Novel FOLR1-specific binding proteins for cancer diagnosis and therapy

Abstract

To provide folate receptor α (FOLR1) binding proteins and methods of producing the same in diagnosis and therapy of FOLR1-related cancer.SOLUTION: A FOLR1 binding protein comprises an amino acid sequence having 70-83% sequence identity to a specific amino acid sequence, where the amino acids corresponding to positions 9, 10, 12, 42, 44, 46, 62, 63, 64, 65, 66, 68 and 70 of the specific amino acid sequence are substituted.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to novel binding proteins specific for folate receptor alpha (FOLR1). The present invention also relates to FOLR1 binding proteins that further comprise a diagnostically or therapeutically effective component. Further aspects of the present invention also include the use of these FOLR1 binding proteins in medicine, for example in the diagnosis and treatment of cancers related to FOLR1. [Background technology]

[0002] 2. Background of the Invention Folate receptor alpha (FOLR1) is a glycosylphosphatidylinositol-anchored membrane protein with high affinity to bind active folate and regulate its transport into the cell interior. FOLR1 is expressed only at low concentrations and has a limited distribution in normal human tissues, such as renal proximal tubule cells.

[0003] FOLR1 is significantly overexpressed in some solid tumors, especially in solid tumor types of epithelial origin. The most frequent cancer types with FOLR1 expression are ovarian, endometrial, brain, lung, and renal cancers. In addition, FOLR1 expression was also found at moderate frequencies in head and neck, breast, gastric, and colorectal cancers.

[0004] Ovarian cancer is the most lethal of all cancers in women. Elevated levels of FOLR1 have been reported in almost all epithelial ovarian cancers. Elevated levels of FOLR1 are associated with high levels of tumor aggressiveness, resulting in shorter disease-free intervals and poorer overall survival in patients. Although most patients initially respond to chemotherapy, they experience disease recurrence due to tumor resistance to chemotherapy. Non-small cell lung cancer accounts for the majority of lung cancers and is associated with poor prognosis for patients, with low 5-year survival rates across all stages. High tissue expression of FOLR1 has been observed in many estrogen receptor / progesterone receptor-negative breast cancers. Additionally, overexpression of FOLR1 has also been detected in patients with rare and aggressive forms of breast cancer and pleural mesothelioma.

[0005] Very few targeted diagnostic or therapeutic agents have been described for tumors with high FOLR1 levels. One example of a potential treatment for chemotherapy (platinum)-resistant ovarian cancer that is linked to FOLR1 is the antibody-drug conjugate mirvetuximab soravtansine, which is currently in clinical trials. Another monoclonal antibody in clinical trials is farletuzumab for ovarian and lung cancer.

[0006] The diagnosis and treatment of FOLR1-associated cancers are not adequately addressed by existing options, and as a result, many patients do not benefit fully from currently used strategies. Needless to say, there is a strong need for novel strategies for diagnosing and treating FOLR1-associated tumors.

[0007] One objective of the present invention is to provide a molecule for specifically targeting FOLR1 to enable targeted diagnostic and therapeutic options, including the detection of FOLR1-positive tumors.Targeting this tumor-associated protein may benefit patients who have unmet needs for novel diagnostic and therapeutic pathways.Because FOLR1 is distributed poorly and limitedly in normal tissues, targeting FOLR1 suggests a potentially non-toxic diagnostic and therapeutic approach.Therefore, binding proteins with specificity for FOLR1 may enable effective medical options for cancer, ultimately improving the quality of life of patients.

[0008] The present invention provides novel FOLR1-binding molecules for new and improved strategies in the diagnosis and treatment of FOLR1-related cancers.

[0009] The foregoing objects and advantages are achieved by the subject matter of the appended claims. The present invention satisfies the above-stated needs by providing exemplary FOLR1 binding proteins. The above summary does not necessarily describe all of the problems solved by the present invention. Summary of the Invention

