies were positive, which were then sequenced and analyzed. Among them, 13 clones were non-monoclonal.

[0184] Based on the results of a phage ELISA with the B7H3-cHis antigen, clones with the best binding to the antigen protein were selected. The protein sequences of each clone were analyzed using the sequence alignment software DNAMAN. Clones with identical CDR1, CDR2, and CDR3 sequences were considered the same antibody strain, while clones with different CDR sequences were considered different. Ultimately, 22 candidate antibodies were selected for subsequent experiments. The specific antibody names and amino acid sequences are shown in Table 3 below.

[0185] 1.4 Preparation of B7H3 Antibody Protein Using Mammalian Cells

[0186] Primers were designed based on the nucleotide sequences of the screened B7H3 single-domain antibodies. Using the plasmid as a template, the nucleotide sequences of each antibody (including the signal peptide and His tag) 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 the PEI (Polyethylenimine) solution required for transformation was added. The plasmid / PEI mixture was added to the HEK293 cell suspension and cultured at 37°C, 10% CO2, and 90 rpm. Four hours later, EX293 medium, 2mM glutamine, and cultured at 135 rpm. After 24 hours, 3.8mM VPA was added. After 6-7 days of culture, the transient expression culture supernatant was collected and purified using a nickel column. Finally, an antibody protein with a purity of more than 90% was obtained.

[0187] Table 3 Screened B7H3 antibodies and their amino acid sequences

[0188] Nucleotide sequence of the B115-cHis antibody (SEQ ID NO: 89):

[0189] Nucleotide sequence of B149-cHis antibody (SEQ ID NO: 90):

[0190] Nucleotide sequence of B65-cHis antibody (SEQ ID NO: 91):

[0191] Nucleotide sequence of B143-cHis antibody (SEQ ID NO: 92):

[0192] Nucleotide sequence of B179-cHis antibody (SEQ ID NO: 93):

[0193] Nucleotide sequence of B75-cHis antibody (SEQ ID NO: 94):

[0194] Nucleotide sequence of B107-cHis antibody (SEQ ID NO: 95):

[0195] Nucleotide sequence of B108-cHis antibody (SEQ ID NO: 96):

[0196] Nucleotide sequence of B106-cHis antibody (SEQ ID NO: 97):

[0197] Nucleotide sequence of the B134-cHis antibody (SEQ ID NO: 98):

[0198] Nucleotide sequence of B154-cHis antibody (SEQ ID NO: 99):

[0199] Nucleotide sequence of B85-cHis antibody (SEQ ID NO: 100):

[0200] Nucleotide sequence of B22-cHis antibody (SEQ ID NO: 101):

[0201] Nucleotide sequence of B2-41-cHis antibody (SEQ ID NO: 102):

[0202] Nucleotide sequence of B2-39-cHis antibody (SEQ ID NO: 103):

[0203] Nucleotide sequence of B2-22-cHis antibody (SEQ ID NO: 104):

[0204] Nucleotide sequence of B4-165-cHis (SEQ ID NO: 105):

[0205] Nucleotide sequence of B4-148-cHis (SEQ ID NO: 106):

[0206] Nucleotide sequence of B2-124-cHis antibody (SEQ ID NO: 107):

[0207] Nucleotide sequence of B6-cHis antibody (SEQ ID NO: 108):

[0208] Nucleotide sequence of B38-cHis antibody (SEQ ID NO: 109):

[0209] Nucleotide sequence of B133-cHis antibody (SEQ ID NO: 110):

[0210] Note: The underlined part is the nucleotide sequence of the target antibody

[0211] Example 2: In vitro verification of B7H3 antibody function

[0212] The B7H3-cHis fusion protein was constructed for detection. The B7H3-cHis fusion protein was transiently expressed in HEK293 cells and affinity purified using nickel filler.

[0213] The amino acid sequence of the B7H3-cHis fusion protein is (SEQ ID NO: 111):

[0214] Note: The underlined characters are signal peptide and His tag

[0215] 2.1. Antibody expression

[0216] Of the 22 antibodies, 5 were not expressed, while 17 were expressed. Expression level calculation: The expression level was calculated based on the total amount of target protein obtained after one-step affinity chromatography purification.

[0217] Table 4. Expression of 17 antibodies

[0218] From the expression level test results, it can be seen that the expression level of most antibodies after one-step affinity purification is around 30 mg / L, and the expression level of some antibodies can reach around 90 mg / L. The corresponding protein can be provided for subsequent testing through transient transfection.

