FAP-binding polypeptide and uses thereof

Abstract

Provided are a specifically FAP-binding polypeptide, a use thereof in the detection of the presence and / or content of FAP in a biological sample, and a use thereof in the detection and / or diagnosis of FAP-related diseases such as cancer or inflammatory diseases.

Description

FAP-binding polypeptides and uses thereof Technical Field

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

[0002] Background of the Invention

[0003] Fibroblast activation protein (FAP), also known as proline endopeptidase FAP or seprase, is a type II transmembrane serine protease. In normal adults, FAP is expressed to a certain extent in the endometrium, cervix, gallbladder, and bladder. It is highly expressed on the surface of activated fibroblasts in the stroma of more than 90% of epithelial malignancies, including breast cancer, colorectal cancer, skin cancer, prostate cancer, and pancreatic cancer. However, it is rarely expressed in epithelial lesions in other tissues and benign or precancerous lesions. In addition, studies have found that FAP is also significantly expressed in diseases related to inflammation and cellular fibrosis, including wound healing, rheumatoid arthritis, osteoarthritis, cirrhosis, pulmonary fibrosis, and ventricular remodeling after myocardial infarction. These differences in expression make FAP a potential imaging and radiotherapy target for cancer and inflammatory diseases. There is still a need in the art for diagnostic and therapeutic agents for FAP-related diseases.

[0004] Summary of the Invention

[0005] The present invention comprises at least the following embodiments:

[0006] Embodiment 1. A FAP-binding polypeptide comprising at least one immunoglobulin single variable domain that specifically binds to FAP, wherein the at least one immunoglobulin single variable domain comprises the CDR1, CDR2, and CDR3 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, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, and 163-184.

[0007] Embodiment 2. The FAP-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:

[0008] (1) CDR1 shown in SEQ ID NO: 2, CDR2 shown in SEQ ID NO: 3, and CDR3 shown in SEQ ID NO: 4;

[0009] (2) CDR1 set forth in SEQ ID NO:6, CDR2 set forth in SEQ ID NO:7, CDR3 set forth in SEQ ID NO:8, SEQ ID NO:185, or SEQ ID NO:186;

[0010] (3) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12;

[0011] (4) CDR1 shown in SEQ ID NO: 14, CDR2 shown in SEQ ID NO: 15, and CDR3 shown in SEQ ID NO: 16;

[0012] (5) CDR1 shown in SEQ ID NO: 18, CDR2 shown in SEQ ID NO: 19, and CDR3 shown in SEQ ID NO: 20;

[0013] (6) CDR1 shown in SEQ ID NO: 22, CDR2 shown in SEQ ID NO: 23, and CDR3 shown in SEQ ID NO: 24;

[0014] (7) CDR1 shown in SEQ ID NO: 26, CDR2 shown in SEQ ID NO: 27, and CDR3 shown in SEQ ID NO: 28;

[0015] (8) CDR1 shown in SEQ ID NO: 30, CDR2 shown in SEQ ID NO: 31, and CDR3 shown in SEQ ID NO: 32;

[0016] (9) CDR1 shown in SEQ ID NO: 34, CDR2 shown in SEQ ID NO: 35, and CDR3 shown in SEQ ID NO: 36;

[0017] (10) CDR1 shown in SEQ ID NO: 38, CDR2 shown in SEQ ID NO: 39, and CDR3 shown in SEQ ID NO: 40;

[0018] (11) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:43, and CDR3 shown in SEQ ID NO:44;

[0019] (12) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:48;

[0020] (13) CDR1 shown in SEQ ID NO: 50, CDR2 shown in SEQ ID NO: 51, and CDR3 shown in SEQ ID NO: 52;

[0021] (14) CDR1 shown in SEQ ID NO: 54, CDR2 shown in SEQ ID NO: 55, and CDR3 shown in SEQ ID NO: 56;

[0022] (15) CDR1 shown in SEQ ID NO: 58, CDR2 shown in SEQ ID NO: 59, and CDR3 shown in SEQ ID NO: 60;

[0023] (16) CDR1 shown in SEQ ID NO: 62, CDR2 shown in SEQ ID NO: 63, and CDR3 shown in SEQ ID NO: 64;

[0024] (17) CDR1 shown in SEQ ID NO: 66, CDR2 shown in SEQ ID NO: 67, and CDR3 shown in SEQ ID NO: 68;

[0025] (18) CDR1 shown in SEQ ID NO: 70, CDR2 shown in SEQ ID NO: 71, and CDR3 shown in SEQ ID NO: 72;

[0026] (19) CDR1 shown in SEQ ID NO: 74, CDR2 shown in SEQ ID NO: 75, and CDR3 shown in SEQ ID NO: 76;

[0027] (20) CDR1 shown in SEQ ID NO: 78, CDR2 shown in SEQ ID NO: 79, and CDR3 shown in SEQ ID NO: 80;

[0028] (21) CDR1 shown in SEQ ID NO: 82, CDR2 shown in SEQ ID NO: 83, and CDR3 shown in SEQ ID NO: 84;

[0029] (22) CDR1 shown in SEQ ID NO: 86, CDR2 shown in SEQ ID NO: 87, and CDR3 shown in SEQ ID NO: 88;

[0030] (23) CDR1 shown in SEQ ID NO:90, CDR2 shown in SEQ ID NO:91, and CDR3 shown in SEQ ID NO:92;

[0031] (24) CDR1 shown in SEQ ID NO:94, CDR2 shown in SEQ ID NO:95, and CDR3 shown in SEQ ID NO:96;

[0032] (25) CDR1 shown in SEQ ID NO:98, CDR2 shown in SEQ ID NO:99, and CDR3 shown in SEQ ID NO:100;

[0033] (26) CDR1 shown in SEQ ID NO: 102, CDR2 shown in SEQ ID NO: 103, and CDR3 shown in SEQ ID NO: 104;

[0034] (27) CDR1 shown in SEQ ID NO: 106, CDR2 shown in SEQ ID NO: 107, and CDR3 shown in SEQ ID NO: 108;

[0035] (28) CDR1 shown in SEQ ID NO: 110, CDR2 shown in SEQ ID NO: 111, and CDR3 shown in SEQ ID NO: 112;

[0036] (29) CDR1 shown in SEQ ID NO: 114, CDR2 shown in SEQ ID NO: 115, and CDR3 shown in SEQ ID NO: 116;

[0037] (30) CDR1 shown in SEQ ID NO: 118, CDR2 shown in SEQ ID NO: 119, and CDR3 shown in SEQ ID NO: 120;

[0038] (31) CDR1 shown in SEQ ID NO: 122, CDR2 shown in SEQ ID NO: 123, and CDR3 shown in SEQ ID NO: 124; and

[0039] (32) CDR1 shown in SEQ ID NO: 126, CDR2 shown in SEQ ID NO: 127, and CDR3 shown in SEQ ID NO: 128.

[0040] Embodiment 3. The FAP-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 NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, and 162-184.

[0041] Embodiment 4. The FAP-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, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, and 162-184.

[0042] Embodiment 5. The FAP-binding polypeptide of any one of embodiments 1-4, wherein the immunoglobulin single variable domain is capable of binding Protein A.

[0043] Embodiment 6. The FAP-binding polypeptide of embodiment 5, wherein the immunoglobulin single 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 SEQ ID NO: 5, and compared to SEQ ID NO: 5, amino acid residue N at position 19 is substituted by R, and amino acid residue F at position 71 is substituted by S.

[0044] Embodiment 7. The FAP-binding polypeptide of embodiment 6, wherein the immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO: 162, 177, 181 or 183.

[0045] Embodiment 8. The FAP-binding polypeptide of any one of embodiments 1-7, wherein the immunoglobulin single variable domain is a VHH.

[0046] Embodiment 9. The FAP-binding polypeptide of any one of embodiments 1-8, wherein the immunoglobulin single variable domain is humanized.

[0047] Embodiment 10. A nucleic acid molecule encoding the FAP-binding polypeptide of any one of embodiments 1-9.

[0048] Embodiment 11. An expression vector comprising the nucleic acid molecule of embodiment 7 operably linked to an expression control element.

