Fusion proteins containing half-life extending domains

Abstract

The present invention relates to a fusion protein comprising at least one albumin binding moiety and further comprising a tumor targeting moiety. The present invention relates to the use of the fusion protein or a composition comprising the fusion protein for medical applications.

Description

[Technical Field]

[0001] The present invention relates to a fusion protein comprising at least one albumin binding moiety and further comprising a tumor targeting moiety. The present invention relates to the use of the fusion protein or a composition comprising the fusion protein for medical applications. [Background technology]

[0002] The present invention provides novel fusion proteins that bind with affinity to serum albumin and tumor targets, and are particularly suitable for medical applications with tailored half-life specific targeting to tumor-associated proteins.

[0003] The above summary does not necessarily describe all of the problems solved by the present invention. Summary of the Invention

[0004] The present disclosure provides, but is not limited to, the following items 1 to 12.

[0005] This summary of the invention is not limiting and other aspects and embodiments of the invention will become apparent from the following description, examples, and drawings. 1. A fusion protein comprising: (a) a binding protein for serum albumin comprising the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence having one or two substitutions, deletions, or insertions relative to SEQ ID NO: 33, and having a binding affinity for serum albumin of less than 25 nM; (b) a targeting moiety having a binding affinity of less than 100 nM, preferably less than 10 nM, for a protein expressed by the tumor; A fusion protein comprising: 2. The fusion protein according to item 1, wherein the binding protein for serum albumin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 32, or an amino acid sequence having one or two substitutions, deletions, or insertions relative to SEQ ID NOs: 1 to 32, and has a binding affinity for serum albumin of less than 25 nM. 3. The fusion protein according to item 1 or 2, comprising a binding protein for human serum albumin or mouse serum albumin. 4. The fusion protein according to any one of items 1 to 3, wherein the targeting moiety is a non-immunoglobulin protein, preferably a ubiquitin mutein, a protein A domain mutein, an ankyrin repeat protein mutein, a lipocalin mutein, a human Fyn SH3 domain mutein, a human fibronectin 10 domain mutein, an FN3 domain mutein, a Kunitz domain mutein, a Sac7d mutein, a chagasin mutein, a multimerized low density lipoprotein receptor A mutein, a cysteine-knot miniprotein mutein, a stefin mutein, an armadillo repeat protein mutein, a tetranectin mutein, a C-type lectin domain mutein, or a CTLA-4 mutein, or wherein the targeting moiety is an immunoglobulin, an immunoglobulin fragment or a variant thereof, a single domain antibody, or a single chain variable fragment (scFv) of an antibody. 5. The fusion protein according to item 4, wherein the non-immunoglobulin protein comprises a ubiquitin mutein exhibiting 80% to 94% identity to ubiquitin (SEQ ID NO: 43). 6. The fusion protein according to any one of items 1 to 5, wherein the targeting moiety binds to a protein expressed by a tumor, such as Her2, FAP, or ED-B. 7. The fusion protein according to any one of items 1 to 6 for use in the diagnosis or treatment of cancer, wherein the half-life (serum or blood) of the targeting moiety of the fusion protein is longer than the half-life (serum or blood) of a targeting moiety that does not comprise a serum albumin binding protein. 8. A nucleic acid molecule encoding the fusion protein according to any one of items 1 to 6. 9. A vector comprising the nucleic acid molecule according to item 8. 10. A host cell or a non-human host comprising a fusion protein as defined in any one of items 1 to 6, a nucleic acid as defined in item 8, and / or a vector as defined in item 9. 11. A composition comprising a fusion protein as defined in any one of items 1 to 6, a nucleic acid as defined in item 8, and / or a vector as defined in item 9. 12. A method for producing a fusion protein as defined in any one of items 1 to 6, comprising culturing a host cell as defined in item 10 under conditions suitable for obtaining said fusion protein, and, if desired, isolating said fusion protein. [Brief explanation of the drawings]

[0006] [Figure 1] Figure 1 shows the affinity of fusion proteins containing an albumin-binding domain and a targeting moiety for human serum albumin and their targets (Figure 1A: fibronectin extra domain B (ED-B), Figure 1B: human epidermal growth factor receptor 2 (Her2), Figure 1C: fibroblast activation protein (FAP)). The KD of each fusion protein was determined by SPR at pH 7.3. All fusion proteins bind to human serum albumin with a KD of less than 25 nM and to their specific targets with a KD of less than 10 nM. [Figure 2] Figure 2 shows the stability of fusion proteins comprising an albumin-binding domain and a targeting moiety to human serum albumin in serum. A FAP-specific targeting moiety fused to the albumin-binding domain of d01 (SEQ ID NO: 9; as a dimer) is shown, with Figure 2A showing the stability of fusion protein 218702 in serum. Figure 2B shows the stability of fusion protein 218690 in serum after prolonged incubation. The affinity of the fusion protein for the target does not decrease significantly after 24 hours of incubation in serum. [Figure 3]Figure 3 shows the biodistribution of fusion proteins containing a Her2-specific targeting moiety and an albumin-binding domain, demonstrating exceptionally high tumor-specific accumulation and excellent tumor-to-blood ratios. Fusion protein 218464, containing a Her2-specific targeting moiety and SEQ ID NO:9 (d01), and fusion protein 218462, containing a Her2-specific targeting moiety and SEQ ID NO:6 (c20), were radiolabeled with 177Lu to a specific activity of approximately 1 MBq / nmol and intravenously injected into mice (SKOV-3 xenograft model). In vivo biodistribution was assessed at five endpoints over a 168-hour period (one mouse per time point). In this in vivo experiment, fusion protein levels were analyzed in the blood, tumor, kidney, and liver. The fusion proteins exhibited exceptional tumor accumulation, due to their longer half-life compared to ubiquitin muteins lacking the albumin-binding domain. The half-life of the fusion proteins was approximately 36 hours. The albumin-binding domain of the fusion protein results in higher tumor accumulation due to a long half-life and excellent tumor / blood ratio. Furthermore, low accumulation in the kidney and liver was observed. Figure 3A: Fusion protein containing the albumin-binding domain of SEQ ID NO: 9 (d01) fused to a Her2-specific ubiquitin mutein exhibits excellent tumor accumulation over time. Figure 3B: Fusion protein containing the albumin-binding domain of SEQ ID NO: 6 (c20) fused to a Her2-specific binding moiety exhibits excellent tumor accumulation over time. [Figure 4] Figure 4 shows the biodistribution of Her2-specific targeting moieties, which exhibit low tumor-specific accumulation due to their short half-life. See Figure 3 for the experimental setup. Light gray bars represent wild-type ubiquitin, and dark gray bars represent ubiquitin muteins as targeting moieties with binding specificity for the cancer target Her2. The half-life (in the blood) of the cancer-specific targeting moieties is very short (i.e., in the absence of fusion to an albumin-binding domain as disclosed herein). [Figure 5]Figure 5 shows an imaging experiment: Fusion protein 218462 (206479_(G4S)4_c20) accumulates in tumors (SK-OV-3 xenograft model) after 72 hours. SPECT / CT and maximum intensity projection (MIP) images are shown 4, 24, and 72 hours after administration of In-111-labeled compound 1 (approximately 30 MBq / mouse). Uptake is expressed as a percentage of the injected dose per gram of tissue (%ID / g). The Her2-specific fusion protein, due to its fusion with the albumin-binding domain, clearly accumulates in tumors after 3 days. [Figure 6] Figure 6 shows an imaging experiment: FAP-specific fusion protein 220521 (SEQ ID NO: 47) with albumin-binding domain d01 accumulates in tumors (hFAP-overexpressing HEK293 cell xenograft model) at 24 and 70 hours. SPECT / CT images and maximum intensity projection (MIP) images are shown 4, 24, and 70 hours after administration of In-111-labeled Compound 1 (approximately 21 MBq / mouse). Uptake is expressed as a percentage of the injected dose per gram of tissue (%ID / g). Due to the fusion with the albumin-binding domain, the FAP-specific fusion protein clearly accumulates in tumors after a long time. [Figure 7] Figure 7 shows the amino acid sequence of an albumin-binding domain with high binding affinity to HSA. High binding affinity refers to a binding affinity with a KD of up to 25 nM. [Figure 8]Figure 8 shows the biodistribution of fusion proteins containing a FAP-specific targeting moiety and one or two albumin-binding domains, demonstrating exceptionally high tumor-specific accumulation. Fusion proteins 220518 (SEQ ID NO: 55), 220519 (SEQ ID NO: 54), and 220521 (SEQ ID NO: 47), each containing a FAP-specific targeting moiety (SEQ ID NO: 42) and SEQ ID NO: 9 (d01), were radiolabeled with 177 Lu and approximately 20 MBq / nmol and intravenously injected at a dose of 100 nmol / kg into female NMRI nude mice (CHO-hFAP overexpressing clone; xenograft model). In this in vivo study, tumor and kidney levels were analyzed at different time points over a 168-hour period. 220518: thick dashed line; 220519: thin line; 220521: thick line. Figure 8A: These fusion proteins demonstrate increasing tumor accumulation (%ID / g) over time. FIG. 8B: These fusion proteins efficiently reduce renal uptake (%ID / g). DETAILED DESCRIPTION OF THE INVENTION

[0007] The present inventors have developed a solution to meet the continuing and urgent need in the art to expand medical options for cancer diagnosis and treatment by providing novel fusion proteins with high affinity for serum albumin and cancer targets. The fusion proteins comprise at least one domain that binds to serum albumin (human or mouse). Furthermore, the fusion proteins defined herein are functionally characterized by specific affinity for human serum albumin and cancer targets (such as Her2, ED-B, or FAP). The fusion of a tumor-specific targeting domain with a human serum albumin-binding domain extends the half-life of the binding protein for the tumor target. In particular, the present invention provides fusion proteins comprising at least one ubiquitin mutein (also known as an Affilin® molecule) and a serum albumin-binding domain described herein.

[0008] Such fusion proteins have the potential to open up previously unmet medical strategies in cancer diagnosis and therapy. In particular, fusion proteins can be used for diagnostic or imaging purposes, e.g., the presence of tumor cells expressing the target of the targeting moiety, and for radiotherapy treatment of tumors expressing the target. The novel proteins are designed to enable all applicable steps for medical use.

[0009] Before describing the present invention in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0010] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W., Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0011] Throughout the text of this specification, several documents are cited (e.g., patents, patent applications, scientific publications, manufacturer's specifications, manuals, etc.). Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as "incorporated by reference." In the event of a conflict between a definition or teaching of such an incorporated reference and a definition or teaching set forth herein, the text of this specification shall control.

[0012] All sequences referred to in this specification are disclosed in the attached sequence listing (WIPO ST.26 compliant xml.-file), which, together with its entire content and disclosure, constitutes part of the disclosure of this specification.

[0013] General definitions of important terms used in this application Throughout this specification and the appended claims, unless the context otherwise requires, the word "comprise", and variations such as "comprises" and "comprising", are understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. The terms "comprise(s)" or "comprising" may include the limitation to "consists of" or "consisting of" wherever for whatever reason and to whatever extent such a limitation is necessary.

[0014] As used herein, the term "about" encompasses the explicitly stated amount as well as deviations therefrom of up to ±20%. More preferably, deviations of up to ±15%, more preferably up to ±10%, and most preferably up to ±5% are encompassed by the term "about." The term "at least about 10, 20, 30, 40, 50, 60, 70, 80 amino acid residues" is not limited to that exact number of amino acid residues, but also includes amino acid stretches containing up to 20% more residues or up to 20% fewer residues.

[0015] The term "fusion protein" refers to a protein comprising at least a first amino acid chain and at least a second amino acid chain genetically linked together. Thus, a fusion protein may comprise a multimer of proteins / peptides expressed as a single linear polypeptide. It may comprise one, two, three, four, or even more proteins / peptides. For example, a fusion protein can be generated by combining two or more genes originally encoding separate proteins / peptides. As described in more detail below, the "fusion protein" of the present invention comprises at least two components: (i) at least one target-binding moiety and (ii) at least one serum albumin-binding moiety.

[0016] The term "moiety" or "domain" refers to a substructure that is part of a protein or fusion protein. The terms "binding protein" and "binding domain" may be used interchangeably herein, for example, the terms "serum albumin binding protein" and "serum albumin binding domain" may be used interchangeably herein.

[0017] The term "fused" means that the components are linked by a peptide bond, either directly or via a peptide linker.

