Specific binding molecules for fibroblast-activating proteins (FAPs)

Abstract

The present invention relates to new ubiquitin-derived molecules that specifically bind to fibroblast activation proteins (FAPs). The invention further relates to FAP-binding ubiquitin-derived molecule (Affilin®) proteins that further comprise a diagnostically or therapeutically active component. Further aspects of the invention cover such FAP-binding proteins for use in medicine, e.g. for use in the diagnosis (including imaging) or treatment of FAP-associated tumors.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to a novel ubiquitin-derived molecule that specifically binds to fibroblast activation protein (FAP). The present invention further relates to an FAP-binding ubiquitin-derived molecule (Affilin®) protein further comprising components of diagnostic or therapeutic activity. Further aspects of the present invention cover such FAP-binding proteins for use in pharmaceuticals, for example, for use in the diagnosis (including imaging) or treatment of FAP-related tumors. [Background technology]

[0002] Background of the Invention Cancer-associated fibroblasts (CAFs) interact with cancer cells. Cancer cells induce activation of cancer-associated fibroblasts, and cancer-associated fibroblasts support tumor growth, metastasis, invasion, and immunosuppression. The proteases expressed by activated CAFs are fibroblast-activating proteins (FAPs; also known as prolyl endopeptidase FAP, dipeptidyl peptidase FAP, integral membrane serine proteases, surface-expressed proteases, etc.). As stromal cell surface proteases, FAPs thereby influence extracellular matrix remodeling, signaling, immunosuppression, and other processes.

[0003] FAP is highly upregulated in numerous cancers, including almost all carcinomas, such as breast, colorectal, pancreatic, lung, brain, intrahepatic bile duct, and ovarian cancers. Additionally, high levels of FAP expression can be detected in some tumors originating from non-epithelial tissues, such as melanoma and myeloma. Overexpression of FAP promotes tumorigenesis and metastasis. It is either not expressed or only weakly in normal adult tissues. Examples include the uterus, cervix, placenta, breast, and skin, which show low to moderate expression compared to tumors. However, high FAP expression occurs in wound healing, inflammation, such as arthritis, atherosclerotic plaques, and fibrosis, as well as in ischemic cardiac tissue after myocardial infarction.

[0004] The application of FAP in the diagnosis and treatment of various cancers has been reported. Several antibodies have been developed as ligands for FAP, such as cibrotuzumab. Despite its tumor-stromal targeting properties, cibrotuzumab failed in a Phase II clinical trial for metastatic colorectal cancer. Additionally, eight of the 26 patients treated with cibrotuzumab developed human-anti-human antibodies along with pharmacokinetic changes, and tumor uptake was reduced in four of those patients. Therefore, further clinical development of cibrotuzumab was halted.

[0005] High levels of FAP expression are associated with a poor prognosis in patients. However, the diagnosis and treatment of FAP-related cancers are not adequately addressed by existing options, and as a result, many patients do not fully benefit from current strategies.

[0006] Needless to say, there is an urgent need for novel strategies for the diagnosis and treatment of tumors with FAP overexpression. [Overview of the project]

[0007] One object of the present invention is to provide molecules for the specific targeting of FAP to enable targeted diagnostic and treatment options, including imaging of FAP-positive tumors, for example, by radiological diagnostic methods and FAP-targeted radiopharmaceuticals. Targeting this tumor-associated protein may benefit patients with unmet needs for novel diagnostic and therapeutic pathways. Specific targeting of FAP suggests potentially non-toxic diagnostic and treatment approaches due to the low and limited distribution of FAP in normal tissues. Thus, binding proteins specific to FAP may enable effective medical options against cancer and ultimately improve the quality of life for patients.

[0008] This invention provides novel FAP-binding molecules for new and improved strategies in the diagnosis and treatment of FAP-related cancers. Furthermore, the novel FAP-binding molecules of this invention provide improved strategies in the diagnosis and treatment of cancers related to FAP overexpression.

[0009] The above-mentioned objectives and advantages are achieved by the subject matter of the appended claims. The present invention satisfies the needs presented above by providing examples of FAP-binding proteins. The above summary does not necessarily describe all the problems that the present invention solves.

[0010] Summary of the Invention This disclosure provides, but is not limited to, the following items 1-11:

[0011] 1. A protein having at least 80% identical amino acid sequences to any one selected from the group of SEQ ID NOs: 1-12 and 15-27, and having a specific binding affinity for human fibroblast-activating protein (hFAP) at a concentration of less than 100 nM. In some embodiments, a protein having at least 90% identical amino acid sequences to any one selected from the group of SEQ ID NOs: 1-12 and 15-27 has a specific binding affinity for hFAP at a concentration of less than 50 nM.

[0012] 2. A multimer of the protein described in item 1. A multimer contains multiple proteins described in item 1. A multimer is a dimer, trimer, or tetramer of the protein described in item 1.

[0013] 3. A fusion protein containing the proteins listed in items 1 and 2.

[0014] 4. Proteins of items 1-3, further comprising at least one additional linking site for linking a chemical moiety. Optionally, the chemical moiety may be selected from chelators, drugs, toxins, dyes, and small molecules.

[0015] 5. A protein described in any one of items 1-4, wherein the fusion protein additionally comprises at least one diagnostically active moiety. Optionally, the diagnostically active moiety may be selected from radionuclides, fluorescent proteins, photosensitizers, dyes, enzymes, magnetic beads, metal beads, colloidal particles, high electron density reagents, biotin, digoxigenin, haptens, CAR-T receptors, or exosomes, or any combination thereof.

[0016] 6. Proteins of items 1-4, further comprising at least one therapeutically active moiety. Optionally, the diagnostically active moiety may be selected from monoclonal antibodies or fragments thereof, binding proteins, receptors or receptor domains, receptor ligands, radionuclides, cytotoxic compounds, cytokines, chemokines, enzymes, CAR-T receptors, or exosomes, or derivatives thereof, or any combination thereof.

[0017] 7. The protein of items 1-6, additionally comprising at least one moiety that modulates pharmacokinetics. Optionally, the moiety that modulates pharmacokinetics is selected from serum albumin, albumin-binding protein, immunoglobulin-binding protein, or immunoglobulin or immunoglobulin fragment, polysaccharide, amino acids alanine, glycine, serine, proline-containing unstructured amino acid sequence, polyethylene glycol, sialic acid, or transferrin.

[0018] 8. The protein of items 1-7 for use in the diagnosis or treatment of FAP-related diseases, such as FAP-related tumors.

[0019] 9. A composition comprising the protein of items 1-8 for use in medicine, preferably for use in the diagnosis or treatment of FAP-related diseases.

[0020] 10. A method for producing the protein of items 1-8, comprising the steps of: a) culturing a host cell under conditions suitable for obtaining said protein and b) isolating the produced said protein.

[0021] 11. A method for detecting FAP constituting a sample using the protein of items 1-8 and detecting the binding of FAP using the protein of items 1-8, by contacting the sample with the protein of items 1-8.

[0022] This summary does not necessarily describe all features of the invention. Other embodiments will become apparent from consideration of the subsequent detailed description.

Brief Description of the Drawings

[0023] [Figure 1]Figure 1 shows the analysis of the binding of Affilin (registered trademark) proteins to hFAP-Fc (label-free interaction assay using SPR). hFAP was immobilized on a CM-5 chip. After fitting the data using a 1:1 Langmuir model, the KD value was calculated. Figure 1A shows Affilin (registered trademark)-217990 (SEQ ID NO: 1) (KD = 14 nM) against hFAP. Figure 1B shows Affilin (registered trademark)-217832 (SEQ ID NO: 4) (KD = 3 nM) against hFAP. Figure 1C shows Affilin (registered trademark)-217993 (SEQ ID NO: 11) (KD = 23 nM) against hFAP. [Figure 2] Figure 2 shows the analysis of the binding of Affilin (registered trademark)-217990, which has high affinity for mFAP-Fc (label-free interaction assay using SPR). mFAP was immobilized on a CM-5 chip. After fitting the data using a 1:1 Langmuir model, a KD value of 90 nM was calculated. [Figure 3] Figures 3, 4, 5, and 6 show the binding affinity of FAP-binding proteins in serum after 24-hour incubation. The KD values for hFAP-Fc were determined after 0-hour serum incubation (black circles) and after 24-hour incubation in serum at 37°C (black triangles) (KD determination by ELISA in Figures 3 - 5, KD determination by flow cytometry in Figure 6). After 24-hour incubation in serum, the KD values showed only minor variations compared to the KD values before incubation in serum, confirming the stability of the FAP-specific Affilin (registered trademark) proteins. Figure 3 shows the binding affinity of the FAP-binding protein Affilin (registered trademark)-217990 (SEQ ID NO: 1) in human serum (Figure 3A) and mouse serum (Figure 3B). [Figure 4] Figure 4 shows the binding affinity of the FAP-binding protein Affilin (registered trademark)-220257 (SEQ ID NO: 22) in human serum (Figure 4A) and mouse serum (Figure 4B). [Figure 5]Figure 5 shows the binding affinity of the FAP-binding protein Affilin®-220164 (SEQ ID NO: 19) in human serum (Figure 5A) and mouse serum (Figure 5B). [Figure 6] Figure 6 shows the binding affinity of FAP-specific Affilin®-217863 (SEQ ID NO: 5) in human serum. [Modes for carrying out the invention]

[0024] Detailed description of the invention The inventors have developed a solution to meet the strong existing need in the art to expand medical options for the diagnosis and treatment of cancer by providing novel FAP-binding proteins. The FAP-specific proteins defined herein are functionally characterized by a high specific affinity for FAP, even on cells expressing FAP. Furthermore, they exhibit a high level of stability in serum (i.e., characterized by their binding affinity to FAP). The present invention provides FAP-binding proteins based on a ubiquitin scaffold (also known as the Affilin® molecule). The FAP-binding proteins described herein thereby provide a molecular format with favorable physicochemical properties, high levels of expression in bacteria, and enable a simple manufacturing method. Novel FAP-binding proteins can expand previously unmet medical strategies for the diagnosis and treatment of FAP-related cancers. In particular, FAP-binding proteins may be used, for example, for imaging purposes, for the presence of tumor cells expressing FAP, and for radiotherapy treatment of FAP-expressing tumors, or for immuno-oncological treatment options.