[0010] The present disclosure provides the following [1] to

[15] , but is not limited to these: [1] A folate receptor alpha (FOLR1) binding protein comprising ubiquitin-derived amino acids as set forth in the amino acid sequence of SEQ ID NO: 46, wherein the amino acids corresponding to positions 9, 10, 12, 42, 44, 46, 62, 63, 64, 65, 66, 68, and 70 of SEQ ID NO: 46 have been substituted. [2] the amino acid corresponding to position 9 of SEQ ID NO: 46 is selected from E, L, S, or N; and the amino acid corresponding to position 10 of SEQ ID NO: 46 is selected from E, Q, Y, or I; and the amino acid corresponding to position 12 of SEQ ID NO: 46 is selected from Y, E, W, or D; and the amino acid corresponding to position 42 of SEQ ID NO: 46 is selected from E, K, Y, Q, or M; and the amino acid corresponding to position 44 of SEQ ID NO: 46 is selected from L, Y, V, or F; and the amino acid corresponding to position 46 of SEQ ID NO: 46 is selected from Y, D, or S; and the amino acid corresponding to position 62 of SEQ ID NO: 46 is selected from L, R, D, or I; and the amino acid corresponding to position 63 of SEQ ID NO: 46 is selected from G, F, L, or A; and the amino acid corresponding to position 64 of SEQ ID NO: 46 is selected from G, D, or Y; and the amino acid corresponding to position 65 of SEQ ID NO: 46 is selected from A, D, Y, G, or M; and the amino acid corresponding to position 66 of SEQ ID NO: 46 is selected from V, H, Y, or T; and the amino acid corresponding to position 68 of SEQ ID NO: 46 is selected from K, D, P, or T; and the amino acid corresponding to position 70 of SEQ ID NO: 46 is selected from Q, P, T, W, or H; and Optionally, one or more additional amino acids of SEQ ID NO: 46 are modified. [1] A FOLR1 binding protein. [3] The FOLR1 binding protein described in [2], comprising amino acids having a sequence identity of 70% to 85% to SEQ ID NO: 46, preferably 75% to 83% to SEQ ID NO: 46, preferably 76% to 83% to SEQ ID NO: 46, and preferably 79% to 83% to SEQ ID NO: 46. [4] A FOLR1 binding protein according to any one of [1] to [3], wherein the amino acid corresponding to position 11 of SEQ ID NO: 46 is selected from K or R, and the amino acid corresponding to position 45 of SEQ ID NO: 46 is selected from W, R, or G. [5] Amino acid residues selected from EEKY, EERY, EQKY, LYKE, SYKW, or NIKD, which correspond to positions 9, 10, 11, and 12 of SEQ ID NO: 46; and amino acid residues selected from ELLWY, KLLWY, KLLRY, YLYWD, YLYGD, QLVWD, or MLFWS corresponding to positions 42, 43, 44, 45, and 46 of SEQ ID NO: 46; and Amino acid residues selected from LGGAVLKLQ, LGDAVLKLQ, LGGAVLKLP, RFGDHLDLT, RFGYHLDLT, DLGGYLPLW, or IAYMTLTLH, corresponding to positions 62, 63, 64, 65, 66, 67, 68, 69, and 70 of SEQ ID NO: 46. The FOLR1 binding protein according to any one of [1] to [4], [6] At one, two, three, four, or five amino acid positions selected from positions corresponding to 6, 8, 13, 14, 20, 23, 24, 25, 29, 30, 31, 32, 33, 34, 48, 49, 51, 52, 58, 59, 60, 71, and 72 of SEQ ID NO: 44, , E24A, N25D, K29R, K29E, K29T, I30V, I30L, Q31R, D32G, D32N, K33R, K33Q, E34A, K48E, Q49R, E51K, E51D, D52G, D58N, Y59H, N60T, N60S, L71P, R72G, R72Y, or R72K. [7] FOLR1 described in any one of [1] to [6], wherein the FOLR1-binding protein is a multimer consisting of multiple FOLR1-binding proteins described in any one of [1] to [6], preferably a dimer of the FOLR1-binding protein described in any one of [1] to [6], more preferably a homodimer of the FOLR1-binding protein described in any one of [1] to [6]. [8] A FOLR1 binding protein according to any one of [1] to [7], comprising or consisting of an amino acid sequence selected from the group consisting of amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 45 and SEQ ID NOs: 69 to 73, or amino acid sequences each having at least 90% identity thereto. [9] A FOLR1 binding protein according to any one of [1] to [8], further comprising one or more coupling domains consisting of 1 to 80 amino acids comprising one or more coupling sites for coupling a chemical moiety, preferably wherein the chemical moiety is selected from the group consisting of a chelating agent, a drug, a toxin, a dye, and a small molecule.

[10] A FOLR1 binding protein described in any one of [1] to [9], further comprising at least one diagnostically effective moiety selected from a radionuclide, a fluorescent protein, a photosensitizer, a dye, or an enzyme, or any combination of the foregoing.

[11] The FOLR1 binding protein of any one of [1] to [9], further comprising at least one therapeutically effective moiety selected from a monoclonal antibody or a fragment thereof, a radionuclide, a cytotoxic compound, a cytokine, a chemokine, an enzyme, or a derivative thereof, or any combination of the foregoing.