[0018] As used herein, a "linker" refers to an amino acid sequence that connects at least two moieties. A linker as understood herein is a peptide linker of up to 30 amino acids. Two or more moieties may be linked via a peptide linker of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. The linker may be composed of amino acids such as glycine and serine, and may be glycine-rich (e.g., more than 50% of the residues in the linker may be glycine residues). However, other linkers for protein fusion are known in the art and may be used herein. In the fusion proteins of the present invention, the serum albumin-binding domain and the tumor-targeting moiety may be operably linked by a linker, particularly a peptide linker. As used herein, the term "operably linked" means positioning components so that they function in their intended manner. The fusion proteins of the present invention may comprise one or more serum albumin binding domains and / or one or more tumor targeting moieties. In various embodiments, the fusion proteins comprise at least two tumor targeting moieties, which may be located at the C-terminus or N-terminus of the fusion protein. In various other embodiments, the fusion proteins comprise or consist of at least two serum albumin binding domains and at least two tumor targeting moieties. The one or more serum albumin binding domains and / or the one or more tumor targeting moieties may be operably linked by a linker, particularly a peptide linker. A linker, particularly a peptide linker, may also be present between the one or more serum albumin binding domains and the one or more tumor targeting moieties. Such a linker operably links the serum albumin binding domains and tumor targeting moieties located at each end. In various embodiments, the fusion protein comprises at least two serum albumin binding domains, which may be located at the C-terminus or N-terminus of the fusion protein.In various embodiments, the fusion protein comprises or consists of at least two serum albumin binding domains and one (or more specifically, one or more) tumor-targeting moiety (or tumor-specific binding protein). The at least two serum albumin binding domains may be operably linked by a linker as described above. Similarly, the at least two tumor-targeting moieties (or tumor-specific binding proteins) may be operably linked by a linker as described above. In various preferred embodiments, the linker may be a peptide linker, more specifically, a Gly-Ser linker. In preferred embodiments, the linker may be a G4S or (G4S)2 linker, preferably a (G4S)2 linker. Thus, in preferred embodiments, the linker may have an amino acid sequence selected from GGGGSGGGGS (SEQ ID NO: 48), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 49), and GGGGSGGGGSGGGGGS (SEQ ID NO: 50).

[0019] 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. Thus, peptide, protein, amino acid chain, or any other term used to refer to a chain of two or more amino acids is included in 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 that are known in the art.

[0020] As used herein, the term "albumin" refers to "serum albumin," more specifically, human serum albumin (HSA) or mouse serum albumin (MSA). HSA is the most abundant protein in blood. The terms "binding protein / domain for albumin" and "binding protein / domain for serum albumin" may be used interchangeably herein. Furthermore, as used herein, "binding protein / domain for serum albumin" refers to an (isolated) non-natural binding domain for albumin, particularly an (isolated) non-natural binding domain for serum albumin. As used herein, the term "non-natural" refers to a synthetic protein or domain, i.e., a protein or domain having an amino acid sequence not found in a natural polypeptide. As used herein, the term "(serum) albumin binding domain" refers to a peptide or polypeptide capable of binding to albumin in vivo and / or in vitro. The albumin-binding domain of the fusion protein of the present invention may bind to (serum) albumin in vivo and / or in vitro with an affinity of less than 25 nM. In the present invention, the albumin (serum albumin) may be derived from any animal species, for example, human, monkey, or rodent, and preferably from human.

[0021] As used herein, a targeting moiety having a binding affinity of less than 100 nM to a protein expressed by a tumor can be considered a tumor targeting moiety (or tumor protein targeting moiety). As used herein, a tumor targeting moiety relates to an (isolated) non-natural targeting moiety having binding affinity to a protein expressed by a tumor. Furthermore, as used herein, a tumor targeting moiety refers to a peptide or polypeptide capable of binding to a tumor protein (i.e., a protein expressed by a tumor) in vivo and / or in vitro. The tumor targeting moiety of the fusion protein of the present invention can bind to a tumor protein in vivo and / or in vitro with an affinity of less than 100 nM. In the present invention, the tumor protein may be derived from any animal species, for example, human, monkey, or rodent, and preferably from human.

[0022] In various embodiments, the tumor targeting moiety of the fusion protein of the invention is a therapeutically effective tumor targeting moiety, i.e., effective for treating cancer. In various other embodiments, the tumor targeting moiety of the fusion protein of the invention is a diagnostically effective tumor targeting moiety, i.e., effective for diagnosing cancer.

[0023] The terms "tumor" and "cancer" may be used interchangeably herein. Similarly, the terms "tumor cell" and "cancer cell" may be used interchangeably herein. As used herein, the terms "tumor" and "cancer" mean or refer to a physiological condition in a mammal, preferably a human, in which a population of cells is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma, and blood cancers such as lymphoma and leukemia. As used herein, the term "tumor" means any mass of tissue resulting from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous), including precancerous lesions. Tumor growth is generally uncontrolled and progressive, without inducing or suppressing the proliferation of normal cells.

[0024] In various embodiments of the invention, the tumor targeting moiety binds to a ligand of an immune checkpoint protein, such as PD-L1 and PD-L2, expressed on cancer cells. In various embodiments of the invention, the tumor targeting moiety binds to the extracellular domain of a protein expressed by a tumor (or tumor cell). In various embodiments of the invention, the protein expressed by a tumor cell is a protein whose expression is upregulated in the tumor (or tumor cell). In various embodiments of the invention, the protein expressed by a tumor cell is the extracellular domain of a protein whose expression is upregulated in the tumor (or tumor cell). In various embodiments of the invention, the protein expressed by a tumor (or tumor cell) is a tumor antigen or tumor-associated / specific antigen expressed by the tumor (or tumor cell). The tumor antigen or tumor-associated / specific antigen is typically an overexpressed antigen (i.e., an antigen whose expression is upregulated). In a preferred embodiment of the invention, the tumor targeting moiety binds to a ligand selected from any one of prostate-specific membrane antigen (PSMA), folate receptors FOLR1 (folate receptor alpha) and FOLR2, extra domain B of oncofetal human fibronectin (ED-B), and epidermal growth factor receptor (EGFR).

[0025] The term "Her2" refers to human epidermal growth factor receptor 2, also known as ErbB-2, Neu, CD340, or p185. Human Her2 is designated by NCBI accession number NP_004439, and the extracellular domain of Her2 (residues 1-652) is designated by UniProt accession number P04626. The term "Her2" includes all polypeptides that exhibit at least 70%, 80%, 85%, 90%, 95%, 96%, or 97% or more, or 100%, sequence identity to NP_004439 and possess Her2 function. Her2 is overexpressed in several types of tumors, for example, in 15-30% of all breast cancers.

[0026] As used herein, the term "FAP" refers to fibroblast activation protein (FAP), also known as prolyl endopeptidase FAP, dipeptidyl peptidase FAP, integral membrane serine protease, surface-expressed protease, etc. The term "FAP" refers to any polypeptide that exhibits at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, or 97% or more, or 100% sequence identity to the FAP of UniProt Accession Number Q12884 and UniProt Accession Number Q12884 (human) and has the functionality of a 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 (residues 26-760). For example, FAP is expressed in epithelial tumors and malignant sarcoma cells. FAP expression has been found in activated stromal fibroblasts in over 90% of all human carcinomas.

[0027] The term "ED-B" or "EDB" refers to the extra domain B of oncofetal human fibronectin (Uniprot accession number P02751-7). ED-B is an extracellular matrix oncoprotein expressed by newly forming blood vessels in (solid) tumors, lymphomas, and some leukemias. ED-B is a fibronectin extra domain B that is expressed by the two fibronectin domains Fn in oncofetal human fibronectin isoforms. III 7 and Fn III It exists between 8.

[0028] As used herein, the term "Affilin" or "Affilin®" (registered trademark of Navigo Proteins GmbH) refers to a binding protein based on a ubiquitin mutein. The term "Affilin" as used herein refers to a derivative of ubiquitin that differs from ubiquitin or a protein having at least 80% and up to 94% amino acid identity to ubiquitin (SEQ ID NO: 43) by amino acid exchanges, insertions, deletions, or any combination thereof, and by specific binding affinity for a target.

[0029] The term "binding" according to the present invention preferably relates to specific binding to a protein target such as serum albumin or a tumor-specific target protein.

[0030] "Dissociation constant" or "K D The term "K" defines the specific binding affinity. D (usually measured in units of "nanomoles / L" and sometimes abbreviated as "nM") is intended to mean the dissociation equilibrium constant of a particular interaction between a binding protein (e.g., a Her2-specific affilin) and a target protein (e.g., Her2). As disclosed herein, a serum albumin binding protein of the invention having a binding affinity for serum albumin of less than 25 nM means that the serum albumin binding protein of the invention has a K D As described elsewhere herein, the present invention encompasses serum albumin-binding proteins having a (moderate) binding affinity for serum albumin of 25 nM or more, particularly 25 nM to 3 μM. Such serum albumin-binding proteins have a K of 25 nM or more. D , especially K values between 25 nM and 3 μM D It binds to serum albumin.

[0031] As further disclosed herein, a tumor targeting moiety of the present invention has a binding affinity for a protein expressed by a tumor of less than 100 nM, which means that the tumor targeting moiety of the present invention has a K D This means that the antibody binds to a protein expressed by the tumor.

[0032] The term "modification" or "amino acid modification" refers to the substitution, deletion, or insertion of a reference amino acid at a particular position in a parent / reference polypeptide sequence with another amino acid. Given the known genetic code and recombinant and synthetic DNA techniques, one skilled in the art can readily construct DNA encoding amino acid variants.

[0033] As used herein, "substitution" is defined as the replacement of an amino acid with another amino acid. With the known genetic code and recombinant and synthetic DNA techniques, one skilled in the art can readily construct DNA encoding amino acid variants. The term "insertion" includes the addition of amino acid residues to the original amino acid sequence, which remains stable without significant structural changes. The term "deletion" means that one or more amino acid residues are removed from the original sequence, and the amino acids originally at the N- and C-termini of the deleted amino acids are then directly connected to form a contiguous amino acid sequence.

[0034] The term "amino acid sequence identity" refers to a quantitative comparison of the identity (or difference) of the amino acid sequences of two or more proteins. "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to maximize the percent sequence identity. To determine sequence identity, the sequence of a query protein is aligned to the sequence of a reference protein. Alignment methods are known in the art. For example, the freely available SIM local similarity program (Xiaoquin Huang and Webb Miller (1991), Advances in Applied Mathematics, vol. 12: 337-357) is preferably used. For multiple sequence alignment analysis, ClustalW is preferably used (Thompson et al. (1994) Nucleic Acids Res., 22(22): 4673-4680).

[0035] Each amino acid in the query sequence that differs from the reference amino acid sequence at a position is counted as one difference. An insertion or deletion in the query sequence also counts as one difference. The sum of the differences is then related to the length of the reference sequence to obtain the percent non-identity.

[0036] Embodiments of the invention The present invention will now be described in further detail. Each embodiment defined below may be combined with any one or more of the other embodiments, unless expressly indicated otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with any one or more of the other features indicated as being preferred or advantageous.

[0037] Fusion protein that binds to HSA with high affinity The present invention relates to a fusion protein comprising a binding protein for albumin, the albumin binding protein comprising the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having one or two substitutions, deletions or insertions thereto, and a targeting moiety selected from a non-immunoglobulin protein or an antibody or antibody fragment capable of binding to a tumor-specific targeting protein.The present invention relates to a fusion protein comprising a binding protein for albumin, the albumin binding protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 33, and a targeting moiety selected from a non-immunoglobulin protein or an antibody or antibody fragment capable of binding to a tumor-specific targeting protein.

[0038] The binding protein for albumin comprises the amino acid sequence of SEQ ID NO: 33, or it comprises an amino acid sequence having one or two substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 33, wherein SEQ ID NO: 33 has the amino acid sequence shown herein: LAEAKVLAX9KELDKX 15 GX 17 SX 19 X 20 YX 22 X 23 LIX 26 X 27 AKTX 31 X 32 GVX 35 ALKX 39 X 40 IX 42 AX 44 , In the sequence, X is L or I, and X 15 is Y or A, and X 17 is I or V, and X 19 is N or D, and X 20 is Y or F, and X 22 is K or F, and X 23 is N or R, and X 26 is N or D, and X 27 is N, K, or R, and X31 is I or V, and X 32 is E or D, and X 35 is K or E, and X 39 is D, A, or E, and X 40 is E, A, or Q, and X 42 is V or L, and / or X 44 is A or R. L = leucine (Leu), A = alanine (Ala), E = glutamic acid (Glu), K = lysine (Lys), V = valine (Val), D = aspartic acid (Asp), G = glycine (Gly), S = serine (Ser), Y = tyrosine (Tyr), I = isoleucine (Ile), T = threonine (Thr), N = asparagine (Asn), F = phenylalanine (Phe), R = arginine (Arg), Q = glutamine (Gln), P = proline (Pro), M = methionine (Met), W = tryptophan (W), H = histidine (His), C = cysteine (C)

[0039] In a preferred embodiment, the fusion protein comprises an albumin binding protein having an amino acid sequence of SEQ ID NO: 33 with one or two substitutions, deletions, or insertions, which has an aromatic amino acid such as Y or F at position 20. In a preferred embodiment, the amino acid sequence of SEQ ID NO: 33 with one or two substitutions, deletions, or insertions does not have G, L, or D at position 20 and has a K binding affinity to human HSA. D is less than 25 nM.