[0049] Embodiment 12. A host cell comprising the nucleic acid molecule of embodiment 10 or transformed with the expression vector of embodiment 11, and capable of expressing the FAP-binding polypeptide.

[0050] Embodiment 13. A method of producing the FAP-binding polypeptide of any one of embodiments 1-9, comprising:

[0051] a) culturing the host cell of embodiment 12 under conditions that allow expression of the FAP-binding polypeptide;

[0052] b) recovering the FAP-binding polypeptide expressed by the host cells from the culture obtained in step a); and

[0053] c) optionally further purifying and / or modifying the FAP-binding polypeptide obtained from step b).

[0054] Embodiment 14. A conjugate molecule comprising the FAP-binding polypeptide of any one of embodiments 1-9, and at least one detectable label and / or therapeutic moiety conjugated to the FAP-binding polypeptide.

[0055] Embodiment 15. The conjugate molecule of embodiment 14, wherein 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.

[0056] Embodiment 16. The conjugate molecule of embodiment 15, wherein the detectable label 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, 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.

[0057] Embodiment 17. The conjugate molecule of any one of embodiments 14-16, wherein the FAP-binding polypeptide is conjugated to the detectable label via a chelator.

[0058] Embodiment 18. The conjugate molecule of embodiment 17, 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.

[0059] Embodiment 19. The conjugate molecule of embodiment 18, wherein the detectable label is 68 Ga and the chelator is NOTA, or the detectable label is 177 Lu and the chelating agent is DOTA.

[0060] Embodiment 20. A method for detecting the presence and / or amount of a FAP in a biological sample, comprising:

[0061] a) contacting the biological sample and the control sample with a FAP-binding polypeptide according to any one of embodiments 1-9 or a conjugated molecule according to any one of embodiments 14-19 under conditions capable of forming a complex between the FAP-binding polypeptide according to any one of embodiments 1-9 or the conjugated molecule according to any one of embodiments 14-19 and FAP;

[0062] b) detecting the formation of a complex,

[0063] wherein the difference in complex formation between the biological sample and the control sample is indicative of the presence and / or amount of FAP in the sample.

[0064] Embodiment 21. A diagnostic agent for detecting and / or diagnosing FAP-related diseases, such as cancer or inflammatory diseases, comprising the FAP-binding polypeptide of any one of embodiments 1-9 and / or the conjugated molecule of any one of embodiments 14-19, and optionally a physiologically acceptable carrier.

[0065] Embodiment 22. The diagnostic agent of embodiment 21, wherein the diagnostic agent is a contrast agent.

[0066] Embodiment 23. The diagnostic agent of embodiment 22, which is a contrast agent.

[0067] Embodiment 24. The diagnostic agent of embodiment 23, wherein the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.

[0068] Embodiment 25. Use of the FAP-binding polypeptide of any one of embodiments 1-9 and / or the conjugated molecule of any one of embodiments 14-19 for the preparation of a diagnostic agent for detecting and / or diagnosing a FAP-related disease, such as cancer or inflammatory disease.

[0069] Embodiment 26. The use of embodiment 25, wherein the diagnostic agent is a contrast agent.

[0070] Embodiment 27. The use according to embodiment 26, which is a contrast agent.

[0071] Embodiment 28. The use of embodiment 27, wherein the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.

[0072] Embodiment 29. A method of detecting and / or diagnosing a FAP-associated disease, such as cancer or an inflammatory disease, in a subject, comprising administering to the subject a FAP-binding polypeptide of any one of embodiments 1-9 and / or a conjugated molecule of any one of embodiments 14-19 and / or a diagnostic agent of any one of embodiments 21-24.

[0073] Embodiment 30. The method of embodiment 29, further comprising the step of imaging the subject, such as ECT imaging.

[0074] Embodiment 31. The method of embodiment 30, wherein the ECT imaging is SPECT imaging, or the ECT imaging is PET imaging.

[0075] Embodiment 32. The diagnostic agent of any one of embodiments 21-24, the use of any one of embodiments 25-28, or the method of any one of embodiments 29-31, wherein the FAP-related disease is selected from cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and scar disease.

[0076] Embodiment 33. The diagnostic agent, use or method of embodiment 32, wherein the cancer is selected from breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, bile duct cell carcinoma, clear cell renal carcinoma, neuroendocrine tumors, carcinogenic osteomalacia, sarcoma, CUP (carcinoma of unknown primary), thymic cancer, glioma, glioma, astrocytoma, cervical cancer and prostate cancer.

[0077] BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 shows the internalization results of eight candidate antibodies.

[0079] Figure 2. 68 Ga-F68, 68 Ga-F425, 68 Ga-F146, 68 Tissue distribution results of Ga-F26 antibody within 1 hour.

[0080] Figure 3. 177 Lu-DOTA-F146 imaging and uptake results in various tissues and organs.

[0081] Figure 4. 177 Lu-DOTA-F425 imaging and uptake results in various tissues and organs.

[0082] Figure 5. 177 Lu-DOTA-F263 imaging and uptake results in various tissues and organs.

[0083] Figure 6. 177 Lu-DOTA-F68 imaging and uptake results in various tissues and organs.

[0084] Figure 7. Binding of F263 and F263-RS to protein A affinity medium.

[0085] Figure 8. F263 antibody variant internalization results.

[0086] Figure 9. 68 Graph showing the antibody distribution results after 1 hour of Ga-NODAGA-FAP modification.

[0087] Detailed Description of the Invention

[0088] definition

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] "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.

[0096] 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.

[0097] 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)).

[0098] 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:

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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).

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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).

[0108] Antibodies can be screened for competitive binding to the same epitope using conventional techniques known to those skilled in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to the antigen. 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, antibodies and antigen-binding fragments thereof that compete with the antibody molecules of the present invention for binding to the same epitope on FAP can be obtained using conventional techniques known to those skilled in the art.

[0109] 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.

[0110] As used herein, the term "FAP-binding protein (FAP-binding polypeptide or FAP-binding molecule)" means any protein that can specifically bind to a FAP protein. FAP-binding proteins can include antibodies against FAP, such as antibodies as defined herein. FAP-binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules. The amino acid sequence of an exemplary FAP is shown in SEQ ID NO: 161.

[0111] The "FAP-binding protein" of the present invention may comprise at least one immunoglobulin single variable domain, such as VHH, that binds to FAP. In some embodiments, the "FAP-binding molecule" of the present invention may comprise 2, 3, 4 or more immunoglobulin single variable domains, such as VHH, that bind to FAP. The FAP-binding protein of the present invention may also comprise a linker and / or a portion with 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 addition to the immunoglobulin single variable domain that binds to FAP. In some embodiments, the "FAP-binding protein" of the present invention also encompasses bispecific antibodies, which contain immunoglobulin single variable domains that bind to different antigens.

[0112] Typically, the FAP 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., FAP 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.

[0113] 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.

[0114] "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.

[0115] 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).

[0116] As used herein, the term "subject" means a mammal, particularly a primate, especially a human.

[0117] FAP-binding polypeptides of the present invention

[0118] The present invention provides a FAP-binding polypeptide comprising at least one immunoglobulin single variable domain that specifically binds to FAP. In some embodiments, the FAP-binding polypeptide is isolated. In some embodiments, the FAP-binding polypeptide specifically binds to FAP.

[0119] In some embodiments, the at least one immunoglobulin single variable domain comprises a CDR1, CDR2, and CDR3 of a VHH shown 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, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, and 163-184. The CDRs may be Kabat CDRs, AbM CDRs, Chothia CDRs, or Contact CDRs. In some embodiments, the CDRs are Kabat CDRs.