[0219] 2.2 Detection of affinity of B7H3 heavy chain single domain antibody to human B7H3 protein

[0220] The B7H3-cHis fusion protein was coated on the plate at 5 μg / mL, 100 μL per well, at 4°C overnight, and after washing, 3% BSA was added for blocking and incubated at 37°C for 1 hour. The antibody to be tested (the heavy chain single domain antibody obtained in Example 1.4) was diluted 4-fold with a starting concentration of 10 μg / mL, and a total of 10 concentrations were diluted. 100 μL was added to each well and reacted at 37°C for 1 hour. After washing, anti-His (HRP) secondary antibody (1:5000 LOT#GR3248851-4) was added and reacted at 37°C for 1 hour. After washing, TMB colorimetric solution was added and the absorbance was read at a wavelength of 450 nm. The software SotfMaxPro v5.4 was used for data processing and graphical analysis, and the antibody binding curve and EC were obtained by four-parameter fitting. 50 In the 17 selected series, the relative activities of different B7H3 heavy chain single domain antibodies were compared with B107-cHis as the control to reflect the affinity of the candidate antibodies to B7H3.

[0221] Table 5 ELISA results of 17 antibodies binding to B7H3

[0222] From the test results in Table 5, it can be seen that some antibodies bind well to the B7H3 antigen. Among the 17 antibodies, B107, B2-124, B2-41, and B38 have good affinity for the B7H3 antigen. Subsequent cell-based assays such as FACS will be used to further verify the binding of the candidate antibodies to B7H3.

[0223] 2.3 ELISA activity screening using alpaca secondary antibody

[0224] The B7H3-cHis fusion protein was coated on the plate at 5 μg / mL, 100 μL per well, and incubated at 4°C overnight. After washing, 3% BSA was added for blocking and incubation 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 the plate, 100 μL was added to each well, and the plate was reacted at 37°C for 1 hour. After washing, anti-camile (HRP) secondary antibody (1:10000 LOT#18E001484) was added and the plate was reacted at 37°C for 1 hour. After washing, TMB colorimetric solution was added and the absorbance was read at a wavelength of 450 nm. The test results are shown in Table 6.

[0225] Table 6 Results of ELISA activity screening of 17 antibodies against alpaca secondary antibodies

[0226] From the test results in Table 6, it can be seen that among the 17 antibodies, 16 antibodies can bind to the alpaca secondary antibody, and 1 antibody binds poorly to the alpaca secondary antibody.

[0227] 2.4 FACS analysis of the binding of B7H3 heavy chain single domain antibody to cell surface B7H3

[0228] A-204 (human rhabdomyosarcoma) and NCI-H322 (human non-small cell lung cancer) cells both express B7H3 antigen. Cells were cultured and collected, and their density was adjusted to 1×10 6 Cells were plated at a concentration of 100 cells / mL. A 100 μL cell aliquot was mixed with 5 μg of primary antibody and incubated on ice for 30 minutes. The cells were washed by centrifugation, resuspended, and 2 μL of Anti-His-PE conjugate was added. The cells were incubated on ice for 30 minutes in the dark. The cells were washed twice with 300 μL of 1× PBS + 0.5% BSA, resuspended, and analyzed on a flow cytometer.

[0229] Eight antibodies with better activity were screened from the above antibodies for NCI-H322 cell binding ability testing, and 8H9 was used as a positive control. Among them, B107, B38, and B2-124 were again tested for binding with A-204 cells.

[0230] The FACS test results are shown in Tables 7 and 8. Among the eight antibodies, only B22 and B38 had poor binding ability to cells, while the positive rates of the remaining antibodies were all above 99%. Among them, the positive control antibody 8H9 had a binding rate of 100% on both A-204 and NCI-H322 cells.

[0231] Table 7 Binding of candidate antibodies to NCI-H322 cells

[0232] Table 8 Binding of candidate antibodies to A204 cells

[0233] 2.5 Investigation of the binding of B7H3 heavy chain single domain antibody to B7H3 using the ForteBio method

[0234] B107, B108, B75, and B179 were selected as detection antibodies. Anti-B7H3 candidate single-domain antibodies (SDOs) were immobilized on SA biosensors. B7H3-cHis was then bound to the Nanobodies at concentrations ranging from 6.25 to 100 nM and subsequently dissociated. Binding kinetics for B107, B108, B75, and B179, including Kon, Koff, and Kd, were evaluated using Octet Data Analysis version 9.0.