[0040] In preferred embodiments, the fusion protein comprises an albumin binding protein having an amino acid sequence of SEQ ID NO: 33 with one or two substitutions, deletions, or insertions, and an aromatic amino acid, such as Y, at position 21. In some embodiments, the amino acid sequence of SEQ ID NO: 33 with one or two substitutions, deletions, or insertions does not have a G at position 21 and has a K binding activity against human HSA. D is less than 25 nM.

[0041] In preferred embodiments, the fusion protein comprises an albumin binding protein having an amino acid sequence with one or two substitutions, deletions, or insertions of SEQ ID NO: 33, which has a G at position 33. In some embodiments, the amino acid sequence with one or two substitutions, deletions, or insertions of SEQ ID NO: 33 does not have an S, L, or V at position 33 and has a K binding affinity to human HSA. D is less than 25 nM.

[0042] In a preferred embodiment, the serum albumin binding protein comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having one or two substitutions, deletions or insertions in the amino acid sequence of SEQ ID NO: 33, wherein the one or two substitutions, deletions or insertions are made at positions other than 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42 and 44 of SEQ ID NO: 33. Thus, in such an embodiment, the substitutions at positions 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42 and 44 in SEQ ID NO: 33 remain unaffected. In other preferred embodiments, one or two substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 33 are made at any one of positions 20, 21, and / or 33 of SEQ ID NO: 33, as described herein above. Thus, in such embodiments, substitutions at positions 9, 15, 17, 19, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44 in SEQ ID NO: 33 remain unaffected.

[0043] In various preferred embodiments, the one or two substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 33 are one or two substitutions or deletions in the amino acid sequence of SEQ ID NO: 33, more preferably one or two substitutions in the amino acid sequence of SEQ ID NO: 33.

[0044] In a preferred embodiment, the serum albumin binding protein comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having one or two substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 33, wherein 31 is I and X 32 is E and X 39 is D and X 40 is E (as shown for SEQ ID NO: 6).

[0045] In a preferred embodiment, the serum albumin binding protein comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having one or two substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 33, wherein 31 is V and X 32 is E and X 39 is E and X 40 is A (as shown for SEQ ID NO: 9).

[0046] In a further preferred embodiment, the serum albumin binding protein comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having one or two substitutions, deletions or insertions in the amino acid sequence of SEQ ID NO: 33, in which X9 is L and more preferably the amino acids at positions 1 to 14 consist of the amino acids LAEAKVLALKELDK (SEQ ID NO: 51). In another further preferred embodiment, the serum albumin binding protein comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having one or two substitutions, deletions or insertions in the amino acid sequence of SEQ ID NO: 33, in which X9 is I and more preferably the amino acids at positions 1 to 14 consist of the amino acids LEAAKVLAIKELDK (SEQ ID NO: 52).

[0047] In some embodiments, the fusion protein comprises an albumin binding protein having an amino acid sequence with one or two substitutions, deletions, or insertions of SEQ ID NO: 33, wherein the amino acids at positions 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 16, 18, 21, 24, 25, 28, 29, 30, 33, 34, 36, 37, 38, 41, and 43 are not substituted or deleted. In such embodiments, the one or two substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 33 are made at one or two positions selected from positions 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44 of SEQ ID NO: 33, and where there are "one or two substitutions," these differ from the substitutions defined for these positions in SEQ ID NO: 33. In a preferred embodiment, the "one or two substitutions" comprises a substitution at position 20 of SEQ ID NO: 33, and such a substitution preferably still provides an aromatic amino acid, as described herein above.

[0048] In some embodiments, the binding protein for albumin comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 33 set forth above. 95% sequence identity allows for up to two modifications, particularly substitutions, in the amino acid sequence of SEQ ID NO: 33 at positions other than 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44. Thus, in such embodiments, the substitutions at positions 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44 in SEQ ID NO: 33 remain unaffected. In various preferred embodiments, the binding protein for albumin comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 33, wherein 95% sequence identity allows for up to two modifications, particularly substitutions, in the amino acid sequence of SEQ ID NO: 33 at any of positions 20, 21, and / or 33 of SEQ ID NO: 33. Thus, in such embodiments, substitutions at positions 9, 15, 17, 19, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44 in SEQ ID NO: 33 remain unaffected.

[0049] The present invention relates to a binding protein for albumin, which comprises an amino acid sequence of SEQ ID NOs: 1 to 32, or an amino acid sequence having one or two substitutions, deletions, or insertions therein, and has a K D The present invention provides a fusion protein comprising a binding protein for albumin, wherein the binding affinity of the binding protein to the albumin is less than 25 nM, and a targeting moiety selected from a non-immunoglobulin protein or an antibody or antibody fragment capable of binding to a tumor-specific targeting protein.

[0050] The present invention provides a fusion protein comprising a binding protein for albumin, wherein the albumin binding protein comprises or consists of an amino acid sequence selected from the group of SEQ ID NOs: 1 to 32, or an amino acid sequence having at least 95% amino acid sequence identity thereto, and has a K D The present invention provides a fusion protein comprising a binding protein for albumin, wherein the albumin binding protein comprises or consists of an amino acid sequence having at least 95% amino acid sequence identity to any one selected from the group consisting of SEQ ID NOs: 1 to 32, and has a K D is less than 25 nM. In preferred embodiments, with 95% sequence identity, up to two modifications, particularly substitutions, are permitted at positions other than 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44 in the amino acid sequence of any one of SEQ ID NOs: 1-32. In various preferred embodiments, with 95% sequence identity, up to two modifications, particularly substitutions, are permitted at positions 20, 21, and / or 33 in the amino acid sequence of any one of SEQ ID NOs: 1-32.

[0051] An embodiment of the present invention relates to a fusion protein comprising a binding protein for albumin, wherein the albumin binding protein consists of an amino acid sequence selected from the group of SEQ ID NOs: 1 to 32, or an amino acid sequence having one or two substitutions, deletions or insertions thereto, wherein the amino acids at positions 1 to 14 consist of the amino acids LEAAKVLAIKELDK (SEQ ID NO: 52) or the amino acids LEAAKVLALKELDK (SEQ ID NO: 51).

[0052] Some embodiments of the present invention relate to a fusion protein comprising a binding protein for albumin, wherein the albumin binding protein consists of an amino acid sequence selected from the group of SEQ ID NOs: 1-32, or an amino acid sequence having one or two substitutions, deletions, or insertions therein, wherein amino acids at positions 1-14 are LEAAKVLAIKELDK.

[0053] Some embodiments of the present invention relate to a fusion protein comprising a binding protein for albumin, wherein the albumin binding protein consists of an amino acid sequence having at least 95%, at least 97%, or 100% identity to SEQ ID NOs: 1-32, and wherein amino acids at positions 1-14 are LEAAKVLALKELDK.

[0054] Some embodiments of the present invention relate to fusion proteins comprising a binding protein for albumin, wherein the albumin binding protein binds to mammalian serum albumin. Some embodiments of the present invention relate to fusion proteins comprising a binding protein for albumin, wherein the albumin binding protein binds to human serum albumin (HSA) and / or mouse serum albumin (MSA).

[0055] In one embodiment of the invention, the fusion protein comprises an albumin binding domain as described above and a tumor target binding domain.

[0056] Some embodiments of the present invention relate to a fusion protein comprising a binding protein for albumin and, in addition, at least one targeting moiety selected from a non-immunoglobulin protein or an antibody or antibody fragment. In some embodiments, the targeting moiety has a dissociation constant K of 100 nM or less. D Some embodiments of the present invention relate to fusion proteins comprising a binding protein for albumin and, in addition, at least one targeting moiety selected from a non-immunoglobulin protein or an antibody or antibody fragment. In some embodiments, the targeting moiety has a dissociation constant K of 10 nM or less. D and capable of binding to the target protein.

[0057] Some embodiments of the present invention relate to fusion proteins comprising a binding protein for albumin and, additionally, at least one targeting moiety, wherein the at least one targeting moiety is selected from a non-immunoglobulin protein that is a ubiquitin mutein, a protein A domain mutein, an ankyrin repeat protein mutein, a lipocalin mutein, a human Fyn SH3 domain mutein, a human fibronectin 10th domain mutein, an FN3 domain mutein, a Kunitz domain mutein, a Sac7d mutein, a chagasin mutein, a multimerized low density lipoprotein receptor A mutein, a cystine-knot protein mutein, a stefin mutein, an armadillo repeat protein mutein, a tetranectin mutein, a C-type lectin domain mutein, or a CTLA4 mutein, or an antibody, or an antibody fragment, or a single domain antibody, or a single chain variable fragment of an antibody.

[0058] Some embodiments of the present invention relate to fusion proteins comprising a binding protein for albumin and, additionally, at least one targeting moiety selected from a non-immunoglobulin protein, wherein the non-immunoglobulin protein is a ubiquitin mutein exhibiting 80%-94% identity to ubiquitin (SEQ ID NO: 43), preferably at least 85%-94% identity to ubiquitin. In various embodiments, the tumor-targeting moiety is a ubiquitin mutein exhibiting 80%-94% identity to ubiquitin, preferably at least 85%-94% identity to ubiquitin, and having a binding affinity of less than 100 nM for a protein expressed by a tumor, as described elsewhere herein. In various embodiments, the tumor-targeting moiety is a ubiquitin mutein that exhibits 80% to 94% identity to ubiquitin, preferably at least 85% to 94% identity to ubiquitin, and has a binding affinity of at least 25 nM, or at least 100 nM, or at least 200 nM, or at least 500 nM for a protein expressed by a tumor, as described elsewhere herein. As will be understood by those skilled in the art, the terms "less than" and "at least" can be used interchangeably herein, as both refer to the binding affinity of a tumor-targeting moiety for a protein expressed by a tumor. The same applies to the terms "less than" and "at least" in the context of the binding affinity of a serum albumin-binding protein / domain for serum albumin, as described elsewhere herein.

[0059] In various embodiments, the tumor targeting moiety is a ubiquitin mutein that exhibits 80% to 94% identity to ubiquitin, preferably at least 85% to 94% identity to ubiquitin, and has a binding affinity of less than 100 nM to any one of PD-L1, PSMA, FOLR1, FOLR2, EGFR, Her2, ED-B, or FAP.

[0060] In various embodiments, the tumor targeting moiety is a ubiquitin mutein that exhibits 80% to 94% identity to ubiquitin, preferably at least 85% to 94% identity to ubiquitin, and has a binding affinity of at least 25 nM, or at least 100 nM, or at least 200 nM, or at least 500 nM to any one of PD-L1, PSMA, FOLR1, FOLR2, EGFR, Her2, ED-B, or FAP.

[0061] Some embodiments of the present invention relate to fusion proteins comprising a binding protein for albumin and additionally at least one targeting moiety, wherein the targeting moiety has a dissociation constant K of 100 nM or less, preferably 10 nM or less. D In some embodiments, the targeting moiety binds to a protein tumor-specific target. In some embodiments, the targeting moiety binds to a protein expressed in a tumor or on a tumor cell. In a specific embodiment, the tumor-specific targeting moiety is specific for the extracellular domain of Her2. In a specific embodiment, the tumor-specific targeting moiety is specific for the extracellular domain of FAP. In a specific embodiment, the tumor-specific targeting moiety is specific for ED-B.

[0062] Further characterization of the fusion proteins of the present invention can be carried out in the form of isolated soluble proteins. Suitable methods are known to those skilled in the art or described in the literature. Such methods include characterization of the physical, biophysical, and functional properties of the protein. The affinity and specificity of the fusion proteins can be detected by standard biochemical methods discussed above and in the Examples and known to those skilled in the art.

[0063] In some embodiments, the fusion proteins described herein have a binding affinity (K) for human serum albumin of less than 25 nM. D). The fusion protein binds to HSA with a measurable binding affinity of less than 25 nM, less than 10 nM, less than 5 nM, and less than 1 nM. Suitable methods are known to those skilled in the art or described in the literature. Methods for determining binding affinity are known per se and can be selected, for example, from the following methods known in the art: enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), equilibrium exclusion binding assay (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 examples below. Typically, the dissociation constant K D is specified in the temperature range of 20°C to 30°C, for example, at 20°C, 25°C, or 30°C. Typically, the dissociation constant K D is specified at pH 7.3. D The lower the value, the higher the binding affinity of the biomolecule for its binding partner. D The higher the value, the weaker the binding between the binding partners. K<25 nM, preferably <10 nM D Binding to HSA at a K of less than 25 nM, preferably less than 10 nM, may be important for targeted therapy applications in cancer treatment. D Fusion proteins that bind to HSA at 25° C. have an extended half-life compared to targeting moieties that are not fused to a serum albumin binding domain.