[0120] In some embodiments, the at least one immunoglobulin single variable domain comprises a CDR1, CDR2, and CDR3 selected from:

[0121] (1) CDR1 shown in SEQ ID NO: 2, CDR2 shown in SEQ ID NO: 3, and CDR3 shown in SEQ ID NO: 4;

[0122] (2) CDR1 set forth in SEQ ID NO:6, CDR2 set forth in SEQ ID NO:7, CDR3 set forth in SEQ ID NO:8, SEQ ID NO:185, or SEQ ID NO:186;

[0123] (3) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12;

[0124] (4) CDR1 shown in SEQ ID NO: 14, CDR2 shown in SEQ ID NO: 15, and CDR3 shown in SEQ ID NO: 16;

[0125] (5) CDR1 shown in SEQ ID NO: 18, CDR2 shown in SEQ ID NO: 19, and CDR3 shown in SEQ ID NO: 20;

[0126] (6) CDR1 shown in SEQ ID NO: 22, CDR2 shown in SEQ ID NO: 23, and CDR3 shown in SEQ ID NO: 24;

[0127] (7) CDR1 shown in SEQ ID NO: 26, CDR2 shown in SEQ ID NO: 27, and CDR3 shown in SEQ ID NO: 28;

[0128] (8) CDR1 shown in SEQ ID NO: 30, CDR2 shown in SEQ ID NO: 31, and CDR3 shown in SEQ ID NO: 32;

[0129] (9) CDR1 shown in SEQ ID NO: 34, CDR2 shown in SEQ ID NO: 35, and CDR3 shown in SEQ ID NO: 36;

[0130] (10) CDR1 shown in SEQ ID NO: 38, CDR2 shown in SEQ ID NO: 39, and CDR3 shown in SEQ ID NO: 40;

[0131] (11) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:43, and CDR3 shown in SEQ ID NO:44;

[0132] (12) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:48;

[0133] (13) CDR1 shown in SEQ ID NO: 50, CDR2 shown in SEQ ID NO: 51, and CDR3 shown in SEQ ID NO: 52;

[0134] (14) CDR1 shown in SEQ ID NO: 54, CDR2 shown in SEQ ID NO: 55, and CDR3 shown in SEQ ID NO: 56;

[0135] (15) CDR1 shown in SEQ ID NO: 58, CDR2 shown in SEQ ID NO: 59, and CDR3 shown in SEQ ID NO: 60;

[0136] (16) CDR1 shown in SEQ ID NO: 62, CDR2 shown in SEQ ID NO: 63, and CDR3 shown in SEQ ID NO: 64;

[0137] (17) CDR1 shown in SEQ ID NO: 66, CDR2 shown in SEQ ID NO: 67, and CDR3 shown in SEQ ID NO: 68;

[0138] (18) CDR1 shown in SEQ ID NO: 70, CDR2 shown in SEQ ID NO: 71, and CDR3 shown in SEQ ID NO: 72;

[0139] (19) CDR1 shown in SEQ ID NO: 74, CDR2 shown in SEQ ID NO: 75, and CDR3 shown in SEQ ID NO: 76;

[0140] (20) CDR1 shown in SEQ ID NO: 78, CDR2 shown in SEQ ID NO: 79, and CDR3 shown in SEQ ID NO: 80;

[0141] (21) CDR1 shown in SEQ ID NO: 82, CDR2 shown in SEQ ID NO: 83, and CDR3 shown in SEQ ID NO: 84;

[0142] (22) CDR1 shown in SEQ ID NO: 86, CDR2 shown in SEQ ID NO: 87, and CDR3 shown in SEQ ID NO: 88;

[0143] (23) CDR1 shown in SEQ ID NO:90, CDR2 shown in SEQ ID NO:91, and CDR3 shown in SEQ ID NO:92;

[0144] (24) CDR1 shown in SEQ ID NO:94, CDR2 shown in SEQ ID NO:95, and CDR3 shown in SEQ ID NO:96;

[0145] (25) CDR1 shown in SEQ ID NO:98, CDR2 shown in SEQ ID NO:99, and CDR3 shown in SEQ ID NO:100;

[0146] (26) CDR1 shown in SEQ ID NO: 102, CDR2 shown in SEQ ID NO: 103, and CDR3 shown in SEQ ID NO: 104;

[0147] (27) CDR1 shown in SEQ ID NO: 106, CDR2 shown in SEQ ID NO: 107, and CDR3 shown in SEQ ID NO: 108;

[0148] (28) CDR1 shown in SEQ ID NO: 110, CDR2 shown in SEQ ID NO: 111, and CDR3 shown in SEQ ID NO: 112;

[0149] (29) CDR1 shown in SEQ ID NO: 114, CDR2 shown in SEQ ID NO: 115, and CDR3 shown in SEQ ID NO: 116;

[0150] (30) CDR1 shown in SEQ ID NO: 118, CDR2 shown in SEQ ID NO: 119, and CDR3 shown in SEQ ID NO: 120;

[0151] (31) CDR1 shown in SEQ ID NO: 122, CDR2 shown in SEQ ID NO: 123, and CDR3 shown in SEQ ID NO: 124; and

[0152] (32) CDR1 shown in SEQ ID NO: 126, CDR2 shown in SEQ ID NO: 127, and CDR3 shown in SEQ ID NO: 128.

[0153] In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence that is at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% identical 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, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, and 162-184. In some embodiments, the immunoglobulin single 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, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125 and 162-184.

[0154] In some preferred embodiments, the immunoglobulin single variable domain is capable of binding to protein A. In some preferred 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 SEQ ID NO: 5, and compared to SEQ ID NO: 5, the amino acid residue N at position 19 is substituted by R, and the amino acid residue F at position 71 is substituted by S. In some particularly preferred embodiments, the immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 162, 177, 181, or 183.

[0155] In some embodiments, the immunoglobulin single variable domain is a VHH. In some embodiments, the immunoglobulin single variable domain is humanized.

[0156] Nucleic acids, vectors, host cells

[0157] In another aspect, the present invention relates to nucleic acid molecules encoding the FAP-binding polypeptides of the present invention. The nucleic acids of the present invention can be RNA, DNA, or cDNA. A skilled artisan can select a nucleic acid molecule encoding a FAP-binding polypeptide of the present invention based on need or conventional methods. In some embodiments, the nucleic acid molecule encoding the FAP-binding polypeptide of the present invention comprises a nucleotide sequence selected from SEQ ID NOs: 129-160.

[0158] The nucleic acids of the present invention may also be in the form of, present in, and / or 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 FAP-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 FAP-binding polypeptide of the present invention include promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.

[0159] 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.

[0160] In another aspect, the present invention relates to a host cell that expresses or is capable of expressing one or more FAP-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.

[0161] 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).

[0162] 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.

[0163] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0164] 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.

[0165] The present invention also provides methods for producing the FAP-binding polypeptides of the present invention, the methods generally comprising the following steps:

[0166] - culturing the host cell of the invention under conditions allowing expression of the FAP-binding polypeptide of the invention; and

[0167] - recovering the FAP-binding polypeptide expressed by the host cells from the culture; and

[0168] - Optionally further purifying and / or modifying the FAP-binding polypeptides of the invention.

[0169] The FAP-binding polypeptides of the invention can be produced intracellularly in the cells as 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.

[0170] 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 FAP-binding polypeptides of the present invention are well known to those skilled in the art.

[0171] However, the FAP-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.

[0172] Conjugated molecules

[0173] In another aspect, the present invention provides a conjugate molecule comprising a FAP-binding polypeptide of the present invention and at least one detectable label and / or therapeutic moiety conjugated to the FAP-binding polypeptide.

[0174] Such detectable labels include, but are not limited to, radionuclides, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, and enzymes.

[0175] 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.

[0176] 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, 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.

[0177] Methods for conjugating detectable labels to polypeptides are well known to those skilled in the art. For example, in some embodiments, the FAP-binding polypeptide can be conjugated to the detectable label via a chelating agent.

[0178] In order to use radionuclides such as 68To label a FAP-binding polypeptide of the invention with Ga, it is necessary to react the FAP-binding polypeptide of the invention with a reagent having a long tail to which are attached a plurality of integrated groups for binding ions. Such a tail can be, for example, polylysine, a polysaccharide, or other polymer having a derivatized or derivatizable chain with a pendant group that can bind a chelating group, 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 FAP-binding polypeptide of the invention via a 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.

[0179] In some embodiments, wherein the detectable label is 68 Ga, and the chelating agent is NOTA. In some embodiments, wherein the detectable label is 177 Lu, and the chelating agent is DOTA.