[12] The FOLR1 binding protein according to any one of [1] to

[11] , further comprising at least one pharmacokinetic modulating moiety arbitrarily selected from polyethylene glycol, human serum albumin, an albumin-binding protein or peptide, an immunoglobulin-binding protein or peptide, or an immunoglobulin or immunoglobulin fragment, a polysaccharide, or a structurally undefined amino acid sequence comprising the amino acids alanine, glycine, serine, and proline.

[13] A FOLR1 binding protein described in any one of [1] to

[12] for use in the diagnosis or treatment of a tumor associated with FOLR1, preferably for tumor imaging and radiotherapy of a tumor associated with FOLR1.

[14] A composition comprising a FOLR1 binding protein described in any one of [1] to

[13] for use in medicine, preferably for use in the diagnosis or treatment of tumors associated with FOLR1, preferably for tumor imaging and radiotherapy of tumors associated with FOLR1.

[15] (a) culturing a host cell under conditions suitable for obtaining a FOLR1 binding protein; and (b) isolating the FOLR1-binding protein. A method for producing a FOLR1 binding protein described in any one of [1] to

[13] , comprising:

[0011] [The present invention 1001] 1. A folate receptor alpha (FOLR1) binding protein comprising an amino acid sequence having 70% to 83% sequence identity to the amino acid sequence of SEQ ID NO: 46, wherein the amino acids corresponding to positions 9, 10, 12, 42, 44, 46, 62, 63, 64, 65, 66, 68, and 70 of SEQ ID NO: 46 are substituted as follows: at position 9 of SEQ ID NO: 46, selected from E, L, S, or N; and at position 10 of SEQ ID NO: 46, selected from E, Q, Y, or I; and at position 12 of SEQ ID NO: 46, selected from Y, E, W, or D; and at position 42 of SEQ ID NO: 46, selected from E, K, Y, Q, or M; and at position 44 of SEQ ID NO: 46, selected from L, Y, V, or F; and position 46 of SEQ ID NO: 46 is selected from Y, D, or S; and position 62 of SEQ ID NO: 46 is selected from L, R, D, or I; and at position 63 of SEQ ID NO: 46, selected from G, F, L, or A; and at position 64 of SEQ ID NO: 46, selected from G, D, or Y; and at position 65 of SEQ ID NO: 46, selected from A, D, Y, G, or M; and at position 66 of SEQ ID NO: 46, selected from V, H, Y, or T; and at position 68 of SEQ ID NO: 46, selected from K, D, P, or T; and Position 70 of SEQ ID NO: 46 is selected from Q, P, T, W, or H. [The present invention 1002] 1001. A FOLR1 binding protein of the present invention, comprising an amino acid sequence having 75% to 83% sequence identity to SEQ ID NO: 46. [The present invention 1003] The FOLR1 binding protein of the present invention 1001 or 1002, wherein the amino acid corresponding to position 11 of SEQ ID NO: 46 is selected from K or R, and the amino acid corresponding to position 45 of SEQ ID NO: 46 is selected from W, R, or G. [The present invention 1004] Amino acid residues selected from EEKY, EERY, EQKY, LYKE, SYKW, or NIKD, which correspond to positions 9, 10, 11, and 12 of SEQ ID NO: 46; and amino acid residues selected from ELLWY, KLLWY, KLLRY, YLYWD, YLYGD, QLVWD, or MLFWS corresponding to positions 42, 43, 44, 45, and 46 of SEQ ID NO: 46; and Amino acid residues selected from LGGAVLKLQ, LGDAVLKLQ, LGGAVLKLP, RFGDHLDLT, RFGYHLDLT, DLGGYLPLW, or IAYMTLTLH, corresponding to positions 62, 63, 64, 65, 66, 67, 68, 69, and 70 of SEQ ID NO: 46. The FOLR1 binding protein of any one of claims 1001 to 1003. [The present invention 1005] At one, two, three, four, or five amino acid positions selected from positions corresponding to 6, 8, 13, 14, 20, 23, 24, 25, 29, 30, 31, 32, 33, 34, 48, 49, 51, 52, 58, 59, 60, 71, and 72 of SEQ ID NO: 46, preferably K6V, K6Q, L8R, L8E, L8Y, I13T, T14A, T14P, S20C, S20G, I23T, I23G, I23H ... The FOLR1 binding protein of any of claims 1001 to 1004, further comprising a substitution selected from any one of V, E24G, E24A, N25D, K29R, K29E, K29T, I30V, I30L, Q31R, D32G, D32N, K33R, K33Q, E34A, K48E, Q49R, E51K, E51D, D52G, D58N, Y59H, N60T, N60S, L71P, R72G, R72Y, or R72K. [The present invention 1006] A FOLR1-binding protein of any one of the present inventions 1001 to 1005, which is a multimer consisting of multiple FOLR1-binding proteins of any one of the present inventions 1001 to 1005, preferably a dimer of a FOLR1-binding protein of any one of the present inventions 1001 to 1005, more preferably a homodimer of a FOLR1-binding protein of any one of the present inventions 1001 to 1005. [The present invention 1007] An amino acid sequence selected from the group consisting of SEQ ID NOs: 1-45, 69-74, or an amino acid sequence having at least 90% identity thereto, respectively. A FOLR1-binding protein according to any one of claims 1001 to 1006, comprising or consisting of the same. [The present invention 1008] further comprising one or more coupling domains of 1 to 80 amino acids comprising one or more coupling sites for coupling a chemical moiety; Preferably, the chemical moiety is selected from the group consisting of a chelator, a drug, a toxin, a dye, and a small molecule. A FOLR1 binding protein according to any one of claims 1001 to 1007. [The present invention 1009] Any of the FOLR1 binding proteins of claims 1001 to 1008, further comprising at least one diagnostically effective moiety selected from a radionuclide, a fluorescent protein, a photosensitizer, a dye, or an enzyme, or any combination of the foregoing. [The present invention 1010] The FOLR1 binding protein of any of claims 1001 to 1008, further comprising at least one therapeutically effective moiety selected from a monoclonal antibody or fragment thereof, a radionuclide, a cytotoxic compound, a cytokine, a chemokine, an enzyme, or a derivative thereof, or any combination of the foregoing. [The present invention 1011] A FOLR1 binding protein of any of the present inventions 1001 to 1010, further comprising at least one pharmacokinetic modulating moiety arbitrarily selected from polyethylene glycol, human serum albumin, an albumin-binding peptide, an immunoglobulin-binding peptide, or an immunoglobulin or immunoglobulin fragment, a polysaccharide, or a structurally undefined amino acid sequence containing the amino acids alanine, glycine, serine, and proline. [The present invention 1012] A FOLR1-binding protein of any of claims 1001 to 1011 for use in the diagnosis or treatment of a tumor associated with FOLR1, preferably for imaging of a tumor associated with FOLR1 and for radiotherapy of a tumor associated with FOLR1. [The present invention 1013] A composition comprising any of the FOLR1 binding proteins of the present inventions 1001 to 1012 for use in medicine, preferably for use in the diagnosis or treatment of tumors associated with FOLR1, preferably for imaging of tumors associated with FOLR1 and radiation treatment of tumors associated with FOLR1. [The present invention 1014] (a) culturing a host cell under conditions suitable for obtaining a FOLR1 binding protein; and (b) isolating the produced FOLR1-binding protein. A method for producing a FOLR1-binding protein of any one of claims 1001 to 1013. This summary does not necessarily describe every aspect of the invention: other aspects will become apparent upon review of the following detailed description. [Brief description of the drawings]