[0064] In various embodiments of the invention, the binding affinity of the serum albumin binding protein to serum albumin is less than 25 nM, preferably less than 10 nM, or less than 5 nM, or less than 1 nM at pH 7.3. In various other embodiments of the invention, the binding affinity of the serum albumin binding protein to serum albumin, as determined by SPR, is less than 25 nM, preferably less than 10 nM, or less than 5 nM, or less than 1 nM, preferably less than 25 nM, or less than 10 nM, or less than 5 nM, or less than 1 nM at pH 7.3.

[0065] In various embodiments of the invention, the binding affinity of the tumor targeting moiety to a protein expressed by a tumor is less than 100 nM, or less than 50 nM, or less than 20 nM at pH 7.3. In various embodiments of the invention, the binding affinity of the tumor targeting moiety to a protein expressed by a tumor is less than 100 nM, or less than 50 nM, or less than 20 nM as determined by SPR, preferably less than 100 nM, or less than 50 nM, or less than 20 nM as determined by SPR at pH 7.3.

[0066] In various embodiments, a fusion protein with high affinity for serum albumin comprising an albumin-binding domain and a tumor-specific binding protein has a half-life in (human or mouse) serum or blood of at least about or greater than about 17 hours, preferably at least about or greater than about 24 hours. In preferred embodiments, the tumor-specific binding protein is a non-immunoglobulin protein, more preferably the tumor-specific binding protein is a non-immunoglobulin protein comprising a ubiquitin mutein exhibiting 80%-94% identity to ubiquitin (SEQ ID NO: 43), and even more preferably the tumor-specific binding protein is a non-immunoglobulin protein comprising a ubiquitin mutein exhibiting 80%-94% identity to ubiquitin (SEQ ID NO: 43) and binding to a protein expressed by a tumor, such as PD-L1, PSMA, FOLR1, FOLR2, EGFR, Her2, FAP, and / or ED-B.

[0067] In various embodiments, a fusion protein with high affinity for serum albumin comprising an albumin-binding domain and a tumor-specific binding protein has a half-life in (human or mouse) serum or blood of at least about or greater than about 30 hours, preferably at least about or greater than about 36 hours. In preferred embodiments, the tumor-specific binding protein is a non-immunoglobulin protein, more preferably the tumor-specific binding protein is a non-immunoglobulin protein comprising a ubiquitin mutein exhibiting 80%-94% identity to ubiquitin (SEQ ID NO: 43), and even more preferably the tumor-specific binding protein is a non-immunoglobulin protein comprising a ubiquitin mutein exhibiting 80%-94% identity to ubiquitin (SEQ ID NO: 43) and binding to a protein expressed by a tumor, such as PD-L1, PSMA, FOLR1, FOLR2, EGFR, Her2, FAP, and / or ED-B.

[0068] In various embodiments, the fusion proteins of the invention comprise or consist of one (or fewer) serum albumin binding domain and one (or fewer) tumor targeting moiety (or tumor-specific binding protein). Such fusion proteins of the invention may have a half-life in serum or blood (human or mouse) of at least about 17 hours (or more than about 17 hours), at least about 24 hours (or more than about 24 hours), at least about 30 hours (or more than about 30 hours), or at least about 36 hours (or more than about 36 hours). In preferred embodiments, one (or less) tumor targeting moiety is a non-immunoglobulin protein, more preferably one (or less) tumor targeting moiety is a non-immunoglobulin protein comprising a ubiquitin mutein exhibiting 80% to 94% identity to ubiquitin (SEQ ID NO: 43), and even more preferably one (or less) tumor targeting moiety is a non-immunoglobulin protein comprising a ubiquitin mutein exhibiting 80% to 94% identity to ubiquitin (SEQ ID NO: 43) and which binds to a protein expressed by a tumor, such as PD-L1, PSMA, FOLR1, FOLR2, EGFR, Her2, FAP, or ED-B.

[0069] In various embodiments, the fusion proteins of the invention comprise or consist of (i) one (or fewer) serum albumin binding domains having at least 90% identity to the amino acid sequence of SEQ ID NO: 9, and (ii) one (or fewer) tumor targeting moiety (or tumor-specific binding protein) having at least 90% identity to the amino acid sequence of SEQ ID NO: 42. Preferably, the one (or fewer) serum albumin binding domains have at least 93% or 94% identity, more preferably 95% or 96% identity, even more preferably 97% or 98% identity, or even 100% identity to the amino acid sequence of SEQ ID NO: 9, and / or the one (or fewer) tumor targeting moiety exhibits at least 93% or 94% identity, more preferably 95% or 96% identity, even more preferably 97% or 98% identity, or even 100% identity to the amino acid sequence of SEQ ID NO: 42. Such fusion proteins of the present invention may have a half-life in serum or blood (human or mouse) of at least about 17 hours (or more than about 17 hours), at least about 24 hours (or more than about 24 hours), at least about 30 hours (or more than about 30 hours), or at least about 36 hours (or more than about 36 hours). Such fusion proteins of the present invention may have an affinity for MSA of less than 100 nM, preferably less than 50 nM, more preferably less than 40 nM (as determined by SPR; pH 7.3). Such fusion proteins of the present invention may have an affinity for FAP of less than 10 nM, preferably 6 nM or less (as determined by SPR). In various embodiments, at least 90% sequence identity with respect to SEQ ID NO: 9 refers to substitutions at positions in the amino acid sequence of SEQ ID NO: 9 other than positions 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44. In various preferred embodiments, at least 90% sequence identity with respect to SEQ ID NO: 9 refers to substitutions at positions including any of positions 20, 21, and / or 33 in the amino acid sequence of SEQ ID NO: 9.Furthermore, in preferred embodiments, one (or less) tumor targeting moiety is a non-immunoglobulin protein, more preferably one (or less) tumor targeting moiety is a non-immunoglobulin protein comprising a ubiquitin mutein exhibiting 80% to 94% identity to ubiquitin (SEQ ID NO: 43), and even more preferably one (or less) tumor targeting moiety is a non-immunoglobulin protein comprising a ubiquitin mutein exhibiting 80% to 94% identity to ubiquitin (SEQ ID NO: 43) and which binds to a protein expressed by a tumor, such as PD-L1, PSMA, FOLR1, FOLR2, EGFR, Her2, FAP, or ED-B.

[0070] In various embodiments, fusion proteins of the invention comprise or consist of one (or fewer) serum albumin binding domain and (at least) two tumor-targeting moieties (or tumor-specific binding proteins). Such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 17 hours (or more than about 17 hours). Such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 24 or 26 hours (or more than about 24 or 26 hours), or at least about 30 hours (or more than about 30 hours). In various embodiments, such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 28 or 29 hours (or more than about 28 or 29 hours). In various other embodiments, such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 36 hours (or more than about 36 hours). In a preferred embodiment, the (at least) two tumor targeting moieties are each a non-immunoglobulin protein, more preferably the (at least) two tumor targeting moieties are each a non-immunoglobulin protein comprising a ubiquitin mutein exhibiting 80% to 94% identity to ubiquitin (SEQ ID NO: 43), and even more preferably the (at least) two tumor targeting moieties are each a non-immunoglobulin protein comprising a ubiquitin mutein exhibiting 80% to 94% identity to ubiquitin (SEQ ID NO: 43) and binding to a protein expressed by a tumor, such as PD-L1, PSMA, FOLR1, FOLR2, EGFR, Her2, FAP, or ED-B.

[0071] In various embodiments, the fusion protein of the invention comprises or consists of (i) one (or less) serum albumin binding domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 9, and (ii) (at least) two tumor targeting moieties (or tumor-specific binding proteins) each exhibiting at least 90% identity to the amino acid sequence of SEQ ID NO: 42. Preferably, the one (or less) serum albumin binding domain has at least 93% or 94% identity, more preferably 95% or 96% identity, even more preferably 97% or 98% identity, or even 100% identity to the amino acid sequence of SEQ ID NO: 9, and / or the (at least) two tumor targeting moieties (or tumor-specific binding proteins) each exhibit at least 93% or 94% identity, more preferably 95% or 96% identity, even more preferably 97% or 98% identity, or even 100% identity to the amino acid sequence of SEQ ID NO: 42. Such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 24 or 26 hours (or more than about 24 or 26 hours), or at least about 30 hours (or more than about 30 hours). In various other embodiments, such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 36 hours (or more than about 36 hours). In various embodiments, such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 28 or 29 hours (or more than about 28 or 29 hours). Such fusion proteins of the invention may have an affinity for MSA of less than 100 nM, preferably less than 70 nM (as determined by SPR; pH 7.3). Such fusion proteins of the invention may have an affinity for FAP of less than 10 nM, preferably 1 nM or less (as determined by SPR). In various embodiments, at least 90% sequence identity with respect to SEQ ID NO:9 means substitutions at positions other than 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44 in the amino acid sequence of SEQ ID NO:9.In various preferred embodiments, at least 90% sequence identity with respect to SEQ ID NO: 9 refers to substitutions at positions in the amino acid sequence of SEQ ID NO: 9, including any of positions 20, 21, and / or 33. Furthermore, in preferred embodiments, the (at least) two tumor targeting moieties are each non-immunoglobulin proteins, more preferably the (at least) two tumor targeting moieties are each non-immunoglobulin proteins comprising a ubiquitin mutein exhibiting 80%-94% identity to ubiquitin (SEQ ID NO: 43), and even more preferably the (at least) two tumor targeting moieties are each non-immunoglobulin proteins comprising a ubiquitin mutein exhibiting 80%-94% identity to ubiquitin (SEQ ID NO: 43) and binding to a protein expressed by a tumor, such as PD-L1, PSMA, FOLR1, FOLR2, EGFR, Her2, FAP, or ED-B.

[0072] In various embodiments, fusion proteins of the invention comprise an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:54 (220519). Preferably, the fusion protein has at least 93% or 94% identity, more preferably 95% or 96% identity, even more preferably 97% or 98% identity, or even 100% identity to the amino acid sequence of SEQ ID NO:54. Such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 24 hours (or more than about 24 hours), or at least about 30 hours (or more than about 30 hours). In various embodiments, such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 28 or 29 hours (or more than about 28 or 29 hours). For example, SEQ ID NO:54 (220519, comprising the albumin-binding domain of SEQ ID NO:9) has a serum half-life of about 28 hours. For example, FAP-specific fusion protein 224216, comprising the albumin-binding domain (c20) of SEQ ID NO: 6 and a FAP-specific binding protein (dimer of SEQ ID NO: 42), has a serum half-life of about 29 hours. In various other embodiments, such fusion proteins of the invention may have a half-life of at least about 36 hours (or more than about 36 hours) in serum or blood (human or mouse). Such fusion proteins of the invention may have an affinity for MSA of less than 100 nM, preferably less than 70 nM (as determined by SPR; pH 7.3). Such fusion proteins of the invention may have an affinity for FAP of less than 10 nM, preferably less than 1 nM (as determined by SPR).

[0073] In various other embodiments, the fusion proteins of the invention comprise or consist of (at least) two serum albumin binding domains and one (or fewer) tumor targeting moiety (or tumor-specific binding protein). Such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 41 hours (or more than about 41 hours). In preferred embodiments, one (or less) tumor targeting moiety (or tumor-specific binding protein) is a non-immunoglobulin protein, more preferably one (or less) tumor targeting moiety (or tumor-specific binding protein) is a non-immunoglobulin protein comprising a ubiquitin mutein exhibiting 80% to 94% identity to ubiquitin (SEQ ID NO: 43), and even more preferably one (or less) tumor targeting moiety (or tumor-specific binding protein) is a non-immunoglobulin protein comprising a ubiquitin mutein exhibiting 80% to 94% identity to ubiquitin (SEQ ID NO: 43) and which binds to a protein expressed by a tumor, such as PD-L1, PSMA, FOLR1, FOLR2, EGFR, Her2, FAP, or ED-B.

[0074] In various other embodiments, fusion proteins of the invention comprise or consist of (at least) two serum albumin binding domains and (at least) two tumor-targeting moieties (or tumor-specific binding proteins). Such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 36 hours (or more than about 36 hours), and in various embodiments, at least about 39 hours (or more than about 39 hours). In some embodiments, such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 47 hours (or more than about 47 hours). In a preferred embodiment, the (at least) two tumor targeting moieties are each a non-immunoglobulin protein, more preferably the (at least) two tumor targeting moieties are each a non-immunoglobulin protein comprising a ubiquitin mutein exhibiting 80% to 94% identity to ubiquitin (SEQ ID NO: 43), and even more preferably the (at least) two tumor targeting moieties are each a non-immunoglobulin protein comprising a ubiquitin mutein exhibiting 80% to 94% identity to ubiquitin (SEQ ID NO: 43) and binding to a protein expressed by a tumor, such as PD-L1, PSMA, FOLR1, FOLR2, EGFR, Her2, FAP, or ED-B.