[0180] In another aspect, the present invention provides a method for preparing a radionuclide of the present invention such as 68 Ga or 177 The invention provides a method for producing a Lu-labeled conjugate molecule, comprising 1) conjugating a FAP-binding polypeptide of the invention to a chelating agent to form a conjugate of the FAP-binding polypeptide and the chelating agent; and 2) conjugating the product of step 1) to a radionuclide such as 68 Ga or 177 Lu contact, whereby radionuclides such as 68 Ga or 177 Lu labels the FAP-binding polypeptide of the present invention through chelation with a chelating agent. In some embodiments, the chelating agent is NOTA, and the conjugate of the FAP-binding polypeptide and NOTA is generated in step 1) by reacting the FAP-binding polypeptide with p-SCN-Bn-NOTA or p-NH2-Bn-NOTA. In some embodiments, the chelating agent is DOTA, and the conjugate of the FAP-binding polypeptide and DOTA is generated in step 1) by reacting the FAP-binding polypeptide with Mal-DOTA.

[0181] In another aspect, the present invention provides a method for preparing the present invention. 125 I-labeled conjugated molecule, comprising 1) allowing the FAP-binding polypeptide of the present invention to react with 125 1 reaction; and 2) terminating the reaction with sodium metabisulfite.

[0182] 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.

[0183] Detection / diagnostic use

[0184] In another aspect, the present invention provides a method for detecting the presence and / or amount of FAP in a biological sample, comprising:

[0185] a) contacting the biological sample and the control sample with a FAP-binding polypeptide of the invention or a conjugate molecule of the invention under conditions allowing formation of a complex between the FAP-binding polypeptide of the invention or the conjugate molecule of the invention and FAP;

[0186] b) detecting the formation of a complex,

[0187] wherein the difference in complex formation between the biological sample and the control sample indicates the presence and / or amount of FAP in the sample.In some embodiments, the biological sample is an ex vivo sample.

[0188] In another aspect, the present invention provides a composition comprising a FAP-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 FAP-related disease.

[0189] In another aspect, the present invention provides a diagnostic agent for detecting and / or diagnosing a FAP-related disease, such as cancer or inflammatory disease, comprising a FAP-binding polypeptide of the present invention and / or a conjugate molecule of the present invention, and optionally a physiologically acceptable carrier. In some embodiments, the diagnostic agent is a contrast agent.

[0190] The FAP-binding polypeptides and / or conjugated molecules of the present invention are particularly suitable for in vivo imaging, for example, for emission computed tomography (ECT). For example, the FAP-binding polypeptides and / or conjugated molecules of the present invention can be used in single-photon emission computed tomography (SPECT) and positron emission tomography (PET) depending on the label. High-resolution tumor imaging can be provided in tumor diagnosis, and quantitative analysis can be performed through the images. The SPECT imaging can also include SPECT / CT imaging, and the PET imaging can also include PET / CT imaging, which can provide better imaging effects.

[0191] Thus, in some embodiments, the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.

[0192] In another aspect, the present invention provides a use of a FAP-binding polypeptide of the invention and / or a conjugate molecule of the invention in the preparation of a diagnostic agent for detecting and / or diagnosing a FAP-related disease, such as cancer or an inflammatory disease. 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.

[0193] In another aspect, the present invention provides a method for detecting and / or diagnosing a FAP-associated disease, such as cancer or an inflammatory disease, in a subject, comprising administering to the subject a FAP-binding polypeptide of the invention and / or a conjugate molecule of the invention and / or a diagnostic agent of the invention.

[0194] 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.

[0195] Diseases that can be detected and / or diagnosed by the FAP-binding polypeptides and / or conjugated molecules of the present invention and / or diagnostic agents of the present invention include diseases in which FAP expression in cells, tissues or organs is abnormally elevated, such as cancer and inflammatory diseases.

[0196] In this context, FAP-related diseases include, but are not limited to, cancer, chronic inflammation (such as rheumatoid arthritis, osteoarthritis), atherosclerosis, fibrosis (such as cirrhosis of the liver or pulmonary fibrosis), tissue remodeling (such as ventricular remodeling after myocardial infarction), and scar disease. In some embodiments, the cancer is selected from breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, bile duct cell carcinoma, clear cell renal carcinoma, neuroendocrine tumors, carcinogenic osteomalacia, sarcoma, CUP (cancer of unknown primary), thymic cancer, glioma, glioma, astrocytoma, cervical cancer, and prostate cancer.

[0197] Pharmaceutical compositions and therapeutic uses

[0198] In another aspect, the present invention provides a pharmaceutical composition comprising a FAP-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 FAP-related disease. The FAP-related disease is as described above, preferably cancer.

[0199] 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).

[0200] In another aspect, the present invention provides use of a FAP-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 FAP-associated disease, wherein the FAP-associated disease is as described above, and is preferably cancer.

[0201] In another aspect, the present invention provides a method for treating a FAP-associated disease in a subject, comprising administering to the subject a therapeutically effective amount of a FAP-binding polypeptide or conjugate molecule of the present invention or a pharmaceutical composition of the present invention. The FAP-associated disease is as described above, preferably cancer.

[0202] Reagent test kit

[0203] In another aspect, the present invention provides a kit comprising a FAP-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

[0204] Example 1. Antibody screening and construction

[0205] 1.1 Library Construction

[0206] The NHis-FAP 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, and PBMCs were isolated according to the instructions of the lymphocyte separation solution. RNA was extracted using RNAiso Plus reagent and PrimeScript was used. 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.

[0207] 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 FAP. The library was then assayed by serial dilution plating, and the library size was calculated to be 3.88×10 9 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.

[0208] 1.2 Selection of heavy chain single domain antibodies targeting FAP

[0209] The NHis-FAP 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% OVA-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 11 cfu, 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 2 times 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 6-8 minutes. The phages specifically bound to FAP are dissociated and transferred to a sterile centrifuge tube, to which 1 / 10 volume 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 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 FAP-specific antibodies from the antibody library using phage display technology. Table 1 shows the conditions for affinity panning.

[0210] Table 1 Affinity panning conditions

[0211] 1.3. Screening of specific single positive clones using phage enzyme-linked immunosorbent assay (ELISA)

[0212] After several rounds of panning, 480 colonies were randomly selected from the panning plates for identification. Each of these 480 randomly selected colonies was cultured, and phage was produced and purified. The NHis-FAP 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 Biological Biotechnology 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. ELISA results revealed 337 positive clones among the 480 clones, which were then sequenced and analyzed. Among them, 65 clones were non-monoclonal and 2 clones were negative in retest.

[0213] 1.4. Detection of binding of phage-positive clones to the FAP-high-expressing cell line U87-MG

[0214] The U87-MG cells were adjusted to a density of 5 × 10 6 cells / mL, 100 μL was added to each well of the ELISA plate and placed in a carbon dioxide incubator for overnight culture. The next day, the cell supernatant was discarded, 125 μL of 2% glutaraldehyde fixative was added to each well, and the cells were reacted at 37°C for 15 minutes, then blocked with 3% BSA and reacted at 37°C for 1 hour. Subsequently, the phage culture medium of the selected positive clone was added, 100 μL was added to each well, and the cells were incubated at 37°C for 1 hour. After washing with PBS, horseradish peroxide-labeled M13 secondary antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was added and the cells were 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.

[0215] Based on the results of phage ELISAs with NHis-FAP and U87-MG cells, clones with good binding to both the protein and cells 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, 32 candidate antibodies were selected for subsequent experiments. The antibody sequences are shown in Table 2 below.

[0216] 1.5. Production of FAP Antibody Protein Using Mammalian Cells

[0217] Primers were designed based on the nucleotide sequence of the FAP single-domain antibody obtained by screening. Using the plasmid as a template, the nucleotide sequence of each antibody (including the signal peptide and His tag) was amplified by PCR, and then cloned into the psna008 (pCDNA4 (Invitrogen, Cat V86220)) vector 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 90rpm. Four hours later, EX293 medium, 2mM glutamine, and cultured at 135rpm. After 24 hours, 3.8mM VPA was added. After 6-7 days of culture, the transient expression culture supernatant was collected and purified with a nickel column. Finally, an antibody protein with a purity of more than 90% was obtained.