[0025] Before the present invention is described in more detail below, it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, and that they may be modified. It should also be understood that the technical terms used herein are for the sole purpose of describing specific aspects and embodiments and are not intended to limit the scope of the present invention as reflected in the appended claims. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. This includes those skilled in the art engaged in the fields of protein manipulation and purification, as well as those skilled in the art engaged in the fields of technical applications and the development of novel target-specific binding molecules for use in therapy and diagnostics.

[0026] Preferably, terms used herein are defined as those described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, HGW, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0027] Throughout this specification and the subsequent claims, unless the context otherwise requires, the word “comprise,” and its variations, e.g., “comprises” and “comprising,” are understood to imply that they include the integers or steps, or groups of integers or steps, described, but not exclude any other integers or steps or groups of integers or steps. The terms “comprise(s)” or “comprising” may also encompass limitations to “consists of” or “consisting of,” where such limitations are necessary for any reason and to any extent.

[0028] Several documents (e.g., patents, patent applications, scientific publications, producer specifications, instructions, UniProt accession numbers, etc.) may be referenced throughout this specification. Nothing in this specification should be construed as an acknowledgment that the present invention has no prior rights to such disclosures on the grounds of prior art. Some of the documents referenced in this specification may be characterized as “incorporated by reference.” In the event of any conflict between the definitions or teachings of such incorporated references and the definitions or teachings described herein, the description herein shall prevail.

[0029] All sequences referenced herein are disclosed in the attached sequence listing (WIPO ST.26 *.xml format), which, together with its contents and the entirety of the disclosure, constitute a part of the disclosures herein. For the avoidance of doubt and as a precautionary measure, the sequence listing in accordance with WIPO ST.25, which constitutes a part of the priority application EP 22 156 353.9, is incorporated herein by reference.

[0030] General definitions of key terms used in this application The term "FAP," as used herein, refers to Uniprot accession number Q12884. It refers to fibroblast-activating proteins (FAPs), also known as prolyl endopeptidase FAP, dipeptidyl peptidase FAP, integral membrane serine proteases, surface-expressed proteases, etc. The term "FAP" includes all polypeptides exhibiting at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, or 97% or more, or 100% sequence identity to Uniprot accession number Q12884 (human) FAP. Human FAP is 89.5% identical to mouse FAP (accession number P97321), 88.6% identical to rat FAP, and 99.6% identical to cynomolgus monkey FAP (accession number A0A2K5VGF4). The term "FAP" includes the extracellular domain of FAP (residues 26-760).

[0031] The terms “FAP-binding protein,” “FAP-specific Affilin® protein,” or “protein containing FAP-binding protein” are used interchangeably herein and refer to proteins that exhibit high affinity binding to FAP.

[0032] The term "Affilin®" is a registered trademark of Navigo Proteins® GmbH and refers to a non-immunoglobulin-derived binding protein. In the context of this invention, the term "Affilin" refers to ubiquitin mutein.

[0033] The terms “protein” and “polypeptide” refer to any chain of two or more amino acids linked by peptide bonds, and do not refer to a specific length of the product. Therefore, “peptide,” “protein,” “amino acid chain,” or any other term used to refer to a chain of two or more amino acids is included within the definition of “polypeptide,” and the term “polypeptide” may be used in place of or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-translational modifications of polypeptides, as is well known in the art.

[0034] The term “modification” or “amino acid modification” refers to the substitution, deletion, or insertion of a reference amino acid by another amino acid at a specific position in the parent polypeptide sequence. With regard to known genetic codes, as well as recombinant and synthetic DNA techniques, those skilled in the art can readily construct DNA encoding amino acid variants.

[0035] The term "ubiquitin" refers to proteins that have at least 95% identity with the amino acid sequence given in Sequence ID No. 13, such as those with point mutations at positions 45, 75, and 76 that do not affect binding to FAP.

[0036] The term "mutein," as used herein, refers to a derivative of ubiquitin, such as the one shown in Sequence ID No. 13, which is determined by the substitution, insertion, deletion, or any combination thereof of amino acids from the aforementioned amino acid sequence, but which has a specific binding affinity to FAP. The FAP-binding protein of the present invention is a ubiquitin mutein protein (ubiquitin mutein). The term "substitution" is understood as the exchange of an amino acid with another amino acid. The term "insertion" includes the addition of an amino acid to the original amino acid sequence.

[0037] The terms “binding affinity” and “binding activity” may be used interchangeably herein and refer to the ability of a polypeptide to bind to another protein, peptide, or a fragment or domain thereof. Binding affinity is measured using the equilibrium dissociation constant (K) used to evaluate and rank the order of strength of bimolecule interactions. D Typically measured and reported by )

[0038] The term "fusion protein" refers to a protein comprising at least one first protein genetically linked to at least one second protein. Fusion proteins are created through the linkage of two or more genes that originally encoded separate proteins. Fusion proteins may further include additional domains not involved in target binding, such as, but not limited to, multimerizing moieties, polypeptide tags, polypeptide linkers, or moieties that bind to targets different from FAPs.

[0039] The term "amino acid sequence identity" refers to the quantitative comparison of the identity (or differences) of the amino acid sequences of two or more proteins. The "amino acid sequence identity percentage (%)" for a reference polypeptide sequence is defined as the percentage of amino acid residues in the sequence that are identical to those in the reference polypeptide sequence, after the sequences have been aligned and gaps introduced where necessary to achieve the maximum sequence identity percentage. To determine sequence identity, the sequence of the query protein is aligned to the sequence of the reference protein or polypeptide. Sequence alignment methods are well known in the art. For example, to determine the degree of amino acid sequence identity of any polypeptide compared to another amino acid sequence, a SIM local similarity program, as known in the art, is preferably used. For multiple alignment analysis, Clustal Omega is preferably used, as known to those skilled in the art.

[0040] As used herein, the terms “percent identical” or “percent amino acid sequence identity (%)” or “identity percentage” refer to two or more sequences or subsequences having, in some embodiments at least 80%, in some embodiments at least 85%, in some embodiments at least 90%, in some embodiments at least 91%, in some embodiments at least 92%, in some embodiments at least 93%, in some embodiments at least 94%, in some embodiments at least 95%, in some embodiments at least 96%, in some embodiments at least 97%, in some embodiments at least 98%, and in some embodiments 100% amino acid residue identity when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For the sake of clarity, for example, a sequence having at least 80% identity includes all sequences having more than 80% identity, such as embodiments having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid identity. For the sake of clarity, for example, a sequence having at least 90% identity includes all sequences having 90% or more identity, such as embodiments having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid identity.

[0041] The term "fused" means that polypeptide components or units are linked by peptide bonds, either directly or via peptide linkers. In various embodiments, the term "fused" may also mean that polypeptide components or units are linked by non-peptide linkers, for example, through chemical conjugation.

[0042] The term "fusion protein" refers to a protein comprising at least one first protein genetically linked to at least one second protein. Fusion proteins are created through the linkage of two or more genes that originally encoded separate proteins. Thus, fusion proteins may contain a multimer of identical or different proteins expressed as a single linear polypeptide. In various embodiments, fusion proteins are created through the linkage of two or more polypeptides via a non-peptide linker, for example, through chemical conjugation. Fusion proteins may further include additional domains not involved in target binding, such as, but not limited to, a multimerizing moiety, a polypeptide tag, a polypeptide linker, or a moiety that binds to a target different from the FAP.

[0043] Detailed description of embodiments of the present invention Structural characterization of FAP-binding proteins. The FAP-binding proteins described herein contain, essentially consist of, or consist of an amino acid sequence selected from any one of the following groups: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or each FAP-binding protein is selected from an amino acid sequence having at least 80% identity to it. In various embodiments, the FAP-binding protein comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of the amino acid sequences of SEQ ID NOs: 1-12 and 15-27. In various embodiments, the FAP-binding protein contains, essentially consists of, or comprises amino acids having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any of the amino acid sequences of SEQ ID NOs: 1-12 and 15-27.

[0044] All FAP-binding proteins are ubiquitin-based and structurally related. All FAP-binding proteins described herein share the same basic protein scaffold.

[0045] In some embodiments, the modification in ubiquitin that results in binding to FAP is located in the region corresponding to amino acids 6-15 and / or 42-46 and / or 62-72 of ubiquitin (SEQ ID NO: 13).