[0012] The drawings show: [Figure 1] Selected amino acid sequence of folate receptor alpha (FOLR1) binding protein. Differences from ubiquitin (SEQ ID NO: 46) are highlighted in grey. Numbers in the top row refer to the corresponding positions in SEQ ID NO: 46. [Diagram 2]Binding of affilin-199490 (homodimer of affilin-197556b) with high affinity to FOLR1. Analysis by label-free interaction assay using SPR (Biacore). FOLR1 was immobilized on a CM-5 chip. Lines represent various concentrations: 0 nM, 0.1 nM, 0.3 nM, 0.93 nM, 2.77 nM, 8.3 nM, 25 nM, and 75 nM. After fitting the data with a 1:1 Langmuir model, the calculated KD value for affilin-199490 was less than 0.5 nM. [Diagram 3] Affilin-199490 does not bind to FOLR2 (folate receptor beta), but specifically binds to FOLR1 (folate receptor alpha). Analysis by label-free interaction assay using SPR (Biacore). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Detailed Description of the Invention The present inventors have created a solution to meet the persistent and strong need in the art for expanding medical options for cancer diagnosis and treatment by providing a novel FOLR1 binding protein. The functional characteristic of the FOLR1 specific protein defined herein is that it has high specific affinity for FOLR1 but not for FOLR2. In particular, the present invention provides a FOLR1 binding protein based on ubiquitin mutein (also known as Affilin® molecule). The FOLR1 binding protein described herein provides a molecular form with favorable physicochemical properties, high level expression in bacteria, and allows for easy production methods. This novel FOLR1 binding protein can open up previously unaddressed medical strategies for the diagnosis and treatment of cancers related to FOLR1. In particular, the FOLR1 binding protein can be used for imaging purposes, for example for the presence of tumor cells expressing FOLR1, and for radiotherapy of tumors expressing FOLR1.