[0218] Table 2. Amino acid sequences of anti-FAP single domain antibodies

[0219] Nucleotide sequence of F1-cHis antibody (SEQ ID NO: 129):

[0220] Nucleotide sequence of F263-cHis antibody (SEQ ID NO: 130):

[0221] Nucleotide sequence of F297-cHis antibody (SEQ ID NO: 131):

[0222] Nucleotide sequence of F146-cHis antibody (SEQ ID NO: 132):

[0223] Nucleotide sequence of F157-cHis antibody (SEQ ID NO: 133):

[0224] Nucleotide sequence of F257-cHis antibody (SEQ ID NO: 134):

[0225] Nucleotide sequence of F413-cHis antibody (SEQ ID NO: 135):

[0226] Nucleotide sequence of F429-cHis antibody (SEQ ID NO: 136):

[0227] Nucleotide sequence of F433-cHis antibody (SEQ ID NO: 137):

[0228] Nucleotide sequence of F144-cHis antibody (SEQ ID NO: 138):

[0229] Nucleotide sequence of F300-cHis antibody (SEQ ID NO: 139):

[0230] Nucleotide sequence of F331-cHis antibody (SEQ ID NO: 140):

[0231] Nucleotide sequence of F388-cHis antibody (SEQ ID NO: 141):

[0232] Nucleotide sequence of F401-cHis antibody (SEQ ID NO: 142):

[0233] Nucleotide sequence of F440-cHis antibody (SEQ ID NO: 143):

[0234] Nucleotide sequence of F416-cHis antibody (SEQ ID NO: 144):

[0235] Nucleotide sequence of F51-cHis antibody (SEQ ID NO: 145):

[0236] Nucleotide sequence of F342-cHis antibody (SEQ ID NO: 146):

[0237] Nucleotide sequence of F25-cHis antibody (SEQ ID NO: 147):

[0238] Nucleotide sequence of F114-cHis antibody (SEQ ID NO: 148):

[0239] Nucleotide sequence of F155-cHis antibody (SEQ ID NO: 149):

[0240] Nucleotide sequence of F292-cHis antibody (SEQ ID NO: 150):

[0241] Nucleotide sequence of F391-cHis antibody (SEQ ID NO: 151):

[0242] Nucleotide sequence of F426-cHis antibody (SEQ ID NO: 152):

[0243] Nucleotide sequence of F456-cHis antibody (SEQ ID NO: 153):

[0244] Nucleotide sequence of F68-cHis antibody (SEQ ID NO: 154):

[0245] Nucleotide sequence of F320-cHis antibody (SEQ ID NO: 155):

[0246] Nucleotide sequence of F167-cHis antibody (SEQ ID NO: 156):

[0247] Nucleotide sequence of F352-cHis antibody (SEQ ID NO: 157):

[0248] Nucleotide sequence of F457-cHis antibody (SEQ ID NO: 158):

[0249] Nucleotide sequence of F126-cHis antibody (SEQ ID NO: 159):

[0250] Nucleotide sequence of F425-cHis antibody (SEQ ID NO: 160):

[0251] Note: The underlined part is the nucleotide sequence of the target antibody

[0252] Example 2: Verification of FAP Antibody Function in Vitro

[0253] The NHis-FAP fusion protein was constructed for detection. The NHis-FAP fusion protein was transiently expressed in HEK293 cells and affinity purified using nickel filler.

[0254] The amino acid sequence of the NHis-FAP fusion protein is SEQ ID NO: 161, as follows:

[0255] Note: The underlined characters are signal peptide and HIS tag

[0256] 2.1. Antibody expression

[0257] Calculation of expression level: The expression level was calculated based on the total amount of target protein obtained after one-step affinity chromatography purification.

[0258] Table 3 Expression of 32 antibodies

[0259] 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 100 mg / L. The corresponding protein can be provided for subsequent testing through transient transfection.

[0260] 2.2. Detection of the affinity of FAP heavy chain single domain antibody to human FAP protein and antibody expression

[0261] The NHis-FAP 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 1.5) was diluted 4-fold with a starting concentration of 10 μg / mL, and a total of 11 concentrations were diluted. 100 μL was added to each well and reacted at 37°C for 1 hour. After washing, anti-camelid VHH secondary antibody (1:5000LOT#2009K104) 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 for FAP were obtained by four-parameter fitting. 50 Among the 32 selected series, the relative activities of different FAP heavy chain single-domain antibodies were compared using F456-cHis as a control to reflect the affinity of the candidate antibodies for FAP. The results are shown in Table 4.

[0262] Table 4 ELISA results of 32 antibodies binding to FAP

[0263] 2.3. FACS analysis of the binding of FAP heavy chain single domain antibody to cell surface FAP

[0264] By constructing a HEK293 cell stably transfected cell line expressing the full-length human FAP protein gene, HEK293 cells stably expressing human FAP protein on the membrane (HEK293-FAP cells) were obtained.

[0265] The cells were cultured and collected, and the density was adjusted to 1×10 6 Cells were plated at a concentration of 100 cells / mL. A 100 μL aliquot of cells was mixed with 0.5 μg of primary antibody and incubated on ice for 30 minutes. Cells were washed by centrifugation, resuspended, and 2 μL of Anti-His-PE conjugate was added. Incubated on ice for 30 minutes in the dark. Cells were washed twice with 300 μL of 1X PBS + 0.5% BSA, resuspended, and analyzed on a flow cytometer.

[0266] The FACS test results are shown in Table 5. 20 of the 32 antibodies had a positive binding rate of over 90% with HEK293-FAP cells in vitro, of which the positive control antibody 28H1 (positive antibody 28H1 is a protein expressed by itself based on the antibody sequence in reference patent AU2020201916B2. The construction and expression process is similar to that of Example 2. It was obtained by transient expression in HEK293 cells and affinity purification using nickel filler) had a binding rate of 99.93%.

[0267] Table 5 Binding of 32 antibodies to HEK293-FAP

[0268] 2.4. Internalization of FAP heavy chain single domain antibody by FACS

[0269] The affinity of 32 candidate antibodies was tested at the protein and cell levels by ELISA and FACS, and eight antibodies, F320, F429, F114, F68, F413, F425, F146 and F263, were initially selected as candidate antibodies for internalization detection.

[0270] Detection was performed using a validated temperature-controlled secondary antibody method: HEK293-FAP cells were collected and divided into 1x10^5 cells / tube, 100μL / tube, and incubated on ice for 20 minutes; the test antibody was added at a final concentration of 1μg / mL and incubated on ice for 30 minutes; after centrifugation and washing three times, the cells were grouped: placed on ice for 0, 1, 2, and 4 hours and incubated at 37°C for 1, 2, and 4 hours; Anti-his-PE secondary antibody was added and incubated on ice for 30 minutes; after centrifugation and washing three times, the cells were resuspended in 200μL and loaded onto the flow cytometer. Flow cytometric analysis was performed to determine the mean fluorescence intensity (MFI). At the same time, (MFI 0℃ -MFI 37℃ ) / MFI 0℃ *100% is the internalization rate of the antibody at time.

[0271] The specific results of internalization are shown in Figure 1. Due to the short half-life of the nanobody, cells may have experienced varying degrees of shedding at 4 hours, so the internalization rate at 4 hours may be higher than that at 2 hours. F429 did not internalize due to poor binding.

[0272] 2.5. Investigating the binding of FAP heavy chain single-domain antibody to FAP using the ForteBio method

[0273] Based on the results of affinity and endocytosis assays, F68, F263, F146, and F425 were selected as detection antibodies. Anti-FAP candidate single-domain antibodies (SDOs) were immobilized on SA biosensors with biotin. NHis-FAP at concentrations of 6.25–100 nM was then bound to the nanobodies and subsequently dissociated. Binding kinetics for F68, F263, F146, and F425, including Kon, Koff, and Kd, were evaluated using Octet Data Analysis version 9.0.

[0274] Table 6 KD affinity test results of 4 antibodies

[0275] As shown in Table 6, the affinity of F263 and F425 antibodies to FAP is about 1 nM, and the affinity of F68 and F146 antibodies is about 5-7 nM. 2 All are greater than 0.99.