[0046] In some embodiments, the FAP-specific ubiquitin mutein has an aromatic amino acid (E64W or E64F) at position 64 of SEQ ID NO: 13.

[0047] In some embodiments, the FAP-specific ubiquitin mutein has proline (P) (S65P) at position 65 of SEQ ID NO: 13.

[0048] In some embodiments, the FAP-specific ubiquitin mutein has an aromatic amino acid (E64W or E64F) at position 64 of SEQ ID NO: 13 and proline (P) (S65P) at position 65 of SEQ ID NO: 13. In some embodiments, the FAP-specific ubiquitin mutein has an aromatic amino acid (K6W or K6Y) at position 6 of SEQ ID NO: 13.

[0049] In some embodiments, the FAP-specific ubiquitin mutein has a basic amino acid (A46K or A46R) at position 46 of SEQ ID NO: 13.

[0050] Some embodiments provide an FAP-specific ubiquitin mutein having at least seven further substitutions at positions 6, 8, 9, 10, 12, 42, 44, 45, 46, 62, 63, 66, 68, and 70 of SEQ ID NO: 13.

[0051] In some embodiments, one or more further substitutions in ubiquitin are provided.

[0052] In some embodiments, FAP-specific ubiquitin muteins have an additional insertion of six amino acids between the positions corresponding to positions 9 and 10 (see, for example, SEQ ID NOs: 1, 2, 15, and 16; Affilin®-217990, Affilin®-217966, Affilin®-219750, and Affilin®-220198, respectively). In some embodiments, FAP-specific ubiquitin muteins having an additional insertion of six amino acids between the positions corresponding to positions 9 and 10 of SEQ ID NO: 13 (ubiquitin) are (i) Alanine (A) at the amino acid position corresponding to position 44 of SEQ ID NO: 13 (exchange I44A; corresponding to position 50 in the Affilin® protein of SEQ ID NOs: 1, 2, 15, 16), and / or (ii) Glycine (G) at the amino acid position corresponding to position 64 of SEQ ID NO: 13 (exchange E64G; corresponding to position 70 in Affilin® protein of SEQ ID NOs: 1, 2, 15, 16), and / or (iii) Lysine (K) at the amino acid position corresponding to position 70 of SEQ ID NO: 13 (V70K; corresponding to position 76 in Affilin® protein of SEQ ID NOs: 1, 2, 15, and 16) It holds.

[0053] In some embodiments, one or more further substitutions in ubiquitin are provided.

[0054] In various embodiments, the FAP-binding protein contains, essentially comprises, or consists of the amino acid sequence of SEQ ID NO: 1, or amino acids having at least 80% identity, at least 85% identity, or at least 90% identity with respect to SEQ ID NO: 1. For example, SEQ ID NO: 1 (217990) is 93.9% identical to SEQ ID NO: 2 (217996) and SEQ ID NO: 16 (220198), respectively. SEQ ID NO: 1 (217990) is 87.8% identical to SEQ ID NO: 15 (217750).

[0055] In various embodiments, the FAP-binding protein comprises, essentially comprises, or consists of the amino acid sequence of SEQ ID NO: 4, or amino acids having at least 80% identity, at least 85% identity, or at least 90% identity with respect to SEQ ID NO: 4.

[0056] In various embodiments, the FAP-binding protein comprises, essentially consists of, or comprises amino acids having at least 80% identity, at least 85% identity, or at least 90% identity with respect to SEQ ID NO: 19.

[0057] Functional Characterization The FAP-binding proteins described herein have a binding affinity (K) of less than 100 nM for FAP. D ) are present. In some embodiments, the FAP-binding proteins described herein bind to human FAP with measurable binding affinities of less than 100 nM, less than 50 nM, less than 25 nM, less than 15 nM, or even less than 10 nM. Suitable methods are known to those skilled in the art or are described in the literature. Methods for determining binding affinity are known in themselves and may be selected from the following methods known in the art: enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), kinetic exclusion analysis (KinExA assay), biolayer interferometry (BLI), flow cytometry, fluorescence spectroscopy, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA), and enhanced chemiluminescence (ECL). Some of these methods are described in the following examples. In some embodiments, the FAP-binding proteins described herein bind to human FAP with measurable binding affinities of less than 100 nM, less than 50 nM, less than 25 nM, less than 15 nM, or even less than 10 nM, as determined by surface plasmon resonance. Typically, the dissociation constant K D This is determined at 20°C, 25°C, or 30°C. DThe lower the value, the higher the binding affinity for that biomolecule's binding partner. D The higher the value, the weaker the binding partners bind to each other (see Figure and Examples). In one embodiment, the FAP-binding protein has a dissociation constant K for human FAP in the range of 0.1 nM to 100 nM, preferably 0.1 nM to 50 nM, more preferably 0.1 nM to 25 nM, and even more preferably 0.1 nM to 15 nM. D It has the following properties. In some embodiments, the FAP-binding protein of the present invention binds to human FAP and cynomolgus monkey FAP. In some embodiments, the FAP-binding protein of the present invention binds to human FAP and mouse FAP.

[0058] A preferred embodiment relates to a protein having specific binding affinity for human fibroblast-activating protein (hFAP) of less than 50 nM, comprising at least 90%, preferably at least 94% or at least 95%, of identical amino acid sequences to any one selected from the group of SEQ ID NOs.

[0059] Other preferred embodiments relate to proteins that contain at least 90%, preferably at least 94%, or at least 95%, identical amino acid sequences to any one selected from the group SEQ ID NOs: 1-12 and 15-27, and have a specific binding affinity for human fibroblast-activating protein (hFAP) of less than 25 nM. Some preferred embodiments relate to proteins that contain at least 90%, preferably at least 94%, or at least 95%, identical amino acid sequences to any one selected from the group SEQ ID NOs: 1-12 and 15-27, and have a specific binding affinity for human fibroblast-activating protein (hFAP) of less than 15 nM. Some preferred embodiments relate to proteins that contain at least 90%, preferably at least 94%, or at least 95%, identical amino acid sequences to any one selected from the group SEQ ID NOs: 1-12 and 15-27, and have a specific binding affinity for human fibroblast-activating protein (hFAP) of less than 10 nM.

[0060] In some embodiments, the FAP-binding proteins described herein are particularly stable under different conditions, as shown in the examples and figures.

[0061] In some embodiments, the FAP-binding protein is stable at 37°C for at least 24 hours in the presence of serum. In some embodiments, as described in more detail in the examples, the FAP-binding protein is stable at 37°C for at least 24 hours in the presence of human serum. In some embodiments, the FAP-binding protein is stable at 37°C for at least 24 hours in the presence of mouse serum. For example, the stability of the FAP-binding protein is determined by its binding affinity (K) after incubation in serum at a high temperature of 37°C for a long period of time (e.g., 24 hours) using the standard methods described above and in the examples and figures herein. D) can be determined by measurement. The binding affinity for FAP can be evaluated for each binding protein before and after the serum incubation described herein. Some preferred embodiments relate to a protein comprising an amino acid sequence having at least 90%, preferably at least 94% or at least 95%, identity to any one selected from the group of SEQ ID NOs: 1-12, 15-27, and having a specific binding affinity for hFAP of less than 5 nM, preferably less than 2 nM, when determined via the ELISA described herein. Some preferred embodiments relate to a protein comprising an amino acid sequence having at least 90%, preferably at least 94% or at least 95%, identity to any one selected from the group of SEQ ID NOs: 1-12, 15-27, and having a specific binding affinity for hFAP of less than 5 nM, preferably less than 2 nM, after incubation at 37 °C for 24 hours in serum when determined via the ELISA described herein.

[0062] FAP-binding proteins that are stable in serum over an extended time period at 37 °C do not exhibit a significant loss in binding affinity for FAP, reflecting the exceptional stability of the FAP-binding proteins described herein. Not having a significant loss means that the binding affinity is not reduced to less than about one-half of the value obtained prior to incubation in serum.

[0063] In some embodiments, the FAP-binding proteins described herein are stable at high temperatures, preferably at least 60 °C, more preferably at least 70 °C. For stability analysis, for example, spectroscopic or fluorescence-based methods associated with chemical or physical unfolding are known to those skilled in the art. For example, the stability of a molecule is the melting temperature (T m ) in degrees Celsius (°C) at which half of the molecules are unfolded, which can be determined by measuring the temperature. Typically, the higher the T m , the more stable the molecule.

[0064] In some embodiments, the specific binding of the FAP-specific Affilin® protein described herein is confirmed by cellular FAP binding analysis using overexpressing cells (see Examples). Cellular FAP binding of the FAP-specific Affilin® protein can be determined by standard methods, including immunofluorescence microscopy and flow cytometry.

[0065] polymer Some embodiments relate to a protein having at least 90% identical amino acid sequences to any one selected from the group SEQ ID NOs: 1-12, 15-27, having a specific binding affinity for human fibroblast-activating protein (hFAP) of less than 50 nM, and being a multimer. In some embodiments, the FAP-binding protein is a multimer comprising, for example, multiple FAP-binding proteins as defined herein, each having at least 90%, preferably at least 94% or at least 95%, identical amino acid sequences to any one selected from the group SEQ ID NOs: 1-12, 15-27, and the protein has a specific binding affinity for human fibroblast-activating protein (hFAP) of less than 50 nM. The multimer may contain two, three, four or more FAP-binding proteins. In one embodiment, the FAP-binding protein comprises two, three, four or more linked FAP-binding proteins, i.e., the FAP-binding protein can be a dimer, trimer, or tetramer, etc. The polymer of the present invention is a fusion protein artificially produced by recombinant DNA technology, which is generally well known to those skilled in the art.