[0014] Before the present invention is described in more detail below, it should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, which may vary.It should also be understood that the terminology used herein is intended to describe only certain aspects and embodiments, and is not intended to limit the scope of the present invention reflected by the appended claims.Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.This includes those skilled in the art of protein manipulation and purification, but also those skilled in the art of developing new target-specific binding molecules for use in technical applications and therapeutic and diagnostic methods.

[0015] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0016] Throughout this specification and the claims which follow, unless the context dictates otherwise, the word "comprise" and variations such as "comprises" and "comprising" are understood to mean the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integers and steps or groups of integers and steps. The terms "comprise(s)" or "comprising" may include a limitation to "consists of" or "consisting of" if such a limitation is necessary to any extent for any reason.

[0017] Several documents (e.g., patents, patent applications, scientific papers, manufacturers' specifications, instruction manuals, GenBank accession number sequence submissions, etc.) may be cited throughout this specification. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by prior invention. Some of the documents cited herein may be characterized as "incorporated by reference." In the event of a conflict between the definitions or teachings of such incorporated references and those set forth herein, the body of this specification shall control.

[0018] All sequences referred to herein are disclosed in the accompanying sequence listing, which, together with its entire contents and disclosure, forms part of the disclosure of the present specification.

[0019] General definitions of important terms used in this application As used herein, the term "FOLR1" or "folate receptor alpha" refers to Uniprot Accession No. P15328, particularly residues 25 to 234. The term "FOLR1" includes any polypeptide that exhibits at least 80%, 85%, 90%, 95%, 96%, or 97% or more, or 100% sequence identity to FOLR1 of Uniprot Accession No. P15328 (human), particularly residues 25 to 234, or FOLR1 of Uniprot Accession No. P35846 (mouse).

[0020] As used herein, the term "FOLR2" or "folate receptor beta" refers to the corresponding protein in Uniprot accession number P14207 or other species.

[0021] The term "FOLR1-binding protein" means a protein that binds with high affinity to FOLR1.

[0022] 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 synonymously with any of these terms. The term "polypeptide" is also intended to refer to the products of post-translational modifications of polypeptides, which are well known in the art.

[0023] The term "modification" or "amino acid modification" refers to the substitution, deletion, or insertion of a reference amino acid with another amino acid at a particular position in a parent polypeptide sequence. Given the known genetic code and recombinant and synthetic DNA techniques, a skilled scientist can readily construct DNA encoding amino acid variants.

[0024] The term "mutein" as used herein means a derivative of ubiquitin or a similar protein, for example as set forth in SEQ ID NO: 46, that differs from the amino acid sequence by amino acid exchanges, insertions, deletions, or any combination thereof, provided that the mutein has specific binding affinity for FOLR1.

[0025] The term "Affilin®" (registered trademark of Navigo Proteins GmbH) refers to a binding protein that is not derived from immunoglobulins.

[0026] The term "substitution" is understood to mean the replacement of one amino acid by another. The term "insertion" includes the addition of an amino acid to the original amino acid sequence.

[0027] The term "ubiquitin" refers to ubiquitin as set forth in SEQ ID NO: 46 and proteins having at least 95% identity, e.g., those with point mutations at positions 45, 75, 76 that do not affect binding to the target (FOLR1).

[0028] The terms "binding affinity" and "binding activity" may be used interchangeably herein and refer to the ability of a polypeptide to bind to another protein, peptide, or fragment or domain thereof. Typically, binding affinity is measured by the equilibrium dissociation constant (K D ), and are used to assess and rank the strength of bimolecular interactions.

[0029] The term "fusion protein" refers to a protein that comprises at least one first protein genetically linked to at least one second protein. Fusion proteins are created by linking two or more genes that originally code for different proteins. Fusion proteins may further comprise additional domains that are not involved in target binding, such as, but not limited to, multimerization moieties, polypeptide tags, polypeptide linkers, or moieties that bind to targets other than FOLR1.

[0030] The term "amino acid sequence identity" refers to the quantitative comparison of the identity (or difference) of amino acid sequences for two or more proteins. "Percentage (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the ratio of amino acid residues in a sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. To determine sequence identity, the sequence of the query protein is aligned to the sequence of the reference protein or polypeptide. Methods for sequence alignment are well known in the art. For example, to determine the degree of amino acid sequence identity of any polypeptide compared to the amino acid sequence of SEQ ID NO: 46, the SIM Local similarity program known in the art is preferably used. For multiple alignment analysis, Clustal Omega is preferably used, as known to those skilled in the art.