[0276] Example 3: Verification of Antibody Function in Vivo

[0277] 3.1. 68 Biodistribution experiment of Ga-labeled FAP single domain antibody in HEK293-FAP tumors

[0278] 3.1.1 Random NOTA coupling of F425, F263, F146 and F68

[0279] The four antibodies F425, F263, F146, and F68 were each exchanged into NaHCO3-Na2CO3 buffer (pH 9.5) using a PD-10 column. p-SCN-Bn-NOTA (Macrocyclics, Catalog No. B-605) was dissolved in DMSO at a concentration of 25 mg / mL. The p-SCN-Bn-NOTA solution was added to the FAP single-domain antibody at a molar ratio of 2 times the number of lysines on the FAP-cHis single-domain antibody. The reaction was carried out at room temperature for a certain period of time. The conjugated product was purified using a PD-10 column (GE) and concentrated by ultrafiltration. The absorbance at 280 nm was measured by spectrophotometry to determine the protein concentration. The protein and SCN-NOTA were mixed at a molar ratio of 1:20 and reacted at 37°C for approximately 24 hours. After the reaction was completed, the solution was exchanged into 50 mM sodium acetate by ultrafiltration.

[0280] 3.1.2 68 Radiolabeling of Ga-NOTA-FAP single-domain antibody

[0281] Eckert & Ziegler IGG100 germanium was rinsed with 0.1 M sterile HCl. 68 / gallium 68 (Ge 68 / Ga 68 ) Generator preparation 68 Ga eluent, add an equal volume of 0.2M sodium acetate solution, and add 1 / 4 volume of 0.1M sodium acetate buffer with a pH of 5.3 containing NOTA-FAP antibody. The pH of the reaction system is between 4.5 and 4.7. React at room temperature for 10 minutes, remove unreacted ionic gallium through a PD-10 column and replace the product buffer system with physiological saline at the same time, and filter with a 0.22μm filter membrane. The product is quality controlled by analyzing pH, Radio-TLC, Radio-HPLC, radioactivity, radiochemical purity, etc. In addition: 0.05M sterile HCl can also be used to elute the ITG germanium 68 / gallium 68 (Ge-68 / Ga-68) generator for preparation. 68For the Ga eluent, 1 / 2 volume of 0.2 M sodium acetate solution was added, and other reaction and quality control conditions were the same.

[0282] 3.1.3 For research 68 Tumor distribution experiment of Ga-labeled FAP single domain antibody

[0283] Twelve female BALBc-Nude mice, 6-8 weeks old, were housed in an SPF-protected environment with free access to food. A standard 12-hour day-night lighting cycle was used. 100 μL of HEK293-FAP cells / PBS were injected subcutaneously into the right axilla of the mice. The cell seeding density was approximately 5-6 × 10 6 cells / mouse. The tumor volume is available after 3-4 weeks, approximately 100-300 mm 3 .

[0284] Will 68 Ga-F68 / F425 / F146 / F263 test articles were administered via tail vein injection to HEK293-FAP tumor-bearing nude mice, with three mice per group receiving a dose of 100 μL / 1 μg / 100 μCi. Sixty minutes after administration, mice were euthanized, and tissues were dissected, weighed, and counted on a gamma counter to determine the % injected dose per gram (%ID / g) for each tissue.

[0285] Figure 2 shows four 68 Ga-labeled FAP single-domain antibodies all showed high radioactivity accumulation in tumors, proving that the selected candidate FAP single-domain antibodies had high affinity for FAP antigen in vivo. In addition to high tumor uptake, the four candidate molecules also showed high radioactivity accumulation in the kidneys, proving that the four 68 Ga-labeled FAP single-domain antibody is metabolized out of the body through the kidneys, maintaining the typical distribution characteristics of single-domain antibodies.

[0286] 3.2. 177 Dynamic imaging of Lu-labeled FAP single-domain antibody in HEK293-FAP tumors

[0287] 3.2.1 Coupling of F425, F263, F146 and F68 with Mal-DOTA

[0288] The four antibody proteins F425, F263, F146 and F68 were mixed with TCEP (tris(2-carboxyethyl)phosphine) reducing agent at a molar ratio of 1:5, and the mixed mixture was placed at 37°C for 2 hours to reduce and open the disulfide bonds in the antibody. The reduced antibody was mixed with Mal-DOTA at a molar ratio of 1:10 and reacted at 37°C for 2 hours to couple the antibody with the chelating agent. After the coupling was complete, the solution was ultrafiltered and exchanged into 25mM sodium acetate to remove residual small molecule chelating agent.

[0289] 3.2.2 177 Radiolabeling of Lu-DOTA-FAP single-domain antibodies

[0290] 20 μg of DOTA-FAP nanobody conjugate was mixed with 10 μL 0.1 M HCl and 50 μL 0.25 M ammonium acetate, and finally 177 LuCl3 solution. React at 45°C for 1 hour. If the reaction volume is small, add 0.25M ammonium acetate to increase the reaction volume.

[0291] After 1 hour of reaction, spot samples for TLC analysis using citric acid as the developing solvent. If the TLC result indicates a labeling efficiency >95%, the sample is released for use. If the labeling efficiency is <95%, purify using a PD-10 column. After saturation with 1% BSA, elute with PBS, collecting 500 μL of each column and measuring the radioactivity. Combine the radioactive eluates and analyze again by TLC. If the radiochemical purity is >95%, the sample is released for use.

[0292] 3.2.3 For research 177 Lu-labeled FAP single domain antibody imaging experiment

[0293] Four female BALBc-Nude mice, 6-8 weeks old, were housed in an SPF-protected environment with free access to food. A standard 12-hour day-night lighting cycle was used. 100 μL of HEK293-FAP cells / PBS were injected subcutaneously into the right axilla of the mice. The cell seeding density was approximately 5-6 × 10 6 cells / mouse. The tumor volume is available after 3-4 weeks, approximately 100-300 mm 3 .

[0294] Will 177 Lu-DOTA-F68 / F425 / F146 / F263 test products were injected into the tail vein of HEK293-FAP tumor-bearing nude mice, one mouse per group, at a dose of 20 μg / 11 Mbq. SPECT / CT imaging was then performed. ROIs were delineated for uptake in different tissues.

[0295] Figures 3 to 6 show the imaging and uptake of the four antibodies in various tissues and organs. The results show that the tumor uptake of F263 is higher than that of the other three antibodies, indicating that it specifically targets the FAP antigen in vivo. In addition, the tumor uptake remains at a relatively high level 72 hours after administration and does not decrease significantly over time. 177 Lu-DOTA-FAP nanobody is mainly metabolized by the kidneys.

[0296] Example 4: Antibody sequence optimization

[0297] 4.1. Sequence optimization of the affinity-binding portion of F263

[0298] After analyzing the results of previous in vitro and in vivo validation, F263-cHis was selected as the final antibody sequence from the candidate antibodies. A tag-free recombinant plasmid, F263, was also constructed. The recombinant single-domain antibody F263 fusion protein plasmid was transfected into HEK293 cells for expression. Purification was performed by Protein A affinity chromatography. However, it was found that the F263 antibody did not bind to the Protein A affinity chromatography medium, making subsequent protein purification difficult. Therefore, the F263 antibody sequence needed to be optimized to enable affinity chromatography purification.

[0299] The FR (Framework region) of the F263 antibody sequence was analyzed, and amino acids related to binding to Protein A affinity filler were selected for mutation. It was finally determined that the mutant F263-RS could bind to Protein A affinity chromatography filler.

[0300] Table 7. Nomenclature and amino acid sequences of F263 and its mutants Note: Bold underlines indicate mutated amino acids; boxes mark CDRs

[0301] The recombinant plasmid F263 and the mutated recombinant plasmid F263-RS were respectively used to express antibodies in HEK293 cells and purified by Protein A affinity chromatography to verify the binding of the mutated recombinant plasmid to the affinity filler.

[0302] Figure 7 shows the SDS-PAGE analysis results of the purified antibodies before and after mutation. The results show that the modified antibodies can bind to the affinity medium Protein A, and the protein is eluted in buffer C.