[0066] The polymer may contain two FAP-binding domains, which preferably contain or essentially consist of the above-described amino acid sequences. In some embodiments, the polymer is a dimer. The present invention provides dimers of the amino acid sequences of SEQ ID NOs: 11, 12, and 22.

[0067] In some embodiments, two or more FAP-binding proteins are directly linked. In some embodiments, two or more FAP-binding proteins are linked by a peptide linker. In various embodiments, two or more FAP-binding proteins are linked via a peptide linker of up to 30 amino acids. In some embodiments, two FAP-binding proteins are directly linked. In some embodiments, two FAP-binding proteins are linked by a peptide linker.

[0068] Fusion protein Some embodiments relate to proteins that contain at least 90%, preferably at least 94% or at least 95%, identical amino acid sequences to any one selected from the group SEQ ID NOs: 1-12, 15-27, have a specific binding affinity for human fibroblast-activating protein (hFAP) of less than 50 nM, and are fusion proteins. Some embodiments relate to proteins that contain at least 90%, preferably at least 94% or at least 95%, identical amino acid sequences to any one selected from the group SEQ ID NOs: 1-12, 15-27, have a specific binding affinity for human fibroblast-activating protein (hFAP) of less than 50 nM, and are polymers of such amino acids, and are fusion proteins.

[0069] In some embodiments, the protein of the present invention is a fusion protein comprising an FAP-binding protein as defined herein and at least a second protein. In some embodiments, the protein of the present invention is a fusion protein comprising at least 90%, preferably at least 94% or at least 95%, of an identical amino acid sequence to any one selected from the group SEQ ID NOs: 1-12, 15-27, having a specific binding affinity for human fibroblast-activating protein (hFAP) of less than 50 nM, and comprising at least a second protein. Thus, some embodiments include fusion proteins comprising one or more FAP-binding proteins and one or more further polypeptides disclosed herein.

[0070] Connection part In some embodiments, the protein of the present invention comprises an amino acid sequence of identity of at least 90%, preferably at least 94% or at least 95%, to any one selected from the group SEQ ID NOs: 1-12, 15-27, wherein the protein has a specific binding affinity for human fibroblast-activating protein (hFAP) of less than 50 nM, or a fusion protein containing such amino acids, and / or a polymer containing the above amino acid sequence, as well as one or more further linking sites for linking chemical moieties. In some embodiments, the protein comprising the FAP-binding protein described herein further comprises one or more linking sites for linking chemical moieties. The linking sites have the ability to react with other chemical groups to link the FAP-binding protein to the chemical moieties. The defined number and defined positions of the linking sites enable site-specific linking of chemical moieties to the FAP-binding proteins described herein. Thus, if required, a number of chemical moieties may be bound to the FAP-binding protein. The number of linking sites can be adjusted to an optimal number for a particular application by those skilled in the art in order to appropriately adjust the amount of chemical moieties. In selected embodiments, the binding sites may be selected from a group of one or more amino acids that can be labeled with a specific chemical, for example, one or more cysteine ​​residues, one or more lysine residues, one or more tyrosine residues, one or more tryptophan residues, or one or more histidine residues. The FAP-binding protein may contain 1 to 20 binding sites, preferably 1 to 6 binding sites, preferably 2 binding sites, or preferably 1 binding site.

[0071] Linked domains One embodiment provides an FAP-binding protein comprising at least one linking domain of 1 to 80 amino acids containing one or more linking sites. In some embodiments, the 1 to 80 amino acid linking domain may contain alanine, proline, or serine, and cysteine ​​as the linking site. In other embodiments, the 5 to 80 amino acid linking domain may consist of alanine, proline, serine, and cysteine ​​as the linking site. In one embodiment, the linking domain consists of 20 to 60% alanine, 20 to 40% proline, 10 to 60% serine, and one or more cysteines as the linking site at the C or N terminus of the FAP-binding protein described herein. In some embodiments, the amino acids alanine, proline, and serine are randomly distributed throughout the entire linking domain amino acid sequence, resulting in at most 2, 3, 4, or 5 identical amino acid residues, preferably at most 3, being adjacent to each other. The composition of the 1 to 20 linking domains may be different or identical.

[0072] In some embodiments, the chemical portion is selected from among chelators, drugs, toxins, dyes, and small molecules. In some embodiments, at least one of the chemical portions is a chelator designed as a complexing agent for linking one or more further portions to a targeted compound or to an FAP-binding protein disclosed herein. One embodiment relates to an FAP-binding protein in which the chelator is a complexing agent for linking one or more radioisotopes or other detectable labels.

[0073] Diagnostic part Various embodiments relate to the FAP-binding protein described herein or a fusion protein and diagnostic moiety comprising the FAP-binding protein described herein. Various embodiments relate to the FAP-binding protein and at least one diagnostic moiety described herein. Various embodiments relate to the FAP-binding protein and diagnostic moiety described herein. Various embodiments relate to a fusion protein comprising the FAP-binding protein and diagnostic moiety (i.e., at least one diagnostic moiety) described herein.

[0074] In some embodiments, such diagnostic components may be selected from radionuclides, fluorescent proteins, photosensitizers, dyes, enzymes, magnetic beads, metal beads, colloidal particles, high electron density reagents, biotin, digoxigenin, haptens, or any combination thereof.

[0075] In some embodiments, the FAP-binding proteins described herein (including fusion proteins described herein) may be used, for example, as imaging agents to assess the presence of tumor cells or metastases, tumor distribution, and / or tumor recurrence. Methods for detecting or monitoring cancer cells are accompanied by imaging methods. Such methods for imaging cancer cells include a protein comprising a FAP-binding protein described herein (including fusion proteins described herein) conjugated to a diagnostic portion for imaging FAP-associated cancer cells, for example, by radiation imaging or photoluminescence or fluorescence. In some embodiments, a method for imaging (detecting) cancer cells includes a FAP-binding protein described herein (including fusion proteins described herein) conjugated or linked to a diagnostic portion for imaging FAP-associated tumor cells, for example, a radionuclide or a fluorescent protein.

[0076] therapeutic part Various embodiments relate to the FAP-binding protein described herein or a fusion protein and therapeutic moiety comprising the FAP-binding protein described herein. Various embodiments relate to the FAP-binding protein and at least one therapeutic moiety described herein. Various embodiments relate to the FAP-binding protein and therapeutic moiety described herein. Various embodiments relate to a fusion protein comprising the FAP-binding protein and therapeutic moiety (i.e., at least one therapeutic moiety) described herein.

[0077] In some embodiments, such therapeutically active moieties may be selected from monoclonal antibodies or fragments thereof, extracellular domains of receptors or fragments thereof, binding proteins, radionuclides, cytotoxic compounds, cytokines, chemokines, enzymes, CAR-T receptors, or exosomes, or derivatives thereof, or any combination thereof.

[0078] In some embodiments, the FAP-binding proteins or fusion proteins described herein, including therapeutic active components, are used in a method of targeted delivery of any of the components listed above to FAP-expressing tumor cells. Thereafter, the FAP-specific binding proteins accumulate in the FAP-expressing tumor cells, and as a result of their high specificity for tumor cells, toxicity is expected to be only at low levels compared to normal cells. In some embodiments, the FAP-binding proteins (including fusion proteins) described herein are conjugated, linked, or attached to the therapeutic portion described herein.

[0079] Radioactive nuclides Suitable radionuclides for applications in imaging (e.g., in vitro) or for treatment methods involving radiotherapy include, but are not limited to, the gamma-emitting isotopes, positron emitters, beta emitters, and alpha emitters. In some embodiments, suitable conjugation partners include chelators, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA) or their activated derivatives, nanoparticles, and liposomes. In various embodiments, DOTA may be suitable as a conjugating agent for radioisotopes and as other agents for imaging.

[0080] The part that modulates pharmacokinetics Various embodiments relate to the FAP-binding protein described herein or a fusion protein comprising the FAP-binding protein described herein and a portion that modulates pharmacokinetics. Various embodiments relate to the FAP-binding protein described herein and at least one portion that modulates pharmacokinetics. Various embodiments relate to the FAP-binding protein described herein and a portion that modulates pharmacokinetics. Various embodiments relate to a fusion protein comprising the FAP-binding protein described herein and a portion that modulates pharmacokinetics (i.e., at least one portion that modulates pharmacokinetics).

[0081] In some embodiments, the FAP-binding protein described herein or a fusion protein comprising the FAP-binding protein of the present invention further comprises at least one moiety modulating the pharmacokinetics, the pharmacokinetic modulating moiety being selected from albumin-binding peptides, albumin-binding proteins, polyethylene glycol, serum albumin (e.g., mouse serum albumin or human serum albumin), immunoglobulin-binding peptides, immunoglobulins, immunoglobulin fragments, sialic acid, or transferrin, polysaccharides (e.g., hydroxyethyl starch), or unstructured amino acid sequences that increase the hydrodynamic radius (e.g., a polymer comprising the amino acids alanine, glycine, serine, and proline). In some embodiments, the FAP-binding protein described herein or a fusion protein comprising the FAP-binding protein described herein is conjugated, linked, or fused to the pharmacokinetic modulating moiety described above.