[0031] DETAILED DESCRIPTION OF EMBODIMENTS OF THEINVENTION Structural characterization of FOLR1-binding proteins FOLR1 binding proteins as defined herein include muteins of ubiquitin. FOLR1 binding proteins include ubiquitin-based amino acids as set forth in SEQ ID NO: 46 with substitutions at least at positions 9, 10, 12, 42, 44, 46, 62, 63, 64, 65, 66, 68, and 70.

[0032] In some embodiments, the FOLR1 binding protein disclosed herein has at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79% sequence identity to the amino acid sequence of SEQ ID NO:46.

[0033] In various embodiments, the FOLR1 binding proteins disclosed herein have at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 46. The FOLR1 binding proteins disclosed herein have at least 81%, 82%, 83%, 84%, or 85% sequence identity to the amino acid sequence of SEQ ID NO: 46.

[0034] In a preferred embodiment, the FOLR1 binding protein disclosed herein may have any amino acid identity between 70% identity and 85% identity to the amino acid sequence of SEQ ID NO: 46. In an even more preferred embodiment, the FOLR1 binding protein disclosed herein may have any amino acid identity between 73% identity and 83% identity to the amino acid sequence of SEQ ID NO: 46. In an even more preferred embodiment, the FOLR1 binding protein disclosed herein may have any amino acid identity between 75% identity and 83% identity to the amino acid sequence of SEQ ID NO: 46. In an even more preferred embodiment, the FOLR1 binding protein disclosed herein may have any amino acid identity between 76% identity and 83% identity to the amino acid sequence of SEQ ID NO: 46. In an even more preferred embodiment, the FOLR1 binding protein disclosed herein may have any amino acid identity between 79% identity and 83% identity to the amino acid sequence of SEQ ID NO: 46.

[0035] FOLR1 binding proteins as defined herein include muteins of SEQ ID NO: 46, i.e., muteins of ubiquitin. FOLR1 binding proteins include amino acids based on SEQ ID NO: 46 with substitutions at least at positions 9, 10, 12, 42, 44, 46, 62, 63, 64, 65, 66, 68, and 70. In a FOLR1 binding protein as defined herein, the amino acid corresponding to position 9 of SEQ ID NO:46 is E, L, S, or N, the amino acid corresponding to position 10 of SEQ ID NO:46 is E, Q, Y, or I, the amino acid corresponding to position 12 of SEQ ID NO:46 is Y, E, W, or D, the amino acid corresponding to position 42 of SEQ ID NO:46 is E, K, Y, Q, or M, the amino acid corresponding to position 44 of SEQ ID NO:46 is L, Y, V, or F, the amino acid corresponding to position 46 of SEQ ID NO:46 is Y, D, or S, the amino acid corresponding to position 62 of SEQ ID NO:46 is L, R, D, or I, the amino acid corresponding to position 63 of SEQ ID NO:46 is G, F, L, or A, and the amino acid corresponding to position 64 of SEQ ID NO:46 is G, D, or Y; In some embodiments, the amino acid corresponding to position 65 of SEQ ID NO: 46 is A, D, Y, G, or M, the amino acid corresponding to position 66 of SEQ ID NO: 46 is V, H, Y, or T, the amino acid corresponding to position 68 of SEQ ID NO: 46 is K, D, P, or T, the amino acid corresponding to position 70 of SEQ ID NO: 46 is Q, P, T, W, or H, and optionally, one or more, preferably one, two, three, four, or five, additional amino acids of SEQ ID NO: 46 are substituted. In some embodiments, the amino acid corresponding to position 11 of SEQ ID NO: 46 is K or R, and the amino acid corresponding to position 45 of SEQ ID NO: 46 is W, R, or G.

[0036] Structural characterization based on amino acid motifs In one embodiment, the FOLR1 binding protein comprises an amino acid residue motif at positions corresponding to positions 9, 10, 11, and 12 of ubiquitin as defined in SEQ ID NO: 46, the amino acid residue motif being selected from the group consisting of EEKY, EERY, EQKY, LYKE, SYKW, or NIKD (SEQ ID NOs: 61-66).

[0037] In one embodiment, the FOLR1 binding protein comprises an amino acid residue motif at positions corresponding to 42, 43, 44, 45, and 46 of SEQ ID NO: 46, wherein the amino acid residue motif is selected from the group consisting of ELLWY, KLLWY, KLLRY, YLYWD, YLYGD, QLVWD, or MLFWS (SEQ ID NOs: 54-60).