[0303] 4.2. Sequence optimization of F263 antibody affinity, humanization, and hydrophobic amino acids

[0304] Camelid antibodies offer advantages over traditional antibodies, such as their small molecular weight, excellent permeability, and ease of passage through blood vessels and tissues to reach their target sites. These advantages have led to their widespread use as diagnostic and detection tools for diseases. However, long-term clinical use can produce varying degrees of immune responses, compromising therapeutic efficacy. Therefore, the F263 antibody required humanization. This humanization was accomplished by humanizing the protein's surface amino acids and transplanting a portion of the VHH humanized universal framework.

[0305] The humanization steps are as follows: obtain the universal humanized VHH framework h-NbBcIII0FGLA (PDB number: 3EAK) designed by Cecile Vincke et al. based on sequence homology. The framework design is based on the nanobody NbBcIII0 antibody (PDB number: 3DWT). The protein surface amino acids are humanized with reference to human antibodies, and the sites that need to be humanized are selected according to the specific situation of the F263 nanobody sequence.

[0306] During radioactive imaging or therapy, nanobodies exhibit nonspecific uptake by nontarget organs, in addition to specific uptake at the tumor site. The presence of hydrophobic amino acids on the nanobody surface may increase uptake by organs like the liver, leading to accumulation of radionuclides in these organs during radiotherapy or imaging, potentially causing hepatotoxicity. To mitigate this, the PDB model of the F263 antibody was analyzed, and several highly hydrophobic amino acids on the antibody surface were selected for mutation testing (see Table 8 for details).

[0307] Antibody affinity directly impacts tumor binding and drug efficacy, so improving antibody affinity is a crucial component of sequence optimization. Typically, certain amino acids in the CDR3 region of an antibody significantly influence antibody affinity. Therefore, we analyzed the PDB model of the F263 antibody to identify key amino acids in the CDR3 region that may influence affinity and attempted mutations.

[0308] Table 8. Nomenclature and amino acid sequences of F263 mutants Note: The bold underline indicates the mutation point, and the box indicates the CDR.

[0309] Each of the above mutants was constructed and expressed. For ease of detection, transient transfection was performed using a recombinant plasmid with a histidine tag. After transient transfection, the recombinant protein was purified by affinity chromatography using the corresponding Ni+ resin gel. The resulting target protein was then used for subsequent detection.

[0310] 4.3. Affinity testing of modified antibodies

[0311] 4.3.1 ELISA test of antibody affinity after modification

[0312] The NHis-FAP fusion protein was coated on the plate at 5 μg / mL, with 100 μL per well, overnight at 4°C, and after washing, 3% BSA was added for blocking and incubated at 37°C for 1 hour. The modified antibody protein was diluted 4-fold with a starting concentration of 10 μg / mL, with a total of 11 concentrations. 100 μL was added to each well and reacted at 37°C for 1 hour. After washing, anti-camelid VHH secondary antibody (1:5000 LOT#2009K104) 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 Using antibody F263-RS as a control, the relative activities of the modified antibodies were compared to reflect the affinity of the modified antibodies for FAP.

[0313] Table 9 ELISA test results of the modified antibodies

[0314] The results are shown in Table 9. Although some modifications reduced the affinity of the antibodies, others significantly improved, with the highest increase being approximately 6-fold (F263-YW-GG). This indicates that the selected sites were relatively appropriate and achieved the goal of optimizing the antibodies.

[0315] 4.3.2 FACS detection of antibody affinity after modification

[0316] Culture and collect HEK293-FAP cells and adjust their density to 2×10 5 A 100 μL cell aliquot was mixed with 10 μg / mL of the engineered antibody, followed by 0.1 μg / mL of F263-cHis antibody and incubation 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 1X PBS + 0.5% BSA, resuspended, and analyzed on a flow cytometer.

[0317] The specific test results are shown in Table 10. Although the affinity of some modified antibodies decreased, the affinity of some modified antibodies was significantly improved, and the results were consistent with the ELISA results, indicating that the optimization was relatively successful and that relatively good antibodies could be selected for further verification.

[0318] Table 10 FACS test results of modified antibodies Note: FACS uses competitive detection. The lower the MFI value, the better the affinity.

[0319] 4.3.3 Antibody internalization detection after modification

[0320] ELISA and FACS analysis of the modified antibodies revealed that some showed significantly improved affinity compared to the pre-modified antibodies. Therefore, antibodies with significantly improved affinity were selected for subsequent internalization testing. Mutating the CDR3 sequences RRWGVSRNGKAWDY (SEQ ID NO: 185) or RRWGVSRNSKAWDY (SEQ ID NO: 186) may help maintain or even improve affinity.

[0321] Detection was performed using a validated temperature-controlled secondary antibody assay: HEK293-FAP cells were harvested and divided into 1x10^5 cells / tube, with 100μL / tube incubated on ice for 20 minutes. F263-RS, F263-YW-GG, hF263-4, and hF263-4-GG were added at a final concentration of 5μg / mL and incubated on ice for 30 minutes. After centrifugation and washing three times, the cells were grouped and incubated on ice for 0, 1, 2, and 4 hours, and at 37°C for 1, 2, and 4 hours. Anti-camelid VHH secondary antibody was added and incubated on ice for 30 minutes. After centrifugation and washing three times, the cells were resuspended in 200μL and loaded onto the flow cytometer. Flow cytometric analysis was performed to determine the mean fluorescence intensity (MFI). At the same time, (MFI 0℃ -MFI 37℃ ) / MFI 0℃ *100% is the internalization rate of the antibody at time.

[0322] The specific results are shown in Figure 8. At 1 hour, the internalization rates of the four candidate antibodies were around 20% to 40%. As time went on, at 4 hours, except for the F263-RS antibody, the internalization rate of the remaining antibodies was 0. This may be because the half-life of the nanobody is short, and some antibodies may fall off the cells over time, resulting in a lower internalization rate of the antibodies at 4 hours.

[0323] 4.4. Antibody transformation 68 Ga distribution experiment

[0324] 4.4.1 Coupling of F263-RS, F263-YW-GG, hF263-4, and hF263-4-GG with NODA-GA

[0325] In order to facilitate site-specific conjugation of antibodies and small molecules and avoid the phenomenon of uneven DAR values ​​or reduced affinity of random conjugation, the four candidate antibodies were constructed by adding GSC to the ends, and HEK293 was used for transient transfection to obtain the corresponding proteins for subsequent experiments.

[0326] F263-RS, F263-YW-GG, hF263-4, and hF263-4-GG proteins were mixed with TCEP (tris(2-carboxyethyl)phosphine) reducing agent at a molar ratio of 1:5. The mixed mixture was reacted at 37°C for 2 hours to reduce and open the disulfide bonds in the antibody. At the same time, NODA-GA chelating agent was dissolved in PBS. The reduced antibody was mixed with NODA-GA at a molar ratio of 1:10 and reacted at 37°C for 2 hours to allow the antibody to couple with the chelating agent. After coupling is complete, the solution was ultrafiltered to remove residual small molecule chelating agent.

[0327] 4.4.2 Conjugated Antibody 68 Ga radiolabeling

[0328] Prepared by rinsing an Eckert & Ziegler IGG100 germanium 68 / gallium 68 (Ge 68 / Ga 68) generator with 0.1 M sterile HCl 68 Ga eluent, add an equal volume of 2M sodium acetate solution, and add 1 / 4 volume of 0.1M sodium acetate buffer with pH 5.3 containing NODA-GA-FAP antibody. The pH of the reaction system is between 4.5 and 4.7. React at room temperature for 10 minutes, remove unreacted ionic gallium through a PD-10 column and replace the product buffer system with physiological saline at the same time, and filter with a 0.22μm filter membrane. The product is quality controlled by analyzing pH, Radio-TLC, Radio-HPLC, radioactivity, radiochemical purity, etc. In addition: 0.05M sterile HCl can also be used to elute the ITG germanium 68 / gallium 68 (Ge-68 / Ga-68) generator for preparation. 68 For the Ga eluent, 1 / 2 volume of 2 M sodium acetate solution was added, and other reaction and quality control conditions were the same.