[0082] Some embodiments include a fusion protein containing the FAP-binding protein described herein and a portion that modulates the pharmacokinetics described above. Some embodiments include a fusion protein containing the FAP-binding protein described herein and an albumin-binding peptide or albumin-binding protein, an immunoglobulin-binding peptide, or an immunoglobulin or immunoglobulin fragment.

[0083] In some embodiments, the FAP-binding protein or fusion protein containing the FAP-binding protein described herein is conjugated, linked, or fused to a diagnostic portion and further conjugated, linked, or linked to the pharmacokinetic modulating portion described above. Some specific embodiments include the FAP-binding protein or fusion protein containing the FAP-binding protein described herein, as well as a radionuclide and a pharmacokinetic modulating portion.

[0084] In some embodiments, the FAP-binding protein or fusion protein containing the FAP-binding protein described herein is conjugated, linked, or fused to a therapeutic portion and further conjugated, linked, or linked to the pharmacokinetic modulating portion described above. Some embodiments include the FAP-binding protein or fusion protein containing the FAP-binding protein described herein, as well as the pharmacokinetic modulating portion and the therapeutic portion described above. Some specific embodiments include the FAP-binding protein or fusion protein containing the FAP-binding protein described herein, as well as the therapeutic portion and the pharmacokinetic modulating portion.

[0085] Several techniques are known in the art for producing proteins comprising FAP-binding proteins or fusion proteins containing FAP-binding proteins described herein having an extended half-life, such as direct fusion or chemical coupling methods of a pharmacokinetic modulating moiety with the FAP-binding proteins described herein or the fusion proteins containing the FAP-binding proteins described above. The pharmacokinetic modulating moiety may be attached, for example, at one or more sites of the FAP-binding proteins or fusion proteins containing FAP-binding proteins described herein via a peptide linker sequence or via the coupling sites described above.

[0086] Further parts In some embodiments, the conjugation of a proteinaceous or non-proteinaceous moiety to the FAP-binding protein or fusion protein containing the FAP-binding protein described herein may be carried out by applying chemical methods well known in the art. In some embodiments, coupling chemistry specific to the derivatization of cysteine ​​or lysine residues may be applied. Chemical linking (chemical coupling) may be carried out by chemistry well known to those skilled in the art, including but not limited to substitution, addition or cyclization, or oxidation chemistry (e.g., disulfide formation).

[0087] Molecules for purification / detection In some embodiments, additional amino acids can be extended at either the N-terminus or C-terminus or both of the FAP-binding protein or fusion protein containing the FAP-binding protein described herein. The additional sequence may include, for example, a sequence introduced for purification or detection. In one embodiment, the additional amino acid sequence includes one or more peptide sequences that confer affinity to a particular chromatography column material. Typical examples of such sequences include, but are not limited to, Strep-tags, oligohistidine-tags, glutathione S-transferase, maltose-binding proteins, inteins, intein fragments, or albumin-binding domains of protein G.

[0088] FAP-binding proteins for pharmaceutical use Various embodiments relate to fusion proteins containing the FAP-binding proteins described herein or disclosed herein, for use in pharmaceuticals. In one embodiment, the FAP-binding proteins described herein or disclosed herein are used in pharmaceuticals to diagnose or treat cancers associated with FAP expression. The FAP-binding proteins described herein or disclosed herein enable the selective diagnosis and treatment of FAP-associated cancer cells or cancer tissues, for example, from breast, colorectal, pancreatic, lung, brain, intrahepatic bile duct, and ovarian cancers, or from tumors originating from non-epithelial tissues, such as melanoma and myeloma. FAP-binding proteins are used in the diagnosis (imaging) and treatment of most epithelial cancers, including breast, lung, colorectal, and pancreatic cancers. It is known that FAP can be upregulated in tumor cells, potentially resulting in uncontrolled proliferation and metastasis of tumor cells. In one embodiment, FAP-binding proteins are used to diagnose FAP-associated tumors by applying in vitro methods.

[0089] One embodiment is a method for diagnosing (including monitoring) a subject having an FAP-associated tumor, the diagnostic (including monitoring / imaging) method comprising administering to the subject an FAP-binding protein described herein or a fusion protein containing an FAP-binding protein described herein, optionally conjugated to a radiomolecule. In various embodiments, the FAP-binding protein described herein or a fusion protein containing an FAP-binding protein disclosed herein may be used in a method for diagnosing an FAP-associated tumor, and optionally, the FAP-binding protein described herein or a fusion protein containing an FAP-binding protein is conjugated to a radiomolecule. In some embodiments, a method for imaging a specific tissue or cells expressing FAP includes the FAP-binding protein described herein or a fusion protein containing an FAP-binding protein described herein. In some embodiments, a method for imaging a specific tissue or cells expressing FAP includes a label conjugated to the FAP-binding protein. In some embodiments, such a label is selected from radioactive or fluorescent molecules. In some embodiments, a method for imaging specific tissues or cells expressing FAP includes a label conjugated to an FAP-binding protein used to visualize FAP on specific tissues or cells for, for example, assessing the presence of FAP-associated tumor cells, FAP-associated tumor distribution, recurrence of FAP-associated tumors, and / or assessing a patient's response to therapeutic treatment. In some embodiments, the method is an in vitro method.

[0090] One embodiment is a method for treating a subject having FAP-associated cancer, the method comprising administering to the subject a described FAP-specific binding protein, optionally conjugated to a radioactive molecule and / or a cytotoxic agent, or as a cancer immunoagent. In various embodiments, the FAP-binding proteins disclosed herein may be used in methods for treating FAP-associated cancer, optionally conjugated to a cytotoxic agent and / or a radioactive molecule, or expressed on the surface of target-specific cancer-associated fibroblasts (CAFs). For example, the cancer cells may be solid tumor cells. Some embodiments relate, in particular, to the use of proteins, including FAP-binding proteins labeled with a suitable radioisotope or cytotoxic compound, in methods for treating FAP-associated tumors to control or kill FAP-associated tumor cells, e.g., malignant cells. In one embodiment, a therapeutic dose of radiation is selectively delivered to FAP-associated tumor cells.

[0091] In some embodiments, a method for treating FAP-related diseases includes an FAP-binding protein described herein or a fusion protein containing an FAP-binding protein disclosed herein. In some embodiments, the treatment of FAP-related diseases includes an FAP-binding protein disclosed herein and further components for promoting an immune response. As described herein, FAP-related cancers may be breast cancer, colorectal cancer, pancreatic cancer, lung cancer, brain cancer, intrahepatic cholangiocarcinoma, epithelial cell carcinoma, squamous cell carcinoma, and ovarian cancer, or tumors of non-epithelial tissue, such as melanoma and myeloma.

[0092] composition Various embodiments relate to compositions comprising FAP-binding proteins described herein or fusion proteins comprising FAP-binding proteins disclosed herein. Compositions comprising proteins comprising the FAP-binding proteins defined above for pharmaceutical use, preferably for use in the diagnosis or treatment of FAP-related cancers. Compositions comprising FAP-binding proteins described herein or fusion proteins comprising the FAP-binding proteins described herein may be used in diagnostic and / or treatment (including imaging) methods for FAP-related diseases. In particular, compositions comprising the FAP-binding proteins described herein may be used for methods of imaging, monitoring, and eliminating or inactivating diseased cells expressing FAP.

[0093] Various embodiments relate to diagnostic compositions for the diagnosis of FAP-associated cancers, comprising FAP-binding proteins as defined herein and diagnostically acceptable carriers and / or diluents. These include, but are not limited to, stabilizers, surfactants, salts, buffers, colorants, and the like. The compositions may be in the form of liquid preparations, lyophilized products, granules, emulsions, or liposome preparations.

[0094] Diagnostic compositions containing FAP-binding proteins described herein may be used for the diagnosis of the FAP-associated cancers described above.

[0095] Various embodiments relate to pharmaceutical (e.g., therapeutic) compositions for the treatment of diseases comprising the FAP-binding proteins disclosed herein, as well as pharmaceutically (e.g., therapeutically) acceptable carriers and / or diluents. The pharmaceutical (e.g., therapeutic) compositions may optionally contain further adjuvants and excipients known on their own, including, but not limited to, stabilizers, surfactants, salts, buffers, and colorants. The therapeutic compositions may also contain the FAP-binding proteins disclosed herein and further agents (immuno-oncological agents) that promote the immune response. The FAP-specific binding proteins disclosed herein as part of the composition may counteract the inhibition of the immune response.

[0096] Pharmaceutical compositions comprising FAP-binding proteins as defined herein may be used for the treatment of the diseases described above.

[0097] The composition contains an effective dose of FAP-binding protein as defined herein. The amount of protein to be administered depends on the organism, the type of disease, the patient's age and weight, and other factors known to the present. Depending on the Galenos preparation, these compositions may be administered parenterally by injection or infusion, systemically, intraperitoneally, intramuscularly, subcutaneously, percutaneously, or by other conventionally used methods of application.