[0075] In various embodiments, the fusion protein of the invention comprises or consists of (i) (at least) two serum albumin binding domains, each exhibiting at least 90% identity to the amino acid sequence of SEQ ID NO: 9(d01), and (ii) (at least) two tumor targeting moieties (or tumor-specific binding proteins), each exhibiting at least 90% identity to the amino acid sequence of SEQ ID NO: 42. Preferably, the (at least) two serum albumin binding domains each have at least 93% or 94% identity, more preferably 95% or 96% identity, even more preferably 97% or 98% identity, or even 100% identity to the amino acid sequence of SEQ ID NO: 9, and / or the (at least) two tumor targeting moieties (or tumor-specific binding proteins) each exhibit at least 93% or 94% identity, more preferably 95% or 96% identity, even more preferably 97% or 98% identity, or even 100% identity to the amino acid sequence of SEQ ID NO: 42. Such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 35 or 36 hours (or more than about 35 or 36 hours), and in various embodiments, at least about 39 hours (or more than about 39 hours). In some embodiments, such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 47 hours (or more than about 47 hours). Such fusion proteins of the invention may have an affinity for MSA of less than 10 nM, preferably 1 nM or less (as determined by SPR; pH 7.3). Such fusion proteins of the invention may have an affinity for FAP of less than 10 nM, preferably 1 nM or less, more preferably 0.5 nM or less (as determined by SPR). In various embodiments, at least 90% sequence identity with respect to SEQ ID NO:9 means substitutions at positions other than 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44 in the amino acid sequence of SEQ ID NO:9.In various preferred embodiments, at least 90% sequence identity with respect to SEQ ID NO: 9 refers to substitutions at positions in the amino acid sequence of SEQ ID NO: 9, including any of positions 20, 21, and / or 33. Furthermore, in preferred embodiments, the (at least) two tumor targeting moieties are each non-immunoglobulin proteins, more preferably the (at least) two tumor targeting moieties are each non-immunoglobulin proteins comprising a ubiquitin mutein exhibiting 80%-94% identity to ubiquitin (SEQ ID NO: 43), and even more preferably the (at least) two tumor targeting moieties are each non-immunoglobulin proteins comprising a ubiquitin mutein exhibiting 80%-94% identity to ubiquitin (SEQ ID NO: 43) and binding to a protein expressed by a tumor, such as PD-L1, PSMA, FOLR1, FOLR2, EGFR, Her2, FAP, or ED-B.

[0076] In various embodiments, the fusion proteins of the invention comprise an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 47 (220521). Preferably, the fusion protein has at least 93% or 94% identity, more preferably 95% or 96% identity, even more preferably 97% or 98% identity, or even 100% identity to the amino acid sequence of SEQ ID NO: 47. Such fusion proteins of the invention may have a half-life in (human or mouse) serum or blood of at least about 35 or 36 hours (or more than about 35 or 36 hours), and in various embodiments, at least about 39 hours (or more than about 39 hours), more preferably at least about 47 hours (or more than about 47 hours). Such fusion proteins of the invention may have an affinity for MSA of less than 10 nM, preferably 1 nM or less (as determined by SPR; pH 7.3). Such fusion proteins of the invention may have an affinity for the FAP of less than 10 nM, preferably 1 nM or less, more preferably 0.5 nM or less (as determined by SPR).

[0077] Some embodiments relate to the above-described fusion proteins for use in the treatment of medical disorders such as cancer. In the treatment of cancer, it is crucial that the therapeutic moiety (targeting moiety) is specifically directed to the tumor cells of interest and that the targeting moiety has a long half-life (e.g., greater than 17 or 24 hours). This is due to a dissociation constant K for human HSA of less than 25 nM as determined using surface plasmon resonance. D This can be achieved by the use of fusion proteins of the invention comprising a targeting moiety of the invention and an albumin-binding domain having the formula: (See Figure 1). In some embodiments, the half-life of the fusion proteins described herein is 17-40 hours, in various preferred embodiments 17-39 hours, and in various further embodiments 28-39 hours. In some embodiments, the half-life of the fusion proteins described herein is approximately 36 hours. In some other embodiments, the half-life of the fusion proteins described herein is approximately 39 hours. In some further embodiments, the half-life of the fusion proteins described herein is approximately 17 hours. Still further, in some embodiments, the half-life of the fusion proteins described herein is approximately 28 or 29 hours. In diagnostic and therapeutic applications, it can be very important to have molecules with extended half-lives. Some embodiments relate to fusion proteins comprising the albumin-binding domain of SEQ ID NO: 33, or a protein at least 95% or at least 97% identical thereto. Some embodiments relate to a fusion protein comprising an albumin binding domain selected from SEQ ID NOs: 1-32, or a protein at least 95% or at least 97% identical thereto, provided that the affinity for human serum albumin is in the range of 0.1 nM to 25 nM.

[0078] Several techniques for producing fusion proteins with extended half-lives are known in the art, such as directly fusing a pharmacokinetic-modifying moiety to the fusion protein described above, or chemical coupling methods. In some embodiments, the pharmacokinetic-modifying moiety can be attached to one or more sites of the fusion protein, for example, directly, via a peptide linker sequence, or via a coupling site as described above.

[0079] Fusion protein that binds to HSA with moderate affinity In one embodiment of the present invention, a fusion protein comprising an albumin binding domain and a targeting domain of the present invention has a dissociation constant K for HSA of 25 nM to 3 μM as determined using surface plasmon resonance. D (See Examples) K of 25 nM to 3 μM, or 50 nM to 3 μM, or 100 nM to 3 μM D The moderate affinity binding of the albumin-binding domain to HSA at 1000 kJ / s may be important for targeted cancer diagnostic applications where a shorter half-life of the fusion proteins of the invention is desirable. For example, fusion proteins containing albumin-binding domains c42, c45, c49, or c50 have a half-life of approximately 6 to 15 hours. The binding affinity of the albumin-binding domain to serum albumin may be within the range of 25 nM to 100 nM, 25 nM to 200 nM, or 25 nM to 500 nM.

[0080] In various embodiments of the invention, the binding affinity of the tumor targeting moiety to a protein expressed by a tumor is at least 25 nM, or at least 100 nM, or at least 200 nM, or at least 500 nM at pH 7.3. In various embodiments of the invention, the binding affinity of the tumor targeting moiety to a protein expressed by a tumor is at least 100 nM, or at least 200 nM, or at least 500 nM, as determined by SPR at pH 7.3, preferably at least 25 nM, or at least 100 nM, or at least 200 nM, or at least 500 nM, as determined by SPR at pH 7.3. The binding affinity of the tumor targeting moiety can be in the range of 25 nM to 100 nM, 25 nM to 200 nM, or 25 nM to 500 nM.

[0081] Some embodiments relate to a fusion protein comprising any one of SEQ ID NOs: 34-38, or a protein at least 95%, 97%, or 100% identical to any one of SEQ ID NOs: 34-38, provided that the affinity for human serum albumin is in the range of 25 nM to 3 μM, or 50 nM to 3 μM, or 100 nM to 3 μM, or 25 nM to 100 nM, or 25 nM to 200 nM, or 25 nM to 500 nM.

[0082] Thus, the present invention provides a fusion protein comprising: (a) a serum albumin binding protein comprising the amino acid sequence of any one of SEQ ID NOS: 34-38, or an amino acid sequence having at least 95% or 97% identity to any one of SEQ ID NOS: 34-38, and having a binding affinity for serum albumin in the range of 25 nM to 3 μM; and (b) a targeting moiety having a binding affinity of at least 25 nM for a protein expressed by a tumor. The binding affinity for serum albumin may follow the definitions / ranges described elsewhere herein for serum albumin binding proteins with moderate affinity for serum albumin. For example, the binding affinity may be in the range of 25 nM to 100 nM, 25 nM to 200 nM, or 25 nM to 500 nM, or may be 50 nM to 3 μM, or 100 nM to 3 μM. The same applies to its binding affinity.

[0083] The present invention provides a fusion protein comprising: (a) a serum albumin binding protein comprising an amino acid sequence set forth in any one of SEQ ID NOS: 34-38, or an amino acid sequence having one or two substitutions, deletions, or insertions in the amino acid sequence set forth in any one of SEQ ID NOS: 34-38, and having a binding affinity for serum albumin in the range of 25 nM to 3 μM; and (b) a targeting moiety having a binding affinity of at least 25 nM for a protein expressed by a tumor. The binding affinity for serum albumin may follow the definitions / ranges described elsewhere herein for serum albumin binding proteins with moderate affinity for serum albumin. For example, the binding affinity may be in the range of 25 nM to 100 nM, 25 nM to 200 nM, 25 nM to 500 nM, 50 nM to 3 μM, or 100 nM to 3 μM.

[0084] In various embodiments, the binding affinity of a serum albumin binding protein to serum albumin refers to the binding affinity as determined at pH 7.3. In various other embodiments of the invention, the binding affinity of a serum albumin binding protein to serum albumin refers to the binding affinity as determined by SPR, preferably as determined by SPR at pH 7.3.

[0085] In various embodiments, the binding affinity of a tumor-targeting moiety to a protein expressed by a tumor refers to the binding affinity as determined at pH 7.3. In various embodiments, the binding affinity of a tumor-targeting moiety to a protein expressed by a tumor refers to the binding affinity as determined by SPR at pH 7.3.

[0086] In some embodiments, the fusion protein comprises an albumin binding domain, wherein the amino acid sequence of the albumin binding domain has 0, 1, or 2 substitutions, deletions, or insertions of SEQ ID NO: 33 and has Y, G, L, or D at position 20.

[0087] In a preferred embodiment, the fusion protein comprises an albumin binding domain, the amino acid sequence of which has 0, 1, or 2 substitutions, deletions, or insertions of SEQ ID NO: 6 (c20) and has G, L, or D at position 20. Specific examples include albumin binding domains of SEQ ID NO: 35 (c45) and SEQ ID NO: 36 (c46), which exhibit K binding to human HSA. D is 25nM to 3µM.

[0088] In a preferred embodiment, the fusion protein comprises an albumin binding domain, the amino acid sequence of which has 0, 1, or 2 substitutions, deletions, or insertions of SEQ ID NO: 7 (c24) and has G, L, or D at position 20. One example is provided by SEQ ID NO: 37 (c49), which binds to human HSA. D is 25nM to 3µM.

[0089] In a preferred embodiment, the fusion protein comprises an albumin binding domain, the amino acid sequence of the albumin binding domain having 0, 1, or 2 substitutions, deletions, or insertions of SEQ ID NO: 33, having G at position 33, and having a K D is 25nM to 3µM.

[0090] In a preferred embodiment, the fusion protein comprises an albumin binding domain, the amino acid sequence of which has 0, 1, or 2 substitutions, deletions, or insertions of SEQ ID NO: 33, and has a G at position 33. One example is provided by SEQ ID NO: 38 (c50), which has a K for human HSA. D is 25nM to 3µM.

[0091] In a preferred embodiment, the fusion protein comprises an albumin binding domain, the amino acid sequence of which has 0, 1, or 2 substitutions, deletions, or insertions of SEQ ID NO: 33, and has an S at position 33. One example is provided by SEQ ID NO: 34 (c42), which has a K for human HSA. D is 25 nM to 3 μM, or 50 nM to 3 μM, or 100 nM to 3 μM.

[0092] In a further preferred embodiment, the serum albumin binding protein comprises the amino acids LEAAKVLALKELDK (SEQ ID NO: 51) at positions 1-14, or the amino acids LEAAKVLAIKELDK (SEQ ID NO: 52) at positions 1-14.

[0093] In some embodiments, the fusion protein comprises an albumin binding domain and a tumor-specific binding protein and has a defined serum half-life of 4 to 15 hours.

[0094] In various embodiments, a fusion protein comprising an albumin binding domain and a tumor-specific binding protein with moderate / low affinity for serum albumin (25 nM to 3 μM) has a half-life in serum or blood (human or mouse) of about 15 hours.

[0095] Some embodiments relate to the above-mentioned fusion proteins for use in the diagnosis of medical disorders such as tumors. In tumor diagnosis, it is crucial that the therapeutic moiety (targeting moiety) is specifically directed to the cells of interest and that the half-life of the targeting moiety is extended for a defined period of time, e.g., 4-15 hours. In some embodiments, the half-life is extended for 6-12 hours. This is due to the fact that the fusion proteins, comprising a targeting moiety and an albumin-binding domain of SEQ ID NOs: 34-38 or at least 95% identical thereto, have a dissociation constant K for human HSA of greater than 25 nM, preferably greater than 50 nM, and more preferably greater than 100 nM, as determined using surface plasmon resonance. D This can be achieved by the use of fusion proteins of the invention having the following structure: The half-life of such fusion proteins described herein is 4-12 hours, and in some embodiments, 6-11 hours.

[0096] In diagnostic and therapeutic applications, it can be very important to have molecules with defined extended half-lives of less than 12 hours. Some embodiments relate to fusion proteins comprising an albumin binding domain selected from SEQ ID NOs: 34-38, or a protein at least 95% or at least 97% identical thereto, more preferably comprising an albumin binding domain selected from SEQ ID NOs: 34-38.