[0329] 4.4.3 For research 68 Tumor distribution experiment of Ga-labeled modified FAP single domain antibody

[0330] Twelve female BALBc-Nude mice, 6-8 weeks old, were housed in an SPF-protected environment with free access to food. A standard 12-hour day-night lighting cycle was used. 100 μL of HEK293-FAP cells / PBS were injected subcutaneously into the right axilla of the mice. The cell seeding density was approximately 5-6 × 10 6 cells / mouse. The tumor volume is available after 3-4 weeks, approximately 100-300 mm 3 .

[0331] Will 68Ga-F263-RS-GSC / F263-YW-GG-GSC / hF263-4-GSC / hF263-4-GG-GSC test products were injected into HEK293-FAP-tumor-bearing nude mice via tail vein, with 3 mice in each group, and each mouse was dosed at 100 μL / 1 μg / 100 uCi. 60 minutes after administration, the mice were euthanized, and the tissues were dissected, weighed, and counted on a gamma counter to determine the % injected dose per gram (% ID / g) of each tissue.

[0332] As shown in Figure 9, the four modified FAP single-domain antibodies were highly enriched in tumors. Tumor uptake of the modified antibodies was significantly increased compared to the unmodified antibodies, while liver uptake was reduced, indicating that the modified antibodies had higher affinity and stronger specificity. The higher kidney uptake indicates that the antibodies are metabolized by the kidneys.

Claims

1. An FAP-binding polypeptide comprising at least one immunoglobulin single variable domain that specifically binds to FAP, 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, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, and 163 - 184.

2. The FAP-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, SEQ ID NO: 185, or SEQ ID NO: 186; (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; (22)CDR1 shown in SEQ ID NO:86, CDR2 shown in SEQ ID NO:87, CDR3 shown in SEQ ID NO:88; (23)CDR1 shown in SEQ ID NO:90, CDR2 shown in SEQ ID NO:91, CDR3 shown in SEQ ID NO:92; (24)CDR1 shown in SEQ ID NO:94, CDR2 shown in SEQ ID NO:95, CDR3 shown in SEQ ID NO:96; (25)CDR1 shown in SEQ ID NO:98, CDR2 shown in SEQ ID NO:99, CDR3 shown in SEQ ID NO:100; (26)CDR1 shown in SEQ ID NO:102, CDR2 shown in SEQ ID NO:103, CDR3 shown in SEQ ID NO:104; (27)CDR1 shown in SEQ ID NO:106, CDR2 shown in SEQ ID NO:107, CDR3 shown in SEQ ID NO:108; (28)CDR1 shown in SEQ ID NO:110, CDR2 shown in SEQ ID NO:111, CDR3 shown in SEQ ID NO:112; (29)CDR1 shown in SEQ ID NO:114, CDR2 shown in SEQ ID NO:115, CDR3 shown in SEQ ID NO:116; (30)CDR1 shown in SEQ ID NO:118, CDR2 shown in SEQ ID NO:119, CDR3 shown in SEQ ID NO:120; (31)CDR1 shown in SEQ ID NO:122, CDR2 shown in SEQ ID NO:123, CDR3 shown in SEQ ID NO:124; and (32)CDR1 shown in SEQ ID NO:126, CDR2 shown in SEQ ID NO:127, CDR3 shown in SEQ ID NO:

128.

3. The FAP-binding polypeptide of claim 1 or 2, wherein the single variable domain of the immunoglobulin 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 to any 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, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, and 162 - 184.

4. The FAP-binding polypeptide of any one of claims 1 - 3, wherein the single variable domain of the immunoglobulin comprises any 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, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, and 162 - 184.

5. The FAP-binding polypeptide of any one of claims 1 - 4, wherein the single variable domain of the immunoglobulin is capable of binding protein A.

6. The FAP-binding polypeptide of claim 5, wherein the single variable domain of the immunoglobulin 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 to the amino acid sequence shown in SEQ ID NO:5, and wherein, compared to SEQ ID NO:5, the amino acid residue N at position 19 is replaced by R, and the amino acid residue F at position 71 is replaced by S.

7. The FAP-binding polypeptide of claim 6, wherein the single variable domain of the immunoglobulin comprises the amino acid sequence shown in any one of SEQ ID NO: 162, 177, 181 or 183.

8. The FAP-binding polypeptide of any one of claims 1-7, wherein the single variable domain of the immunoglobulin is a VHH.

9. The FAP-binding polypeptide of any one of claims 1-8, wherein the single variable domain of the immunoglobulin is humanized.

10. A nucleic acid molecule encoding the FAP-binding polypeptide of any one of claims 1-9.

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

12. A host cell comprising the nucleic acid molecule of claim 10 or transformed with the expression vector of claim 11 and capable of expressing the FAP-binding polypeptide.

13. A method for producing the FAP-binding polypeptide of any one of claims 1-9, comprising: a) culturing the host cell of claim 12 under conditions permitting expression of the FAP-binding polypeptide; b) recovering the FAP-binding polypeptide expressed by the host cell from the culture obtained in step a); and c) optionally further purifying and / or modifying the FAP-binding polypeptide obtained in step b).

14. A conjugate molecule comprising the FAP-binding polypeptide of any one of claims 1-9 and at least one detectable label and / or therapeutic moiety conjugated to the FAP-binding polypeptide.

15. The conjugate molecule of claim 14, wherein the detectable label is selected from radionuclides, fluorophores, chemiluminescent agents, bioluminescent agents, paramagnetic ions, and enzymes.

16. The conjugate molecule of claim 15, wherein the detectable label 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, 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 label is 68 Ga or 177 Lu.

17. The conjugate molecule of any one of claims 14-16, wherein the FAP-binding polypeptide is conjugated to the detectable label through a chelator.

18. The conjugate molecule of claim 17, 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.

19. The conjugate molecule of claim 18, wherein the detectable label is 68 Ga and the chelator is NOTA, or the detectable label is 177 Lu and the chelator is DOTA.

20. A method for detecting the presence and / or amount of FAP in a biological sample, comprising: a) contacting the biological sample and a control sample with the FAP-binding polypeptide of any one of claims 1-9 or the conjugate molecule of any one of claims 14-19 under conditions that permit formation of a complex between the FAP-binding polypeptide or conjugate molecule and FAP; b) detecting the formation of the complex, wherein a difference in complex formation between the biological sample and the control sample indicates the presence and / or amount of FAP in the sample.

21. A diagnostic agent for detecting and / or diagnosing FAP-related diseases such as cancer or inflammatory diseases, comprising an FAP-binding polypeptide of any one of claims 1-9 and / or a conjugate molecule of any one of claims 14-19, and optionally a physiologically acceptable carrier.

22. The diagnostic agent of claim 21, wherein the diagnostic agent is a contrast agent.

23. The diagnostic agent of claim 22, which is a contrast agent.

24. The diagnostic agent of claim 23, wherein the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.

25. Use of an FAP-binding polypeptide of any one of claims 1-9 and / or a conjugate molecule of any one of claims 14-19 in the preparation of a diagnostic agent for detecting and / or diagnosing FAP-related diseases such as cancer or inflammatory diseases.

26. The use of claim 25, wherein the diagnostic agent is a contrast agent.

27. The use of claim 26, which is a contrast agent.

28. The use of claim 27, wherein the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.

29. A method for detecting and / or diagnosing FAP-related diseases such as cancer or inflammatory diseases in a subject, comprising administering to the subject an FAP-binding polypeptide of any one of claims 1-9 and / or a conjugate molecule of any one of claims 14-19 and / or a diagnostic agent of any one of claims 21-24.

30. The method of claim 29, further comprising the step of imaging the subject, such as ECT imaging.

31. The method of claim 30, wherein the ECT imaging is SPECT imaging, or the ECT imaging is PET imaging.

32. The diagnostic agent of any one of claims 21-24, the use of any one of claims 25-28, or the method of any one of claims 29-31, wherein the FAP-related diseases are selected from cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloid disease.

33. The diagnostic agent, use, or method of claim 32, wherein the cancer is selected from breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocarcinoma, clear cell renal cell carcinoma, neuroendocrine tumors, oncogenic osteomalacia, sarcoma, CUP (cancer of unknown primary), thymic cancer, glioma, glioblastoma, astrocytoma, cervical cancer, and prostate cancer.

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