[0098] The composition may be in the form of a liquid preparation, lyophilized product, cream, topical lotion, aerosol, powder, granule, emulsion, or liposome preparation. The type of preparation depends on the type of disease, route of administration, severity of the disease, and other factors known to the patient and those skilled in the pharmaceutical field.

[0099] The various components of the composition may be packaged as a kit along with instructions for use.

[0100] Preparation of FAP-binding proteins The FAP-binding proteins described herein may be prepared by any of many conventional and well-known techniques, such as conventional organic synthesis strategies, solid-phase assisted synthesis techniques, fragment ligation techniques, or commercially available automated synthesis equipment. Alternatively, they may also be prepared by conventional recombination techniques, either alone or in combination with conventional synthesis techniques. Furthermore, they may also be prepared by cell-free in vitro transcription / translation. Various embodiments relate to polynucleotides encoding the FAP-binding proteins disclosed herein. One embodiment further provides an expression vector containing the polynucleotide, and a host cell containing the isolated polynucleotide or expression vector.

[0101] Various embodiments of the method disclosed herein for producing FAP-binding proteins include culturing host cells under suitable conditions that enable the expression of the FAP-binding protein and optionally isolating the FAP-binding protein.

[0102] For example, one or more polynucleotides encoding an FAP-binding protein may be expressed in a suitable host, and the resulting FAP-binding protein can be isolated. The host cell comprises the nucleic acid molecule or vector. Suitable host cells include prokaryotes or eukaryotes. A vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) that can be used to transfer protein-coding information into a host cell. Various cell culture systems, including but not limited to mammals, yeast, plants, or insects, can also be used to express recombinant proteins. Suitable conditions for culturing prokaryotic or eukaryotic host cells are well known to those skilled in the art. Cell culture and protein expression for the purpose of protein production can be carried out on any scale, starting from small volume shaker flasks to large fermenters, by applying techniques well known to those skilled in the art.

[0103] One embodiment is a method for preparing the binding protein detailed above, which is directed to include the following steps: (a) preparing a nucleic acid encoding the FAP-binding protein as defined herein; (b) introducing the nucleic acid into an expression vector; (c) introducing the expression vector into host cells; (d) culturing the host cells; (e) subjecting the host cells to culture conditions under which the FAP-binding protein is expressed, thereby producing the FAP-binding protein as defined herein; (f) optionally isolating the FAP-binding protein produced in step (e); and (g) optionally conjugating the FAP-binding protein with a further functional moiety as defined herein.

[0104] Generally, the isolation of purified FAP-binding proteins from culture mixtures can be performed by applying conventional methods and techniques well known in the art, such as centrifugation, precipitation, cotton-like precipitation, different embodiments of chromatography, filtration, dialysis, concentration and combinations thereof, and others. Chromatographic methods are well known in the art and include, but are not limited to, ion-exchange chromatography, gel filtration chromatography (size exclusion chromatography), hydrophobic interaction chromatography, or affinity chromatography.

[0105] For the sake of simplifying purification, the FAP-binding protein may be fused to another peptide sequence that has increased affinity to the separation material. Preferably, a fusion is selected that does not have an adverse effect on the functionality of the FAP-binding protein or that can be separated after purification due to the introduction of a specific protease cleavage site. Such methods are also known to those skilled in the art.

[0106] FAP detection method Some embodiments relate to a method for detecting (human) FAP in a sample, comprising contacting the sample with the FAP-binding protein of the present invention as described herein. In a preferred embodiment, the sample is a sample obtained from a subject, which is preferably a human subject. In a preferred embodiment, the sample is, but is not limited to, one of blood, plasma, serum, tissue, tissue fluid, and urine. In a preferred embodiment, the sample is cancerous tissue or cancer biopsy, or obtained therefrom, where the cancer is an FAP-associated cancer as described elsewhere herein. [Examples]

[0107] The following examples are provided for further embodiment of the present invention. The present invention is particularly exemplified by the modification of ubiquitin resulting in binding to FAP. The present invention, however, is not limited thereto, and the following examples merely illustrate the feasibility of the present invention based on the above description.

[0108] Example 1: Target mammalian expression Expi293-F cells were cultured at 37°C, 8% CO2, and 95% humidity in Expi293-(trademark) Expression medium (Fisher Scientific, 13469756) in a 135 rpm shake flask at a density of 500,000 to 1,000,000 cells / ml. One day before transfection, cells were seeded at a density of 2,000,000 cells / ml. On the day of transfection, cells were seeded at a density of 2,500,000 cells / ml. Plasmid DNA of hFAP-Fc, hCD26-Fc, or mCD26-Fc, or biotinylated hFAP-His, was diluted in Opti-MEM I Reduced Serum Medium (Life Technologies, 31985-062) using 1 μg of hFAP-Fc, hCD26-Fc, or mCD26-Fc plasmid DNA or biotinylated hFAP-His with 0.5 μg of hFAP-AviHis and 0.5 μg of hBirA per 1 ml of culture volume. ExpiFectamine (Thermo Fisher, A14524) was diluted in Opti-MEM I Reduced Serum Medium according to the producer's instructions and incubated at room temperature for 5 minutes. Subsequently, the DNA solution was added to the ExpiFectamine mixture and incubated at room temperature for 20 minutes before being added to the cells. Cells were incubated at 37°C, 8% CO2, and 95% humidity for protein expression. Enhancer (Thermo Fisher, A14524) was added to the transfection mixture after 16 hours. The supernatant was collected after 96–120 hours, centrifuged, and filtered through a 0.45 μm membrane.

[0109] For the production of mFAP-Fc, ExpiCHO cells were cultured in ExpiCHO Expression Medium (Thermo Fisher Scientific, A2910001) at a density of 500,000 cells / ml (see above). Cells were seeded at a density of 4 million / ml one day before transfection. For transfection, cells were seeded at a density of 6 million / ml. 1 μg of mFAP-Fc plasmid DNA per 1 ml of culture volume was diluted in OptiPRO SFM (Thermo Fisher Scientific, 12309019). ExpiFectamine (Thermo Fisher, A29129) was diluted in OptiPRO SFM and mixed with the DNA solution. After incubation at room temperature for 3 minutes, the transfection mix was added to the cells. Cells were incubated at 37°C for 24 hours. After 24 hours of incubation at 37°C, ExpiFectamine Enhancer and ExpiFectamine Feed were added, and cells were incubated at 32°C. The supernatant was collected after 120 hours, centrifuged, and filtered through a 0.45 μm membrane.

[0110] Example 2. Identification of FAP-binding proteins Library construction and library cloning Different proprietary libraries containing randomized amino acid positions in ubiquitin and / or inserts were synthesized in-house using randomized oligonucleotides generated by synthetic trinucleotide phosphoramidites (ELLA Biotech) to achieve a well-balanced amino acid distribution with the simultaneous exclusion of cysteine ​​and other amino acid residues at randomized positions.

[0111] The corresponding cDNA library was amplified by PCR and ligated with a modified pCD87SA phagemide (referred to herein as pCD12) using standard methods known to those skilled in the art. The pCD12 phagemide contained a modified torA reader sequence (deletion of amino acid sequence QPAMA) to achieve protein processing without additional amino acids at the N-terminus. Aliquots of the ligation mixture were used for electroporation of E. coli ER2738 (Lucigen). Unless otherwise indicated, established recombinant genetic methods were used.

[0112] target Selection was performed using the extracellular domains of human FAP proteins and / or mouse FAP proteins as targets. On one hand, an IgG1-Fc fusion protein was applied, and on the other hand, a biotinylated AviHis fusion protein was applied (cloning and expression were performed in-house as described in Example 1).

[0113] Primary selection by TAT phage display A naive library for FAP was enriched using phage display as a selection system. After transformation of competent bacterial ER2738 cells (Lucigene) with the phagemide pCD12 containing the library, phage amplification and purification were performed using standard methods known to those skilled in the art. Target proteins were immobilized on magnetic beads for selection. Target proteins fused to IgG1-Fc were immobilized on Protein A Dynabeads. Site-directed biotinylated target proteins fused to AviHis tags were immobilized on M-270 Epoxy Dynabeads. FAP concentrations during phage incubation were decreased from 140 nM (round 1) to 20 nM (round 3) or 5 nM (round 4) for biotinylated AviHis-fused target proteins containing target switches in each round between human FAP and mouse FAP, starting with human FAP. The FAP concentration for IgG1-Fc fusion targets was reduced from 100 nM (Round 1) to 20 nM (Round 3) or 5 nM (Round 4) using only human FAP.

[0114] Selection of IgG1-Fc fusion targets was performed in 3–4 rounds, depending on the library, and included preliminary selection of gammanorm (Octopharma, catalog number PZN 13336380) on Protein A Dynabeads in round 2, and off-target extracellular domains of human CD26 fused to IgG1-Fc on Protein A Dynabeads (AcroBiosystems, catalog number DP4-H5266) in rounds 3 and 4. Additional pre-incubation of phage particles in mouse serum at 37°C for 23 hours was performed prior to rounds 3 and 4.

[0115] Selection for biotinylated AviHis fusion targets was also carried out in 3-4 rounds, depending on the library, and included preliminary selection in round 3 using only human CD26 fused to IgG1-Fc on Protein A Dynabeads (AcroBiosystems, catalog no. DP4-H5266).