[0038] In one embodiment, the FOLR1 binding protein comprises an amino acid residue motif at positions corresponding to positions 62, 63, 64, 65, 66, 67, 68, 69, and 70 of SEQ ID NO: 46, wherein the amino acid residue motif is selected from LGGAVLKLQ, LGDAVLKLQ, LGGAVLKLP, RFGDHLDLT, RFGYHLDLT, DLGGYLPLW, or IAYMTLTLH (SEQ ID NOs: 47-53).

[0039] In one embodiment, the FOLR1 binding protein comprises an amino acid residue motif at positions corresponding to positions 9, 10, 11, and 12 of ubiquitin as defined in SEQ ID NO: 46, selected from the group consisting of EEKY, EERY, EQKY, LYKE, SYKW, or NIKD; and an amino acid residue motif at positions corresponding to positions 42, 43, 44, 45, and 46 of SEQ ID NO: 46, selected from the group consisting of ELLWY, KLLWY, KLLRY, YLYWD, YLYGD, QLVWD, or MLFWS; and an amino acid residue motif at positions corresponding to positions 42, 43, 44, 45, and 46 of SEQ ID NO: 46, selected from the group consisting of LGGAVLKLQ, LGDAVLKLQ, LGGAVLKLP, RFGDHLDLT, RFGYHLDLT, DLGGYLPLW, or IAYMTLTLH. It contains amino acid residue motifs at positions corresponding to positions 62, 63, 64, 65, 66, 67, 68, 69, and 70 of SEQ ID NO:46.

[0040] In one embodiment, the FOLR1 binding protein comprises an amino acid motif selected from EEKY, ELLWY, and LGGAVLKLQ; EEKY, KLLWY, and LGGAVLKLQ or LGGAVLKLP or LGDAVLKLQ; EERY, KLLWY, and LGGAVLKLQ or LGDAVLKLQ; EEKY, KLLRY, and LGGAVLKLQ; RLYKE, YLYWD, and RFGDHLDLT or RFGYHLDLT; RLYKE, YLYGD, and RFGYHLDLT; ESYKW, QLVWD, and DLGGYLPLW; or YNIKD, MLFWS, and IAYMTLTLH at positions corresponding to positions 9-12, 42-46, and 62-70 of ubiquitin as defined in SEQ ID NO: 46, as shown in Table 1.

[0041] Table 1. Amino acids at selected positions in FOLR1-binding proteins Numbers in the top row refer to corresponding positions in SEQ ID NO:46. TIFF0007608538000001.tif147128

[0042] In one embodiment, the FOLR1 binding protein comprises additional substitutions at one, two, three, four, five amino acid positions selected from positions corresponding to 6, 8, 13, 14, 20, 23, 24, 25, 29, 30, 31, 32, 33, 34, 48, 49, 51, 52, 58, 59, 60, 71, and 72 of SEQ ID NO:46. In some embodiments, these substitutions are selected from any one of the group consisting of K6V, K6Q, L8R, L8E, L8Y, I13T, T14A, T14P, S20C, S20G, I23T, I23V, E24G, E24A, N25D, K29R, K29E, K29T, I30V, I30L, Q31R, D32G, D32N, K33R, K33Q, E34A, K48E, Q49R, E51K, E51D, D52G, D58N, Y59H, N60T, N60S, L71P, R72G, R72Y, and R72K, and preferably, the FOLR1 binding protein has a 70% to 85% or 90% substitutions of SEQ ID NO: 46, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 59, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO In some embodiments, the additional substitution is selected from Y59H (see, e.g., SEQ ID NO: 7). In some embodiments, the additional substitution is selected from E24A (see, e.g., SEQ ID NO: 1; 81.6% sequence identity to SEQ ID NO: 46; 14 of the 76 amino acids are modified). In some embodiments, the additional substitution is selected from any one of the group consisting of K29R and K48E (see, e.g., SEQ ID NO: 4; 80.3% sequence identity to SEQ ID NO: 46; 15 of the 76 amino acids are modified).In some embodiments, the further substitution is selected from any one of the group consisting of T14A, I30V, and K48E (see, e.g., SEQ ID NO: 2) (78.9% sequence identity to SEQ ID NO: 46; 16 amino acids have been altered; see, e.g., the dimer of SEQ ID NO: 2 shown in SEQ ID NO: 30). In some embodiments, the further substitution is selected from any one of the group consisting of K6V, L8R, and R72G (see, e.g., SEQ ID NO: 24; 78.9% sequence identity to SEQ ID NO: 46; 16 amino acids have been altered). In some embodiments, the further substitution is selected from any one of the group consisting of K6V, L8R, W45G, R72G (see, e.g., SEQ ID NO: 25; 77.6% sequence identity to SEQ ID NO: 46; 17 amino acids have been altered). In some embodiments, the additional substitution is selected from any one of the group consisting of K6V, L8R, I13T, T14A, R72G (see, e.g., SEQ ID NO: 27; 76.3% sequence identity to SEQ ID NO: 46; 18 amino acids altered).