[0097] Further moieties: In some embodiments, the attachment of proteinaceous or non-proteinaceous moieties to the fusion protein can be performed by applying chemical methods known in the art. In some embodiments, coupling chemistries specific to the derivatization of cysteine or lysine residues can be applied. Chemical coupling can be performed by chemistries known to those skilled in the art, including, but not limited to, substitution, addition or cycloaddition, or redox reactions (e.g., disulfide formation).

[0098] Some embodiments relate to fusion proteins further comprising at least one diagnostically active moiety. Such diagnostically active moieties may be selected from radionuclides, fluorescent proteins, photosensitizers, chelators, dyes, or enzymes, or any combination of the above. In other embodiments, the fusion protein further comprises two or more diagnostic moieties. In some embodiments, fusion proteins comprising at least one diagnostic moiety may be used as imaging agents, for example, to assess the presence of tumor cells or metastases, tumor distribution, and / or tumor recurrence. Methods for detecting or monitoring cancer cells include imaging methods. Such methods include, for example, radioimaging or imaging of cancer cells by photoluminescence or fluorescence. Some embodiments relate to methods for imaging at least a portion of a subject, comprising administering to the subject a composition, the composition comprising a fusion protein described herein and an imaging agent. In some embodiments, the method further comprises diagnosing the subject with target-expressing cancer. In some embodiments, the method further comprises monitoring the distribution of the composition within the subject.

[0099] Radionuclides suitable for in vivo or in vitro imaging applications or for radiotherapy include, but are not limited to, gamma-emitting radioisotopes, positron emitters, beta emitters, and alpha emitters. In some embodiments, suitable binding partners include chelators such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), or DATA (6-pentanoic acid-6-amino-1,4-diazapine-triacetic acid), or activated derivatives thereof. In various embodiments, DOTA may be suitable as a complexing agent for radioisotopes and other imaging agents, as described in more detail in the Examples. Some embodiments relate to methods of imaging at least a portion of a subject, comprising administering to the subject a composition comprising a fusion protein described herein and a radionuclide loaded onto a suitable binding partner, e.g., DOTA. In some embodiments, the method further comprises diagnosing the subject with target-expressing cancer. In some embodiments, the method further comprises monitoring the distribution of the composition within the subject.

[0100] In some embodiments, additional amino acids can be extended at either the N-terminus or C-terminus, or both, of the fusion protein. For example, additional sequences can include sequences introduced for purification or detection. In one embodiment, the additional amino acid sequence comprises 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, a Strep tag, an oligohistidine tag, glutathione S-transferase, maltose binding protein, an intein, an intein fragment, or the albumin-binding domain of protein G.

[0101] Uses in Medicine: Various embodiments relate to the fusion proteins disclosed herein for use in medicine. In one embodiment, the fusion proteins are used in medicine to diagnose or treat tumors associated with target (e.g., Her2, FAP, or ED-B) expression. The fusion proteins disclosed herein allow for selective diagnosis and treatment of tumor cells or tumor tissue.

[0102] The present invention encompasses a method for diagnosing cancer in a subject, the method comprising obtaining a sample from the subject and detecting a protein expressed by tumor cells using a fusion protein of the present invention. The sample may be an invasive or non-invasive sample. The detection is performed ex vivo. Preferably, the subject is a mammalian subject, and more preferably, the subject is a human subject.

[0103] The present invention encompasses a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a fusion protein of the present invention. Preferably, the subject is a mammalian subject, and more preferably, the subject is a human subject.

[0104] For example, the membrane protein Her2 is known to be upregulated in tumor cells, resulting in uncontrolled tumor cell growth and the formation of metastases. Overexpression of Her2 has been reported in a wide variety of cancers. For example, Her2 overexpression occurs in approximately 15% to 30% of breast cancers and 10% to 30% of gastric / gastroesophageal cancers, and has also been observed in other cancers, such as ovarian cancer, endometrial cancer, bladder cancer, lung cancer, colon cancer, and head and neck cancer. Therefore, pharmaceutical compositions containing the fusion proteins described herein can be used to treat cancers in which Her2 is involved in the development of the disease, including, but not limited to, breast cancer, ovarian cancer, and gastric cancer, but also lung cancer, head and neck cancer, cervical cancer, prostate cancer, and pancreatic cancer.

[0105] The FAP-binding proteins described herein or fusion proteins containing the FAP-binding proteins disclosed herein enable selective diagnosis and treatment of FAP-associated cancer cells or tissues from tumors derived from non-epithelial tissues, such as breast cancer, colorectal cancer, pancreatic cancer, lung cancer, brain cancer, intrahepatic cholangiocarcinoma, and ovarian cancer, or melanoma and myeloma. FAP-binding proteins are used in the diagnosis (imaging) and treatment of most epithelial cancers, including breast cancer, lung cancer, colorectal cancer, and pancreatic cancer. FAP is known to be upregulated in tumor cells, which can result in uncontrolled tumor cell growth and the formation of metastases. In one embodiment, the FAP-binding proteins are used to diagnose FAP-associated tumors by applying in vitro methods.

[0106] ED-B-specific fusion proteins with coupled chelators, drugs, toxins, and small molecules may be particularly useful for use in the diagnosis or treatment of cancers, including breast cancer, ovarian cancer, prostate cancer, non-small cell lung cancer, colorectal cancer, pancreatic cancer, human skin cancer, hepatocellular carcinoma, intracranial meningioma, glioblastoma, or for use in the diagnosis or treatment of cardiovascular diseases, including atherosclerotic plaques, myocardial infarction, or inflammation. For example, fusion proteins with dyes coupled to the coupling moiety may be useful for cancer diagnosis. For example, fusion proteins with chelators coupled to the coupling moiety may be useful for diagnostic or therapeutic applications; for example, additional substances, such as radioisotopes, may be coupled to the chelators, preferably for use in molecular imaging.

[0107] One embodiment is a method for diagnosing (including monitoring) a subject having a tumor with expression of a specific target, the method comprising administering to the subject a described fusion protein, optionally conjugated to a radioisotope. In various embodiments, the fusion proteins disclosed herein can be used for diagnosing tumors with a specific target protein, optionally conjugated to a radioisotope. In some embodiments, imaging methods using fusion proteins with labels, such as radioactive or fluorescent labels, can be used to visualize the target protein on specific tissues or cells, for example, to assess the presence of target-specific tumor cells, the distribution of target-specific-associated tumors, the recurrence of target-specific tumors, and / or to assess the patient's response to therapeutic treatment.

[0108] One embodiment is a method of treating a subject with a target-specific cancer, the method of treatment comprising administering to the subject a described target-specific binding protein, optionally conjugated to a radioisotope and / or a cytotoxic agent. In various embodiments, the fusion proteins disclosed herein can be used for target-specific cancer treatment, optionally conjugated to a cytotoxic agent and / or a radioisotope. Some embodiments relate to the use of fusion proteins labeled with an appropriate radioisotope or cytotoxic compound for the treatment of tumor cells, particularly to control or kill target-specific tumor cells, e.g., malignant cells. In one embodiment, a curative dose of radiation is selectively delivered to target-specific tumor cells but not to normal cells.

[0109] Compositions: Various embodiments relate to compositions comprising the fusion proteins disclosed herein. Some embodiments relate to compositions comprising the fusion proteins defined above for use in medicine, preferably for use in the diagnosis or treatment of the various tumors described above. Compositions comprising the fusion proteins described above can be used in clinical applications for both diagnostic and therapeutic purposes. In particular, compositions comprising the fusion proteins described herein can be used in clinical applications for imaging, monitoring, and eliminating or inactivating pathological cells expressing tumor targets or adjacent tumor-associated structures via a bystander effect thereon.

[0110] Various embodiments relate to diagnostic compositions for target-specific cancer diagnosis, comprising a fusion protein as defined herein and a diagnostically acceptable carrier and / or diluent, such as, but not limited to, stabilizers, surfactants, salts, buffers, colorants, etc. The composition may be in the form of a liquid formulation, a lyophilizate, a granule, an emulsion, or a liposomal formulation.

[0111] Diagnostic compositions comprising the fusion proteins described herein can be used for target-specific cancer diagnosis, as described above.

[0112] Various embodiments relate to pharmaceutical (e.g., therapeutic) compositions for the treatment of diseases, comprising a fusion protein disclosed herein and a pharmaceutically (e.g., therapeutically) acceptable carrier and / or diluent. The pharmaceutical (e.g., therapeutic) composition may further contain auxiliary substances and excipients known per se, as desired. These include, but are not limited to, stabilizers, such as radical quenchers, surfactants, salts, buffers, colorants, etc.

[0113] Pharmaceutical compositions comprising the fusion proteins defined herein can be used for the treatment of diseases as described above.

[0114] The compositions comprise an effective dose of the fusion protein as defined herein. The amount of protein to be administered varies depending on the organism, the type of disease, the age and weight of the patient, as well as other factors known per se. Depending on the galenic preparation, these compositions can be administered parenterally by injection or infusion, systemically, intraperitoneally, intramuscularly, subcutaneously, transdermally, or by other conventional application methods.

[0115] The composition may be in the form of a liquid formulation, a lyophilisate, a cream, a lotion for topical administration, an aerosol form, a powder, a granular form, an emulsion or a liposomal formulation. The type of formulation will vary depending on the type of disease, the route of administration, the severity of the disease, the patient, and other factors known to those skilled in the medical arts.

[0116] The various components of the composition may be packaged as a kit together with instructions for use.

[0117] Fusion Protein Production: The fusion proteins described herein may be produced by any of a number of conventionally known techniques, such as simple organic synthesis strategies, solid-phase synthesis techniques, fragment ligation techniques, or commercially available automated synthesizers. On the other hand, fusion proteins may also be produced by conventional recombinant techniques alone or in combination with conventional synthetic techniques. Furthermore, fusion proteins may also be produced by cell-free in vitro transcription / translation.

[0118] Some embodiments relate to nucleic acid molecules encoding the fusion proteins described above.

[0119] An embodiment further provides an expression vector comprising the nucleic acid molecule, and a host cell comprising the isolated polynucleotide or expression vector.

[0120] In one embodiment, the present invention relates to a vector comprising the nucleic acid molecule described above. A vector refers to any molecule or entity (e.g., a nucleic acid, a plasmid, a bacteriophage, or a virus) that can be used to transfer protein-encoding information into a host cell. In one embodiment, the vector is an expression vector.

[0121] In one embodiment, the invention relates to a host cell or non-human host comprising a fusion protein described herein, a nucleic acid described herein, and / or a vector described herein.

[0122] Various embodiments relate to methods of producing the fusion proteins disclosed herein, which methods include culturing host cells under appropriate conditions to allow expression of the fusion protein, and, if desired, isolating the fusion protein.

[0123] For example, one or more polynucleotides encoding a fusion protein can be expressed in a suitable host, and the produced fusion protein can be isolated. Host cells include the nucleic acid molecules or vectors described above. Suitable host cells include prokaryotes and eukaryotes. A vector refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) that can be used to transfer protein-encoding information into a host cell. Various cell culture systems, including but not limited to mammalian, yeast, plant, or insect, can also be used to express recombinant proteins. Suitable conditions for culturing prokaryotic or eukaryotic host cells are known to those skilled in the art. Cell culture and protein expression for protein production can be carried out at any scale, starting from small shaker flasks to large fermenters, using techniques known to those skilled in the art.

[0124] One embodiment relates to a method for producing a binding protein as detailed above, said method comprising the following steps: (a) preparing a nucleic acid encoding a fusion protein as defined herein; (b) introducing said nucleic acid into an expression vector; (c) introducing said expression vector into a host cell; (d) culturing the host cell; (e) subjecting the host cell to culture conditions in which the fusion protein is expressed, thereby producing a fusion protein as defined herein; (f) optionally isolating the fusion protein produced in step (e); and (g) optionally conjugating the fusion protein to a further functional moiety as defined herein.

[0125] Generally, isolation of the purified fusion protein from the culture mixture may be carried out by applying conventional methods and techniques known in the art, such as centrifugation, precipitation, flocculation, different embodiments of chromatography, filtration, dialysis, concentration, and combinations thereof. Chromatographic methods are 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.

[0126] For convenient purification, the fusion protein may be fused to other peptide sequences that enhance its affinity to the separation material. Preferably, the fusion is selected so that it does not adversely affect the function of the fusion protein or so that it can be separated after purification by introducing a specific protease cleavage site. Such methods are also known to those skilled in the art.

[0127] Further aspects of the present invention include: 1. A binding protein for serum albumin comprising the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having one or two substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 33, and having a binding affinity for serum albumin of less than 25 nM.