[0235] Table 9 KD affinity test results of 4 antibodies

[0236] As shown in Table 9, the KD values ​​of the four candidate antibodies for B7H3 were all less than 1 nM, indicating that they all had good affinity for the B7H3 antigen.

[0237] Example 3: In vivo verification of B7H3 antibody function

[0238] 3.1 99m Imaging of Tc-labeled B7H3 single-domain antibody on MC38-B7H3 tumors

[0239] By analyzing the results of in vitro validation, the B107 antibody was finally selected as a candidate antibody for in vivo validation. At the same time, the positive antibody 8H9 (positive antibody 8H9 is the antibody sequence in the reference patent US20200197546A1, and the protein is expressed by itself. The construction and expression process is similar to that of Example 2. It is obtained by transient expression in HEK293 cells and affinity purification with nickel fillers) was labeled for in vivo development.

[0240] Take 200 μL 99m TcO4 (PH ~ 5.0) was added to the ISOLINK KIT (21.7mCi), 200μL of normal saline was added, and the mixture was incubated at 100℃ for 30min; 100μL (4.05mCi) was removed. 99m Mix Tc(H2O)3(CO)3 (pH 12.5) with 400 μg (150 μL) of 8H9 protein and incubate at 50°C for 90 min. Take out 100 μL (4.04 mCi) 99m Tc(H2O)3(CO)3 (PH~12.5) was mixed with 300μg (164μL) of 8H9 protein and incubated at 50℃ for 90min. After labeling, it was purified with PD10 column and then developed with PBS and measured by TLC. 99m The labeling efficiency of B107 and 8H9 with Tc(H2O)3(CO)3 was 93% for 8H9 and greater than 99% for B107.

[0241] Six female BALBc-Nude mice, 6 to 8 weeks old, were housed in an SPF-protected environment with free access to food. A standard 12-hour light-dark cycle was used. 100 μL of MC38-B7H3 / PBS was injected subcutaneously into the right axilla of the mice. The cell seeding density was approximately 5 to 6 × 10 5 cells / mouse. The tumor volume is available after 3-4 weeks, approximately 100-300 mm 3 .

[0242] Will 99mTc-B107 / 8H9 test products were injected into the tail vein of MC38-B7H3 tumor-bearing nude mice, with 3 mice in each group. The dose of B107 was 802μCi / 59.5μg, and the dose of 8H9 was 786μCi / 77.6μg. SPECT / CT imaging was then performed, and ROI was used to outline the uptake of different tissues.

[0243] Figure 1 shows 99m The uptake of Tc-labeled B7H3 single-domain antibody and positive antibody 8H9 in various organs showed that both B107 antibody and 8H9 antibody had high radioactivity concentration in tumors, and the tumor uptake of B107 was higher than that of 8H9, which proved that the selected candidate B7H3 single-domain antibody B107 had a higher affinity for B7H3 antigen in vivo. In addition to the high uptake in tumors, B107 single-domain antibody also had high radioactivity concentration in the kidneys, which proved that 99m Tc-labeled B7H3 single-domain antibody is metabolized out of the body through the kidneys, maintaining the typical distribution characteristics of single-domain antibodies.

[0244] 3.2 177 Lu-labeled B7H3 single-domain antibody imaging on MC38-B7H3 tumors

[0245] The B107 antibody was exchanged into NaHCO3-Na2CO3 buffer (pH 9.5) using a PD-10 column. The SCN-Bn-DOTA solution was added to the B107 antibody at a molar ratio of 5 times the number of lysine on the B107 antibody and mixed. The mixture was reacted at 37°C for 21 hours. After the reaction, the solution was exchanged and concentrated by ultrafiltration. The absorbance at 280 nm was measured using a spectrophotometer to determine the protein concentration.

[0246] 250 μg of DOTA-B107 nanobody conjugate was mixed with 100 μL 0.1 M HCl and 500 μL 0.25 M sodium acetate, and finally 1.91 mCi 177 LuCl3 solution. React at 45°C for 1 hour. If the reaction volume is small, add 0.25M sodium acetate to increase the reaction volume.