[0116] All selection rounds were performed using an automated KingFisher System (Thermo Fisher) for isolation, washing, and capture of the desired phage-target complex on magnetic beads. FAP-bound phages were eluted with trypsin.

[0117] To identify target-specific phage pools, eluted and re-amplified phages from each selected round were analyzed by phage pool ELISA. Wells on a medium-binding microtiter plate (Greiner Bio-One) were coated with human FAP (2.5 μg / ml) or mouse FAP (2.5 μg / ml), gammanorm (2.5 μg / ml), and human CD26 (2.5 μg / ml). Biotinylated AviHis-fused FAP was coated via Streptavidin. Binding phages were detected using α-M13 HRP conjugate antibody (GE Healthcare).

[0118] Cloning of a target-binding phage pool to an expression vector A selection pool showing specific binding to FAP in a phage pool ELISA was amplified by PCR according to methods known in the art, cleaved with an appropriate restriction nuclease, and ligated to a derivative of the expression vector pET-28a (Merck, Germany) containing Strep-Tag II (IBA GmbH).

[0119] Single Colony Hit Analysis After transformation of BL21(DE3) cells, kanamycin-resistant single colonies were automatically picked using a Qpix2 colony picker. FAP-binding protein expression was achieved by culturing in a 384-well plate (Greiner Bio-One) using auto-induction medium (Studier, 2005, Protein Expr. Purif. 41(1):207-234). Cells were harvested and subsequently mechanically thawed by freeze / thaw cycles. After centrifugation, the resulting supernatant was first screened by ELISA using immobilized ProtA / FAP-Fc on a high-binding 384 ELISA microtiter plate (Greiner Bio-One). Proteins bound to FAP-Fc were detected using Strep-Tactin HRP Conjugate (IBA GmbH) in combination with TMB-Plus substrate (Biotrend, Germany). The reaction was stopped by adding a 0.2 M H2SO4 solution and measured at 450 nm vs. 620 nm using a plate reader. In confirmatory screening, the binding of initial hits was analyzed against FAP-Fc (on-target) and IgG-Fc (off-target). Specific hits were selected for μ-scale purification, SPR analysis, sequencing, and further laboratory-scale expression and analysis.

[0120] Example 3A: Purification of target molecule Cell culture supernatants expressing hFAP-Fc-His, mFAP-Fc-His, hCD26-Fc-His, and mCD26-Fc-His were centrifuged and filtered for application to affinity chromatography on a HisTrap excel 1 mL column or HiTrap Protein A HP 5 ml (Cytiva, buffered according to manufacturer's instructions). Cell culture supernatants expressing hFAP-Avi-His, mFAP-Avi-His, hCD26-Avi-His, and mCD26-Avi-His were centrifuged and filtered for application to affinity chromatography on a HisTrap excel 1 mL column (Cytiva, buffered according to manufacturer's instructions).

[0121] The eluted target protein was applied to a Superdex 200 XK 16 / 600 gel filtration column. The purity of the recovered target protein was analyzed and confirmed by SDS-PAGE and SE-HPLC. Enzyme activity was confirmed by fluorescence assays based on the ability of the FAP target protein to convert the substrate benzyloxycarbonyl-Gly-Pro-7-amide-4-methylcoumarin (Z-GP-AMC) to 7-amino-4-methylcoumarin (AMC) and the ability of the CD26 off-target to convert H-Gly-Pro-7-amino-4-methylcoumarin (GP-AMC) to AMC.

[0122] Example 3B: Enzyme activity test of hFAP-Fc-His in the presence of Affilin® protein To investigate whether the Affilin® protein affects the enzymatic activity of hFAP-Fc-His, an activity assay was performed as described in Example 3A, but in the presence of the Affilin® protein. Affilin®-217990 (SEQ ID NO: 1), Affilin®-217862 (SEQ ID NO: 3), Affilin®-217832 (SEQ ID NO: 4), Affilin®-217917 (SEQ ID NO: 6), Affilin®-217993 (SEQ ID NO: 11), Affilin®-219750 (SEQ ID NO: 15), Affilin®-219235 (SEQ ID NO: 17), Affilin®-220134 (SEQ ID NO: 18), Affilin®-220164 (SEQ ID NO: 19), and Affilin®-220257 (SEQ ID NO: 22) were tested. The reaction mixture contained 0.9 nM hFAP-Fc-His and 1 μM Affilin® protein. None of the Affilin® proteins tested reduced the enzymatic activity of hFAP-Fc-His.

[0123] Example 4. Expression and purification of FAP-binding protein Genes for FAP-binding proteins were cloned into expression vectors using standard methods known to those skilled in the art, purified, and analyzed as described below. All FAP-specific proteins were expressed and highly purified by affinity chromatography and gel filtration. Following affinity chromatography using a Strep-Tactin Superflow high-capacity column, the eluted proteins were applied to size exclusion chromatography using an AKTA xpress system (GE Healthcare) (Superdex 75 HiLoad 16 / 600 or Sephacryl S200HR 16 / 600 column). Elution in PBS containing 500 mM NaCl (pH 7.4) was performed in three column volumes. After SDS-PAGE analysis, the positive fractions were pooled and their protein concentrations were measured.

[0124] Further analysis included SDS-PAGE, RP-HPLC, and SE-HPLC. Protein concentration was determined by absorbance measurement at 280 nm using a specific molar extinction coefficient. Reverse-phase chromatography (RP-HPLC) was performed using an Ultimate 3000 HPLC system (Thermo Fisher Scientific) and a PLRP-S (5 μm, 300 Å) column (Agilent). Purity results were >78%. Analytical size exclusion chromatography (SE-HPLC) was performed using an Ultimate 3000 HPLC system (Thermo Fisher Scientific) and a Superdex75 increase 5 / 150 GL (Cytiva). No aggregation was detected.

[0125] Example 5. Analysis of FAP-binding proteins (surface plasmon resonance, SPR) Recombinant protein A was immobilized onto a high-capacity amino acid sensor chip (Broker) after NHS / EDC activation, and the volume was increased to approximately 2000 RU using a Sierra SPR-32 system (Broker). The chip was equilibrated with SPR running buffer (PBS 0.05%, Tween pH 7.3). Unreacted NHS groups were blocked using ethanolamine injection after ligand immobilization. Fc-tagged FAP and CD26 target molecules were injected at 60 nM, followed by injection of FAP-binding protein. Upon binding, the target analyte accumulated on the surface, increasing the refractive index. This change in refractive index was measured in real time and plotted as a response to time or as resonance units. FAP-binding protein was applied to the chip at a flow rate of 30 μl / min in serial dilutions. Association was performed for 120 seconds and dissociation for 180 seconds. After each run, the chip surface was regenerated with 30 μl of regenerating buffer (10 mM glycine, pH 2.0) and equilibrated with running buffer. Binding studies were performed using the Sierra SPR-32 system (Bruker); data evaluation was performed using the Langmuir 1:1 model (RI=0) via the Sierra Analyser software provided by the producer. The evaluated dissociation constant (K) was... D The ) were standardized and indicated for immobilized proteins. Table 1 shows the binding affinity of FAP-binding proteins to hFAP.

[0126] TIFF0007854218000001.tif109170

[0127] Affilin(registered trademark)-217990 (SEQ ID NO: 1), Affilin(registered trademark)-217862 (SEQ ID NO: 3), Affilin(registered trademark)-217832 (SEQ ID NO: 4), Affilin(registered trademark)-217863 (SEQ ID NO: 5), Affilin(registered trademark)-219235 (SEQ ID NO: 17), Affilin(registered trademark)-220164 (SEQ ID NO: 19), Affilin(registered trademark)-223078 (SEQ ID NO: 26), and Affilin(registered trademark)-223077 (SEQ ID NO: 27) bind (cross-specifically) to mouse FAP (mFAP) and human FAP (hFAP). The affinity of Affilin(registered trademark)-220164 to mFAP is 3.1 nM. The affinity of Affilin(registered trademark)-223078 to mFAP is 3 nM. The affinity of Affilin(registered trademark)-223077 for mFAP is 5 nM. The affinity of Affilin(registered trademark)-219235 for mFAP is 21.6 nM. The affinity of Affilin(registered trademark)-217990 for mFAP is 90 nM. Affilin®-217990 and Affilin®-217832 cross-specifically bind to cynomolgus monkey FAP (cFAP) and human FAP (hFAP), respectively. The FAP-specific Affilin® disclosed herein does not bind to either hCD26 or mCD26.

[0128] Example 6. Competition of binding proteins for epitopes The competitive binding of two isolated Affilin® proteins to hFAP-Fc was investigated as follows: The first Affilin® protein was immobilized on a CM5 Biacore chip using NHS / EDC chemistry (approximately 200 RU). 250 nM hFAP-Fc was injected with or without a 4-fold excess of a second Affilin® protein. The results are shown in Table 2. In Table 2, "competition" means that the binding of the first Affilin® is affected by the presence of the second Affilin®, and vice versa. Affilin®-217993, Affilin®-217990, and Affilin®-217832 bind to the same or overlapping epitopes, i.e., the same or overlapping surface-exposed amino acids.