[0128] 2. A binding protein for serum albumin (or a serum albumin binding protein according to item 1), wherein the serum albumin binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 32, or an amino acid sequence having one or two substitutions, deletions, or insertions in the amino acid sequences of SEQ ID NOs: 1 to 32, and has a binding affinity for serum albumin of less than 25 nM.

[0129] 3. A serum albumin binding protein according to item 1 or 2, comprising a binding protein for human serum albumin or mouse serum albumin.

[0130] 4. The serum albumin binding protein according to any one of items 1 to 3, wherein one or two substitutions, deletions, or insertions in any one of SEQ ID NOs: 1 to 33 are made at positions other than 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44. Accordingly, in such embodiments, the substitutions at positions 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44 defined in SEQ ID NO: 33 remain unaffected. In another preferred embodiment, one or two substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 33 are made at any one of positions 20, 21, and / or 33 of SEQ ID NO: 33, as described herein above. Thus, in such embodiments, the substitutions at positions 9, 15, 17, 19, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44 in SEQ ID NO: 33 remain unaffected.

[0131] In various preferred embodiments, the one or two substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 33 are one or two substitutions or deletions in the amino acid sequence of SEQ ID NO: 33, more preferably one or two substitutions in the amino acid sequence of SEQ ID NO: 33.

[0132] In a preferred embodiment, the serum albumin binding protein comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having one or two substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 33, wherein 31 is I and X 32 is E and X 39 is D and X 40 is E (as shown for SEQ ID NO: 6).

[0133] In a preferred embodiment, the serum albumin binding protein comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having one or two substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 33, wherein 31 is V and X 32 is E and X 39 is E and X 40 is A (as shown for SEQ ID NO: 9).

[0134] In a further preferred embodiment, the serum albumin binding protein comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having one or two substitutions, deletions or insertions in the amino acid sequence of SEQ ID NO: 33, in which X9 is L and more preferably the amino acids at positions 1 to 14 consist of the amino acids LAEAKVLALKELDK (SEQ ID NO: 51). In another further preferred embodiment, the serum albumin binding protein comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having one or two substitutions, deletions or insertions in the amino acid sequence of SEQ ID NO: 33, in which X9 is I and more preferably the amino acids at positions 1 to 14 consist of the amino acids LEAAKVLAIKELDK (SEQ ID NO: 52).

[0135] 5. The serum albumin-binding protein according to any one of Items 1 to 3, wherein the amino acids at positions 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 16, 18, 21, 24, 25, 28, 29, 30, 33, 34, 36, 37, 38, 41, and 43 of any one of SEQ ID NOs: 1 to 33 are not substituted or deleted. In such embodiments, the one or two substitutions, deletions, or insertions to any of SEQ ID NOs: 1-33 are made at one or two positions selected from positions 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44 of any of SEQ ID NOs: 1-33, and where there are "one or two substitutions," these are different from the substitutions defined for these positions in SEQ ID NO: 33. In a preferred embodiment, "one or two substitutions" includes a substitution at position 20 of SEQ ID NO: 33, and such substitutions preferably still provide an aromatic amino acid, as described elsewhere herein.

[0136] 6. The serum albumin protein according to item 1, wherein the albumin-binding protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 33. With at least 95% sequence identity, up to two modifications, particularly substitutions, are permitted at positions other than 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44 in the amino acid sequence of SEQ ID NO: 33. Accordingly, in such an embodiment, the substitutions at positions 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44 in SEQ ID NO: 33 remain unaffected. In various preferred embodiments, the binding protein for albumin comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 33, wherein 95% sequence identity allows for up to two modifications, particularly substitutions, in the amino acid sequence of SEQ ID NO: 33 at any of positions 20, 21, and / or 33 of SEQ ID NO: 33. Thus, in such embodiments, substitutions at positions 9, 15, 17, 19, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44 in SEQ ID NO: 33 remain unaffected.

[0137] In preferred embodiments, with at least 95% sequence identity, up to two modifications, particularly substitutions, are permitted in the amino acid sequence of any one of SEQ ID NOs: 1 to 32 at positions other than 9, 15, 17, 19, 20, 22, 23, 26, 27, 31, 32, 35, 39, 40, 42, and 44. In various preferred embodiments, with at least 95% sequence identity, up to two modifications, particularly substitutions, are permitted in the amino acid sequence of any one of SEQ ID NOs: 1 to 32 at any of positions 20, 21, and / or 33.

[0138] 7. A serum albumin binding protein according to any one of items 1 to 6 for use in diagnosis or therapy.

[0139] 8. A nucleic acid molecule encoding the serum albumin-binding protein according to any one of items 1 to 6.

[0140] 9. A vector comprising the nucleic acid molecule according to item 8.

[0141] 10. A host cell or non-human host comprising a serum albumin binding protein defined in any one of items 1 to 6, a nucleic acid defined in item 8, and / or a vector according to item 9.

[0142] 11. A composition comprising a serum albumin binding protein as defined in any one of items 1 to 6, a nucleic acid as defined in item 8, and / or a vector as defined in item 9.

[0143] 12. A method for producing a serum albumin binding protein as defined in any one of items 1 to 6, comprising culturing a host cell according to item 10 under conditions suitable for obtaining said serum albumin binding protein, and, if desired, isolating said serum albumin binding protein.

[0144] 13. A binding protein for serum albumin, comprising the amino acid sequence of any one of SEQ ID NOs: 34 to 38, or an amino acid sequence having at least 95% or 97% identity to any one of SEQ ID NOs: 34 to 38, and having a binding affinity for serum albumin in the range of 25 nM to 3 μM.

[0145] 14. The binding affinity for serum albumin may follow the definitions / ranges described elsewhere herein for serum albumin-binding proteins with moderate affinity for serum albumin. For example, the binding affinity may be within the range of 25 nM to 100 nM, 25 nM to 200 nM, or 25 nM to 500 nM, or may be 50 nM to 3 μM, or 100 nM to 3 μM.

[0146] 15. A serum albumin binding protein (or a serum albumin binding protein according to item 13), wherein the serum albumin binding protein comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 34 to 38, or an amino acid sequence having one or two substitutions, deletions, or insertions in the amino acid sequence of any one of SEQ ID NOs: 34 to 38, and has a binding affinity for serum albumin within the range of 25 nM to 3 μM. The binding affinity for serum albumin may follow the definitions / ranges described elsewhere herein for serum albumin binding proteins with moderate affinity for serum albumin. For example, the binding affinity may be within the range of 25 nM to 100 nM, 25 nM to 200 nM, or 25 nM to 500 nM, or may be 50 nM to 3 μM, or 100 nM to 3 μM.

[0147] 16. A serum albumin binding protein according to item 13 or 14, comprising a binding protein for human serum albumin or mouse serum albumin.

[0148] 17. The serum albumin binding protein according to any one of items 13 to 15, wherein the amino acids at positions 1 to 14 comprise or consist of the amino acids LEAAKVLALKELDK (SEQ ID NO: 51) or comprise or consist of the amino acids LEAAKVLAIKELDK (SEQ ID NO: 52).

[0149] 18. A serum albumin binding protein according to any one of items 13 to 16 for use in diagnosis or therapy.

[0150] 19. A nucleic acid molecule encoding a serum albumin-binding protein according to any one of items 13 to 16.

[0151] 20. A vector comprising the nucleic acid molecule according to item 18.

[0152] 21. A host cell or a non-human host comprising a serum albumin binding protein defined in any one of items 13 to 16, a nucleic acid defined in item 18, and / or a vector according to item 19.

[0153] 22. A composition comprising a serum albumin binding protein as defined in any one of items 13 to 16, a nucleic acid as defined in item 18, and / or a vector as defined in item 19.

[0154] 23. A method for producing a serum albumin-binding protein defined in any one of items 13 to 16, comprising culturing a host cell according to item 20 under conditions suitable for obtaining said serum albumin-binding protein, and, if desired, isolating said serum albumin-binding protein. [Example]

[0155] The following examples are provided to further illustrate the present invention. However, the present invention is not limited thereto, and the following examples merely demonstrate the feasibility of the present invention based on the above description. For a complete disclosure of the present invention, reference is also made to the documents cited in this application, which are fully incorporated by reference into this application.

[0156] Example 1. Expression and purification of fusion proteins Genes for the fusion proteins were cloned into expression vectors using standard methods and purified and analyzed as described below. All fusion proteins containing a Strep tag were expressed in E. coli and highly purified by affinity chromatography and gel filtration. After affinity chromatography using a Strep-Tactin® Superflow® high-capacity column, the eluted proteins were subjected to size-exclusion chromatography (Superdex™ 75 HiLoad 16 / 600, Superdex™ 200 HiLoad 16 / 600, or Sephacryl S200HR 16 / 600 column) using an AKTAxpress system (GE Healthcare).

[0157] The tag-free fusion protein was purified using a Praesto-HSA column followed by gel filtration (Superdex™ 200 HiLoad 26 / 600 or Superdex™ 75 HiLoad 26 / 600). After SDS-PAGE analysis, positive fractions were pooled and their protein concentrations were determined by absorbance measurement at 280 nm using the specific molar extinction coefficient.

[0158] Further analyses included reversed-phase chromatography (RP-HPLC) and analytical size-exclusion chromatography (SE-HPLC). RP-HPLC was performed using an Ultimate 3000 HPLC system (Thermo Fisher Scientific) and a PLRP-S (5 μm, 300 Å) column (Agilent). SE-HPLC was performed using an Ultimate 3000 HPLC system (Thermo Fisher Scientific) and a Superdex 75 or Superdex 200 increase 5 / 150 GL (Cytiva). No aggregation was detected.

[0159] Example 2. Functional characterization: specific binding of fusion proteins to serum albumin and targets (surface plasmon resonance, SPR) Recombinant Protein A, HSA, MSA, or target (either ED-B or Her2) was immobilized onto a High Capacity Amine sensor chip (Bruker) after NHS / EDC activation using a Sierra SPR-32 system (Bruker). The chip was equilibrated with SPR running buffer (PBS 0.05%, Tween pH 7.3). After target immobilization, free NHS groups were blocked using an injection of ethanolamine.

[0160] To characterize the binding of the fusion protein to FAP, the Fc-tagged target was injected at 60 or 30 nM over a Protein A coupling spot, followed by the fusion protein.

[0161] Binding proteins were serially diluted and applied to the chip at a flow rate of 30 μl / min. Upon binding, the analyte accumulated on the surface, increasing the refractive index. This change in refractive index was measured in real time and plotted as response or resonance units versus time. Association was performed for 120 s, and dissociation for 180 s. After each run, the chip surface was regenerated with 30 μl of regeneration buffer (10 mM glycine, pH 2.0 or pH 1.5) and equilibrated with running buffer. Binding experiments were performed using a Sierra SPR-32 system (Bruker), and data evaluation was performed using the Sierra Analyser software provided by the manufacturer, using a Langmuir 1:1 model (RI = 0). Figure 1 shows the binding affinities of the fusion proteins to specific domains of HSA and tumor targets (Figure 1A: ED-B, Figure 1B: Her2, Figure 1C: FAP).

[0162] All fusion proteins showed binding affinity to mouse serum albumin (MSA).

[0163] Fusion proteins having albumin-binding domains of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and 32 showed high specific binding affinity of less than 16 nM to MSA.

[0164] Fusion proteins having albumin-binding domains of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 17, 20, 23, 26, 27, 28, 29, 30, and 32 showed high specific binding affinity of less than 10 nM to MSA.

[0165] Fusion proteins having albumin-binding domains of SEQ ID NOs: 3, 4, 8, 9, 10, 11, 17, 20, 27, 28, 29, 30, and 32 exhibited high specific binding affinities of less than 5 nM to MSA.

[0166] Fusion proteins comprising a FAP-specific binding protein (SEQ ID NO: 42) and an albumin binding domain of SEQ ID NO: 9 have an affinity for MSA of less than 1 nM (eg, fusion proteins of SEQ ID NO: 54 and SEQ ID NO: 47).

[0167] Ubiquitin (wild type) does not bind to Her2, EDB, or FAP.

[0168] Fusion proteins containing albumin-binding domains c42 (SEQ ID NO: 34), c45 (SEQ ID NO: 35), c49 (SEQ ID NO: 37), and c50 (SEQ ID NO: 38) showed low affinity binding to HSA or MSA, down to 100 nM.

[0169] Example 3. Functional characterization: specific binding of fusion proteins to cell surface-expressed hFAP (flow cytometry) Flow cytometry was used to analyze the interaction of the fusion protein (and, as a control, a FAP-specific targeting moiety lacking the albumin-binding domain) with cell surface-exposed hFAP. The human embryonic kidney cell line HEK293, which overexpresses FAP, and HEK293-pEntry cells, an empty vector control, were used. Anti-hFAP antibody (R&D Systems, MAB3715-100) at a concentration of 1 μg / ml combined with anti-mouse IgG-Alexa488 (Invitrogen, A10680) at a 1:1000 dilution was used as a positive control for hFAP-expressing cells. The results are summarized in Table 1.