[0247] After 1 hour of reaction, spot samples for TLC analysis using citric acid as the developing solvent. If the TLC result shows a labeling efficiency >95%, release the sample for use. If the labeling efficiency is <95%, purify the sample using a PD-10 column. After saturation with 1% BSA, elute with PBS, collecting and measuring radioactivity in 500 μL fractions. Combine the radioactive eluates and perform TLC analysis again. If the radiochemical purity is >95%, release the sample for use. Testing has verified that the labeling efficiency of B107 is greater than 99%.

[0248] Three female BALBc-Nude mice, 6-8 weeks old, were housed in an SPF-protected environment with free access to food. A standard 12-h light-dark cycle was used. 100 μL of MC38-B7H3 / PBS was injected subcutaneously into the right axilla of the mice. The cell seeding density was approximately 5-6 × 10 5 cells / mouse. The tumor volume is available after 3-4 weeks, approximately 100-300 mm 3 .

[0249] Will 177 The Lu-B107 test sample was injected into the tail vein of MC38-B7H3 tumor-bearing nude mice, with 3 mice in each group. The B107 dose was 270μCi / 51.3μg. 8mg of lysine was also given at the same time. SPECT / CT imaging was then performed, and ROI was used to outline the uptake of different tissues.

[0250] Figure 2 shows the imaging results of B107 antibody for 2 hours and the uptake of various tissues and organs. B107 antibody is significantly enriched in tumors, which improves the specific targeting of B7H3 antigen in vivo. The high uptake in the kidneys suggests 177 Lu-DOTA-B7H3 nanobody is mainly metabolized by the kidneys.

Claims

1. A B7H3-binding polypeptide comprising at least one immunoglobulin single variable domain that specifically binds to B7H3, wherein the at least one immunoglobulin single variable domain comprises 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.

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: 2, CDR2 shown in SEQ ID NO: 3, CDR3 shown in SEQ ID NO: 4; (2) CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, CDR3 shown in SEQ ID NO: 8; (3) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, CDR3 shown in SEQ ID NO: 12; (4) CDR1 shown in SEQ ID NO: 14, CDR2 shown in SEQ ID NO: 15, CDR3 shown in SEQ ID NO: 16; (5) CDR1 shown in SEQ ID NO: 18, CDR2 shown in SEQ ID NO: 19, CDR3 shown in SEQ ID NO: 20; (6) CDR1 shown in SEQ ID NO: 22, CDR2 shown in SEQ ID NO: 23, CDR3 shown in SEQ ID NO: 24; (7) CDR1 shown in SEQ ID NO: 26, CDR2 shown in SEQ ID NO: 27, CDR3 shown in SEQ ID NO: 28; (8) CDR1 shown in SEQ ID NO: 30, CDR2 shown in SEQ ID NO: 31, CDR3 shown in SEQ ID NO: 32; (9) CDR1 shown in SEQ ID NO: 34, CDR2 shown in SEQ ID NO: 35, CDR3 shown in SEQ ID NO: 36; (10) CDR1 shown in SEQ ID NO: 38, CDR2 shown in SEQ ID NO: 39, CDR3 shown in SEQ ID NO: 40; (11) CDR1 shown in SEQ ID NO: 42, CDR2 shown in SEQ ID NO: 43, CDR3 shown in SEQ ID NO: 44; (12) CDR1 shown in SEQ ID NO: 46, CDR2 shown in SEQ ID NO: 47, CDR3 shown in SEQ ID NO: 48; (13)CDR1 shown in SEQ ID NO:50, CDR2 shown in SEQ ID NO:51, CDR3 shown in SEQ ID NO:52; (14)CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:55, CDR3 shown in SEQ ID NO:56; (15)CDR1 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:59, CDR3 shown in SEQ ID NO:60; (16)CDR1 shown in SEQ ID NO:62, CDR2 shown in SEQ ID NO:63, CDR3 shown in SEQ ID NO:64; (17)CDR1 shown in SEQ ID NO:66, CDR2 shown in SEQ ID NO:67, CDR3 shown in SEQ ID NO:68; (18)CDR1 shown in SEQ ID NO:70, CDR2 shown in SEQ ID NO:71, CDR3 shown in SEQ ID NO:72; (19)CDR1 shown in SEQ ID NO:74, CDR2 shown in SEQ ID NO:75, CDR3 shown in SEQ ID NO:76; (20)CDR1 shown in SEQ ID NO:78, CDR2 shown in SEQ ID NO:79, CDR3 shown in SEQ ID NO:80; (21)CDR1 shown in SEQ ID NO:82, CDR2 shown in SEQ ID NO:83, CDR3 shown in SEQ ID NO:84; and (22)CDR1 shown in SEQ ID NO:86, CDR2 shown in SEQ ID NO:87, CDR3 shown in SEQ ID NO:

88.