[0129] TIFF0007854218000002.tif43170

[0130] Example 7. Functional Characterization: FAP-binding proteins are stable at high temperatures. The thermal stability of FAP-specific Affilin® protein was determined by differential scanning fluorescence (DSF) or circular dichroism (CD). For DSF measurements, each probe was transferred to a LightCycler 480 Multiwell Plate 96 (Roche) at a concentration of 0.25 μg / μL, and SYPRO Orange dye was added at a suitable dilution. A temperature gradient from 20°C to 90°C was programmed with a heating rate of 1°C / min (LightCycler480 RT-PCR-System, Roche). Fluorescence was continuously measured at an excitation wavelength of 465 nm and an emission wavelength of 580 nm. For CD measurements, the sample was desalted in 20 mM NaH2PO4 (pH 7.0) using a HiTrap Desalting 5 ml column (Cytiva). FAP-binding proteins were diluted to concentrations of 0.2–0.4 μg / μL. A temperature gradient from 20°C to 90°C was performed with a heating rate of 0.5°C / min (J-815, Jasco).

[0131] Midpoint of transition due to thermal denaturation (T m The melting point was determined and is shown in Table 3.

[0132] TIFF0007854218000003.tif50170

[0133] Example 8. Functional Characterization: Specific binding to hFAP expressed on the cell surface (flow cytometry) Flow cytometry was used to analyze the interaction of FAP-binding proteins with hFAP and mFAP exposed on the cell surface. FAP-overexpressing human embryonic kidney cell line HEK293, CD26-overexpressing HEK293 cells, native FAP-expressing cell line Wi38, and empty vector control HEK293-pEntry cells were used. A 1 μg / ml anti-hFAP antibody (R&D Systems, MAB3715-100) combined with a 1:1000 dilution of anti-mouse IgG-Alexa488 (Invitrogen, A10680) was used as a positive control for hFAP-expressing cells. A 1 μg / ml anti-mFAP antibody (R&D Systems, MAB9727) combined with a 1:1000 dilution of anti-rat IgG-Alexa488 (Invitrogen, A11006) was used as a positive control for mFAP-expressing cells.

[0134] Cells were trypsinized, resuspended in a medium containing FCS, washed, and stained in pre-cooled FACS blocking buffer (3% FCS / PBS). 1 × 10 6 Cell concentration suspensions were prepared for cell staining at a cell / ml concentration, and 100 μl / well was placed in a triple-row 96-well plate (Greiner) for each cell line.

[0135] Affilin® protein was tested at concentrations of 1 μM, 100 nM, 10 nM, or 1 nM in FAP-expressing cells HEK293-hFAP, HEK293-mFAP, and Wi38 cells. To exclude nonspecific binding, Affilin® protein was incubated at the same concentrations in CD26-expressing cells HEK293-hCD26 and HEK293-mCD26, as well as in control cells HEK293-pEntry. An equivalent amount of wild-type ubiquitin (clone 139090) was used as a negative control. The supernatant was removed after 45 minutes, cells were washed in a blocking buffer, and 100 μl / well of rabbit anti-StrepTag antibody (GenScript; A00626), diluted 1:300 in FACS blocking buffer, was added. After removal of the primary antibody, goat anti-rabbit IgG-Alexa Fluor 488 antibody (Invitrogen; A11008) was applied at a dilution of 1:1000. Flow cytometry was performed on a Guava easyCyte 5HT device from Merck-Millipore at an excitation wavelength of 488 nm and an emission wavelength of 525 / 30 nm.

[0136] FACS experiments demonstrate that all Affilin® proteins disclosed herein bind to hFAP expressed on the cell surface of HEK293 cells. A characteristic strong binding (+++) was observed for Affilin®-217863, Affilin®-217862, Affilin®-217832, Affilin®-220164, Affilin®-223078, Affilin®-223077, Affilin®-223019, Affilin®-220257, and Affilin®-220198 (see Table 4).

[0137] Binding of Affilin®-217990, Affilin®-220164, Affilin®-223078, Affilin®-223077, and Affilin®-219235 to the hFAP-expressing cell line HEK293-hFAP and the mFAP-expressing cell line HEK293-mFAP was confirmed. Binding to control cells HEK293-hCD26, HEK293-mCD26, or HEK293-pEntry was not observed for FAP-binding Affilin® proteins.

[0138] Furthermore, all Affilin® proteins disclosed herein showed binding to native hFAP-expressing Wi38 cells. The anti-FAP antibody shows positive staining on all FAP-expressing cells. Wild-type ubiquitin did not show binding on FAP-expressing cells.

[0139] TIFF0007854218000004.tif116170

[0140] Example 9. Binding affinity (ELISA) of FAP in serum after long-term incubation. High-binding plates (Greiner, 781061) were immobilized with 2.5 μg / ml of hFAP-Fc overnight at 4°C. A dilution series of Affilin® protein, ranging from 3 μM to 0.07 pM, was incubated in 100% human serum or 100% mouse serum for 24 hours at 37°C. ELISA plates were washed three times with PBST (PBS + 0.1% Tween) and blocked at room temperature for 1 hour with 3% BSA / 0.5% Tween / PBS. After 0 or 24 hours of pre-incubation in the presence of serum, the dilution series was incubated on the ELISA plate for 30 minutes at room temperature (rt). Wells were washed with PBST and incubated with biotinylated anti-ubiquitin antibody (1:300) for 30 minutes at room temperature. Binding was visualized with Streptavidin-HRP (1:5.000). Affilin® protein was converted to K after 24 hours of incubation in human or mouse serum. D No significant change was observed in (see Figures 3-5). D The values ​​are summarized in Table 5. ELISA analysis confirmed the high stability of FAP-binding proteins in serum.

[0141] TIFF0007854218000005.tif89170

[0142] Example 10. Binding affinity of FAP in serum after long-term incubation (cell binding assay - flow cytometry) Dilution series of 30 μM to 2.1 pM of Affilin®-217966 (SEQ ID NO: 2), Affilin®-217862 (SEQ ID NO: 3), and Affilin®-217863 (SEQ ID NO: 5) were incubated in 100% human serum for 24 hours at 37°C. hFAP-expressing HEK293 cells were thawed, washed in FCS-containing medium, followed by washing with FACS blocking buffer (PBS / 0.1% sodium azide / 3% FCS), and 100 μl was placed in 1 × 10⁶ wells of a 96-well round-bottom plate. 6Cells were seeded at a density of cells / ml. A dilution series of Affilin protein was incubated with human serum for 24 hours and 0 hours (control) at 37°C. HEK293-hFAP cells were incubated with the dilution series for 45 minutes at 4°C. Cells were centrifuged and the supernatant was removed. Cells were washed with FACS blocking buffer, and 100 μl / well of rabbit anti-Strep-Tag antibody (GenScript; A00626), diluted 1:300 in FACS blocking buffer, was added. After removal of the primary antibody, goat anti-rabbit IgG Alexa Fluor 488 antibody (Invitrogen; A11008) was applied in FACS blocking buffer at a dilution of 1:1000. Flow cytometry measurements were performed on a Guava easyCyte 5HT device from Merck-Millipore at excitation wavelength 488 nm and emission wavelength 525 / 30 nm. The results are shown in Figure 6. K of Affilin(registered trademark)-217863 D The results showed no significant difference in binding to FAP even after 24 hours of serum incubation. Affilin®-217863 is stable in human serum. Similar results were obtained with Affilin®-217862 and Affilin®-217966.

Claims

1. A protein having the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 (provided that the following three amino acid positions: alanine (A) at position 50, glycine (G) at position 70, and lysine (K) at position 76 are maintained), The protein is characterized by having a specific binding affinity for human fibroblast-activating protein (hFAP) with a mass of less than 50 nM, and not binding to human CD26.

2. The protein according to claim 1, having a specific binding affinity for hFAP less than 25 nM.

3. The protein according to claim 1, which is a polymer.

4. The protein according to claim 1, further comprising one or more linking sites for linking chemical moieties, wherein the chemical moieties are selected from among chelators, drugs, toxins, dyes, and small molecules.

5. The protein according to claim 1, further comprising at least one diagnostically active moiety, wherein the diagnostically active moiety is selected from radionuclides, fluorescent proteins, photosensitizers, dyes, enzymes, magnetic beads, metal beads, colloidal particles, high electron density reagents, biotin, digoxigenin, haptens, CAR-T, or exosomes, or any combination thereof.

6. The protein according to claim 1, further comprising at least one therapeutically active moiety, wherein the therapeutically active moiety is selected from a monoclonal antibody, a receptor or receptor domain, a receptor ligand, a radionuclide, a cytotoxic compound, a cytokine, a chemokine, an enzyme, CAR-T, or an exosome, or any combination thereof.

7. The protein according to claim 1, further comprising at least one portion that modulates pharmacokinetics, wherein the portion that modulates pharmacokinetics is selected from serum albumin, albumin-binding protein, immunoglobulin-binding protein, immunoglobulin, polysaccharide, polyethylene glycol, sialic acid, or transferrin.

8. A composition for use in the diagnosis or treatment of FAP-related diseases, comprising the protein described in any one of claims 1 to 7.

9. A method for producing a protein according to any one of claims 1 to 7, comprising the steps of: a) culturing host cells under conditions suitable for obtaining the protein; and b) isolating the produced protein.

10. A method for detecting FAP constituting a sample using the protein described in any one of claims 1 to 7, the method comprising contacting the sample with the protein described in any one of claims 1 to 7.

Images