[0170] Cells were trypsinized, resuspended in FCS-containing medium, washed, and stained in pre-chilled FACS blocking buffer (3% FCS / PBS). Cell concentration was 1 × 10 6 A suspension of cells / ml was prepared for cell staining and loaded into 96-well plates (Greiner) at 100 μl / well in triplicate for each cell line.

[0171] Fusion proteins were tested in hFAP-expressing HEK293-hFAP cells at concentrations of 1 μM, 100 nM, 10 nM, 1 nM, or 0.1 nM. To exclude nonspecific binding, the fusion proteins were also incubated at the same concentrations in control cells, HEK293-pEntry. An equivalent amount of wild-type ubiquitin (clone 139090) was used as a negative control. After 45 min, the supernatant was removed, the cells were washed with blocking buffer, and 100 μl / well of rabbit anti-Strep tag antibody (GenScript; A00626) diluted 1:300 in FACS blocking buffer was added. After removing the primary antibody, goat anti-human IgG-Alexa Fluor 488 antibody (Invitrogen; A4008) was applied at a 1:1000 dilution. Flow cytometry measurements were performed with a Guava easyCyte 5HT instrument from Merck-Millipore at an excitation wavelength of 488 nm and an emission wavelength of 525 / 30 nm.

[0172] FACS experiments showed that all fusion proteins bound to hFAP expressed on the cell surface of HEK293 cells. Strong binding was observed for all fusion proteins (Table 1; indicated by +++). None of the fusion proteins showed binding to control cells (HEK293-pEntry). Anti-hFAP antibodies showed positive staining on hFAP-expressing cells. TIFF2025525305000002.tif93170

[0173] Example 4: Binding affinity of fusion proteins in target-expressing cells Her2-expressing Sk-Br-3 cells and HEK293-hFAP-overexpressing cells were thawed and washed with FCS-containing medium, then washed with FACS blocking buffer (PBS / 0.1% sodium azide / 3% FCS), and 100 μl of each was added to 1 × 10 6Cells were seeded at a density of 1000 cells / ml into a 96-well round-bottom plate. Dilution series ranging from 10 μM to 8.6 fM (CID217781, CID217787) and 3 μM to 0.2 pM (CID218466, CID218467, 218512, CID218513, CID218514, CID218515, CID218516) were used for Her2-expressing Sk-Br-3 cells, and dilution series of fusion proteins 218690 and 218702 were used for hFAP-overexpressing HEK293 cells at 1 μM to 0.07 pM for 45 min at 4°C. After centrifugation, the supernatant was removed, the 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 removing the primary antibody, goat anti-rabbit IgG-Alexa Fluor 488 antibody (Invitrogen; A4008) was applied at a 1:1000 dilution in FACS blocking buffer. Flow cytometry measurements were performed using a Guava easyCyte 5HT instrument from Merck-Millipore at an excitation wavelength of 488 nm and an emission wavelength of 525 / 30 nm. The results are shown in Table 2. * means a fusion protein comprising an albumin binding domain as a dimer (homodimer) and / or a targeting moiety as a dimer. TIFF2025525305000003.tif119170

[0174] Example 5. Long-term stability of fusion proteins in serum (determining binding affinity to target by flow cytometry) Fusion protein 218690 and fusion protein 218702 were diluted in a dilution series ranging from 1 μM to 0.07 pM and incubated in 100% mouse serum at 37°C for 24 hours. hFAP-expressing HEK293 cells were thawed, washed with FCS-containing medium, then washed with FACS blocking buffer (PBS / 0.1% sodium azide / 3% FCS), and 100 μl of each was added to a 1 × 10 6Cells were seeded into 96-well round-bottom plates at a density of 1000 cells / ml. A dilution series of the fusion protein was incubated with human serum at 37°C for 24 hours and 0 hours (control). HEK293-hFAP cells were then incubated with the dilution series at 4°C for 45 minutes. The cells were centrifuged, and the supernatant was removed. The 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 removing the primary antibody, goat anti-rabbit IgG-Alexa Fluor 488 antibody (Invitrogen; A4008) was applied at a 1:1000 dilution in FACS blocking buffer. Flow cytometry measurements were performed using a Guava easyCyte 5HT instrument (Merck-Millipore) at an excitation wavelength of 488 nm and an emission wavelength of 525 / 30 nm. The results are shown in Table 2. No significant difference in binding to hFAP was observed even after 24 hours of serum incubation. The fusion protein is stable in mouse serum.

[0175] Example 6. Long-term stability of fusion proteins in serum (determining binding affinity to target by ELISA) 2.5 μg / ml hFAP-Fc or 1.2 μg / ml Her2-Fc was immobilized on high-binding plates (Greiner, 781061) overnight at 4°C. A dilution series of fusion proteins specifically binding to Her2, ranging from 3 μM to 0.07 pM, was incubated in 100% human serum or 100% mouse serum at 37°C for 24 hours. A dilution series of fusion proteins specifically binding to hFAP, ranging from 3 μM to 0.2 pM or 1 μM to 0.01 fM, was incubated in 100% human serum or 100% mouse serum at 37°C for 24 hours. ELISA plates were washed three times with PBST (PBS + 0.1% Tween) and blocked with 3% BSA / 0.5% Tween / PBS at room temperature (rt) for up to 2 hours. After preincubation in the presence of serum for 0 or 24 hours, the dilution series was incubated on an ELISA plate for up to 1 hour at room temperature. Wells were washed with PBST and incubated with biotinylated anti-ubiquitin antibody (1:300) for up to 1 hour at room temperature. Binding was visualized with streptavidin-HRP (1:5,000).

[0176] Tables 3 and 4 show the binding affinity for each target after 0 and 24 hours of incubation in serum, as shown in K D The fusion proteins contain an albumin binding domain (referred to as HED) and a targeting moiety. The targeting moieties of SEQ ID NOs: 41 and 42 bind to hFAP, respectively, and SEQ ID NO: 40 binds to Her2. * means a dimer, e.g., 9 * means SEQ ID NO: 9 as a dimer in the fusion protein, and 40 * refers to SEQ ID NO: 40 as a dimer in the fusion protein. D No significant shift in binding was observed, and no significant decrease or increase in maximum binding was observed. The fusion protein is stable in human and mouse serum. TIFF2025525305000004.tif144170TIFF2025525305000005.tif153170

[0177] Example 7: Long-term stability of fusion protein in serum (Western blot analysis) Fusion proteins containing the ED-B-specific targeting moiety of SEQ ID NO: 39 and the albumin-binding domains c45 (SEQ ID NO: 35; fusion protein 221202), c49 (SEQ ID NO: 37; fusion protein 221203), c50 (SEQ ID NO: 38; fusion protein 221204), or c46 (SEQ ID NO: 36; fusion protein 221248) were tested for stability in human serum by prolonged incubation (up to 2 days). These fusion proteins were incubated at 37°C in 100% human serum and at 37°C in 100% mouse serum for fusion proteins 221248 and 221204 at a concentration of 3.6 ng / μl for 48 hours. 30 ng was removed from the mixture for analysis after 0, 6, 24, and 48 hours. The protein-serum mixtures were boiled at 95°C for 5 minutes. Thirty nanograms of the fusion protein-serum mixture was applied to an unstained gel (4-20% Mini-PROTEAN® TGX Stain-Free™ Protein Gels, #4568094, Bio-Rad). Proteins were transferred to a PVDF membrane (Trans-Blot Turbo Midi 0.2 μM PVDF Transfer Packs, #1704157, Bio-Rad) using a Gel Doc EZ System (#1708270, Bio-Rad) according to the manufacturer's instructions. The membrane was blocked overnight at 4°C in blocking buffer (PBS, 0.1% Tween, 3% BSA). After three washes with PBST (PBS, 0.1% Tween), the membrane was incubated with biotinylated anti-ubiquitin antibody (1:2000) in blocking buffer for 1 hour at room temperature, followed by three washes and further incubation with StreptAvidin-HRP (#554066, BD Pharmingen, Becton Dickinson) at a dilution of 1:5000 in blocking buffer. Proteins were visualized using Pierce ECL Western Blotting Substrate (#32209, Thermo Fisher Scientific) according to the manufacturer's instructions.The blot showed that the fusion protein was stable in human or mouse serum for as long as 48 hours.

[0178] Example 8: DOTA coupling of fusion proteins 218462, 218464, 220521 and loading with Lu or In Fusion proteins comprising the Her2-specific targeting moiety of SEQ ID NO: 40 and the albumin-binding domain c20 (SEQ ID NO: 6; fusion protein 218462) or d01 (SEQ ID NO: 9; fusion protein 218464), as well as fusion protein 220521 comprising the hFAP-specific targeting moiety (SEQ ID NO: 42, as a dimer) and d01 (SEQ ID NO: 9, as a dimer), fusion protein 220520 (SEQ ID NO: 53) comprising the hFAP-specific targeting moiety (SEQ ID NO: 42) and d01 (SEQ ID NO: 9, as a dimer), fusion protein 220519 (SEQ ID NO: 54) comprising the hFAP-specific targeting moiety (SEQ ID NO: 42, as a dimer) and d01 (SEQ ID NO: 9), or fusion protein 220518 (SEQ ID NO: 55) comprising the hFAP-specific targeting moiety of SEQ ID NO: 42 and d01 (SEQ ID NO: 9) were coupled with DOTA and loaded with Lu or In. The protein was incubated with a 20-fold excess of maleimide-DOTA (Chematech) in 50 mM Hepes, 5 mM EDTA, 150 mM NaCl, pH 7.0, for 3 hours at room temperature. Uncoupled maleimide-DOTA was removed with Resource Q or Resource S, followed by gel filtration (Superdex™ 75 HiLoad 16 / 600). To load DOTA with Lu or In, the protein was incubated with equimolar concentrations of indium(III) chloride or lutetium(III) chloride (Sigma) in 100 mM NaAc, pH 5.5, for 60 minutes at 50°C. After centrifugation, the protein was desalted in PBS, pH 7.3, and analyzed by MALDI-TOF, RP-HPLC, SE-HPLC, and SPR. TIFF2025525305000006.tif159170

Claims

1. (a) a binding protein for serum albumin comprising the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence having one or two substitutions, deletions, or insertions relative to SEQ ID NO: 33, and having a binding affinity for serum albumin of less than 25 nM; (b) a targeting moiety having a binding affinity of less than 100 nM for a protein expressed by the tumor; A fusion protein comprising:

2. 2. The fusion protein of claim 1, wherein the binding protein for serum albumin comprises an amino acid sequence selected from the group of SEQ ID NOs: 1-32, or comprises an amino acid sequence having one or two substitutions, deletions, or insertions relative to SEQ ID NOs: 1-32, and has a binding affinity for serum albumin of less than 25 nM.

3. 3. The fusion protein of claim 1 or 2, comprising a binding protein for human serum albumin or mouse serum albumin.

4. 3. The fusion protein of claim 1, wherein the targeting moiety is a non-immunoglobulin protein, preferably a ubiquitin mutein, a protein A domain mutein, an ankyrin repeat protein mutein, a lipocalin mutein, a human Fyn SH3 domain mutein, a human fibronectin domain 10 mutein, an FN3 domain mutein, a Kunitz domain mutein, a Sac7d mutein, a chagasin mutein, a multimerized low density lipoprotein receptor A mutein, a cystine-knot protein mutein, a stefin mutein, an armadillo repeat protein mutein, a tetranectin mutein, a C-type lectin domain mutein, or a CTLA-4 mutein, or wherein the targeting moiety is an immunoglobulin, an immunoglobulin fragment or variant thereof, a single domain antibody, or a single chain variable fragment (scFv) of an antibody.

5. The fusion protein of claim 4, wherein the non-immunoglobulin protein comprises a ubiquitin mutein exhibiting 80% to 94% identity to ubiquitin (SEQ ID NO: 43).

6. 3. The fusion protein of claim 1 or 2, wherein the targeting moiety binds to a protein expressed by tumors, such as Her2, FAP, or ED-B.

7. 3. The fusion protein of claim 1 or 2 for use in the diagnosis or treatment of cancer, wherein the half-life (serum or blood) of the targeting moiety of the fusion protein is longer than the half-life (serum or blood) of a targeting moiety that does not include the serum albumin binding protein.

8. A nucleic acid molecule encoding the fusion protein of claim 1.

9. A vector comprising the nucleic acid molecule of claim 8.

10. A host cell or non-human host comprising a fusion protein as defined in claim 1 or 2, a nucleic acid as defined in claim 8, and / or a vector as defined in claim 9.

11. A composition comprising a fusion protein as defined in claim 1 or 2, a nucleic acid as defined in claim 8, and / or a vector as defined in claim 9.

12. 11. A method for producing a fusion protein as defined in claim 1 or 2, comprising culturing a host cell as defined in claim 10 under conditions suitable for obtaining said fusion protein, and, if desired, isolating said fusion protein.

Images