3. The B7H3-binding polypeptide of claim 1 or 2, wherein the single immunoglobulin variable domain comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the amino acid sequence shown in 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.

4. The B7H3-binding polypeptide of any one of claims 1-3, wherein the single immunoglobulin variable domain comprises the amino acid sequence shown in 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.

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

6. A B7H3-binding polypeptide of any one of claims 1-5, wherein the single variable domain of the immunoglobulin is humanized.

7. A nucleic acid molecule encoding the B7H3-binding polypeptide of any one of claims 1-6.

8. An expression vector comprising the nucleic acid molecule of claim 7 operably linked to an expression control element.

9. A host cell comprising the nucleic acid molecule of claim 7 or transformed with the expression vector of claim 8 and capable of expressing the B7H3-binding polypeptide.

10. A method for producing a B7H3-binding polypeptide of any one of claims 1-6, comprising: a) culturing the host cell of claim 9 under conditions permitting expression of the B7H3-binding polypeptide; b) recovering the B7H3-binding polypeptide expressed by the host cell from the culture obtained in step a); and c) optionally further purifying and / or modifying the B7H3-binding polypeptide obtained in step b).

11. A conjugate molecule comprising a B7H3-binding polypeptide of any one of claims 1-6, and at least one detectable label and / or therapeutic moiety conjugated to the B7H3-binding polypeptide.

12. The conjugate molecule of claim 11, wherein the detectable label is selected from radionuclides, fluorophores, chemiluminescent agents, bioluminescent agents, paramagnetic ions, and enzymes; or the therapeutic moiety is selected from radionuclides, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide glucopyranoside, etoposide thioglycoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, anthramycin, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, calicheamicin, maytansine, alisatin, nitrogen mustard, chlorambucil, phenylalanine mustard, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin, bleomycin, anthramycin, abrin, 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.

13. The conjugate molecule of claim 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, e.g., the detectable label is 68 Ga or 177 Lu.

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

15. The conjugate molecule of claim 14, wherein the chelating agent is selected from DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG 3 , MAG 2 , HYNIC, DADT, EC, NS 3 , H2dedpa, HBED, DFO, PEPA or HEHA and their derivatives.

16. The conjugate molecule of claim 15, wherein the detectable label is 99m Tc, or the radionuclide is 177 Lu and the chelating agent is DOTA.

17. A method for detecting the presence and / or amount of B7H3 in a biological sample, comprising: a) Under the conditions where a complex can be formed between the B7H3-binding polypeptide of any one of claims 1-6 or the conjugate molecule of any one of claims 11-16 and B7H3, contacting the biological sample and the control sample with the B7H3-binding polypeptide of any one of claims 1-6 or the conjugate molecule of any one of claims 11-16; b) Detecting the formation of the complex, wherein a difference in the formation of the complex between the biological sample and the control sample indicates the presence and / or amount of B7H3 in the sample.

18. A diagnostic agent for detecting and / or diagnosing B7H3-related diseases such as cancer, comprising the B7H3-binding polypeptide of any one of claims 1-6 and / or the conjugate molecule of any one of claims 11-15, and optionally a physiologically acceptable carrier.

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

20. The diagnostic agent of claim 19, which is a contrast agent.

21. The diagnostic agent of claim 20, wherein the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.

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

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

24. The use of claim 23, which is a contrast agent.

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

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 the B7H3-binding polypeptide of any one of claims 1-6 and / or the conjugate molecule of any one of claims 11-16 and / or the diagnostic agent of any one of claims 18-21.

27. The method of claim 26, further comprising the step of imaging the subject, such as ECT imaging.

28. The method of claim 27, wherein the ECT imaging is SPECT imaging, or the ECT imaging is PET imaging.

29. The diagnostic agent of any one of claims 18-21, the use of any one of claims 22-25, or the method of any one of claims 26-28, wherein the B7H3-related disease is cancer, such as the cancer is 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 (such as oligodendroglioma, anaplastic astrocytoma, glioblastoma multiforme (GBM), ependymoma, and diffuse intrinsic pontine glioma (DIPG)).

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