[0043] Another example is shown in Figure 1.

[0044] Structural characterization based on unsubstituted positions in SEQ ID NO: 46 In various embodiments, the FOLR1 binding proteins disclosed herein are further structurally characterized in that certain residues are not subject to mutation, e.g., amino acid residues corresponding to positions 1, 2, 3, 4, 5, 7, 15, 16, 17, 18, 19, 21, 22, 26, 27, 28, 35, 36, 37, 38, 39, 40, 41, 43, 47, 50, 53, 54, 55, 56, 57, 61, 67, 69, 73, 74, 75, and 76 of SEQ ID NO: 46 (ubiquitin). Thus, in various embodiments, the amino acid residues at positions 1-5, 7, 15-19, 21, 22, 26-28, 35-41, 43, 47, 50, 53-57, 61, 67, 69, 71, and 73-76 correspond to the amino acids set forth in SEQ ID NO:46.

[0045] Multimers In a preferred embodiment, the FOLR1 binding protein is a multimer consisting of a plurality of FOLR1 binding proteins as defined herein. The multimer may contain two, three, four or more FOLR1 binding proteins. In one embodiment, the FOLR1 binding protein contains two, three, four or more FOLR1 binding proteins linked to each other. That is, the FOLR1 binding protein can be a dimer, trimer, tetramer, etc. In some embodiments, the multimer is a dimer of the FOLR1 binding proteins as defined above. In various embodiments, the FOLR1 binding protein is a homodimer. A homodimeric FOLR1 binding protein is a protein in which two FOLR1 binding proteins having the same amino acid sequence are linked to each other. A homodimer can be made by fusing two identical proteins of any one of the group consisting of SEQ ID NOs: 1-29 or two identical proteins having an amino acid sequence having at least 90% identity thereto. For example, the FOLR1 binding protein of SEQ ID NO: 33 is a homodimer consisting of two identical amino acid sequences of SEQ ID NO: 4. Selected examples of homodimers are shown in SEQ ID NOs: 30-43 and Table 2.

[0046] Table 2. Homodimeric FOLR1-binding proteins TIFF0007608538000002.tif104168

[0047] In other embodiments, the multimer is a heterodimer, eg, the two amino acid sequences of the affilin protein specific for FOLR1 have different amino acid sequences from each other.

[0048] In some embodiments, two or more FOLR1 binding proteins are directly linked. In some embodiments, two or more FOLR1 binding proteins are linked by a peptide linker. In various embodiments, two or more FOLR1 binding proteins are linked by a peptide linker of up to 30 amino acids. In other embodiments, two or more FOLR1 binding proteins are linked by a peptide linker of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids. In one embodiment, two FOLR1 binding proteins are linked by 16 amino acids, preferably two identical FOLR1 binding proteins are linked by 16 amino acids.

[0049] One embodiment relates to a linker composed of amino acids such as glycine, serine, alanine, or proline. The linker may consist of glycine and serine, and may be rich in glycine (e.g., more than 50% of the residues in the linker may be glycine residues). In some embodiments, two or more FOLR1 binding proteins are linked by a peptide linker having an amino acid sequence set forth in any one of SEQ ID NO: 67 or a peptide linker having 90% identity thereto. Other linkers for fusing proteins are known in the art and can be used.

[0050] The FOLR1 binding protein described herein comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-45 and SEQ ID NOs: 69-74, or amino acid sequences having at least 90% identity thereto, respectively. In some embodiments, the FOLR1 binding protein comprises an amino acid sequence that exhibits at least 90% identity to the amino acid sequence of SEQ ID NO: 2 (Affilin-197556b). For example, SEQ ID NOs: 1-23 and SEQ ID NOs: 30-43 and SEQ ID NOs: 69-74 comprise an amino acid sequence that exhibits at least 90% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the FOLR1 binding protein comprises an amino acid sequence that exhibits at least 90% identity to the amino acid sequence of SEQ ID NO: 24 (Affilin-189864). For example, SEQ ID NOs: 25-27 are at least 90% identical to SEQ ID NO: 2. In some alternative embodiments, the FOLR1-binding protein comprises an amino acid sequence that exhibits at least 90% identity to the amino acid sequence of SEQ ID NO: 29 (Affilin-187803) or SEQ ID NO: 28 (Affilin-187770). For example, and without limitation, selected examples of FOLR1-binding proteins are shown in FIG.

[0051] Functional characterization In some embodiments, the FOLR1 binding proteins described herein bind to FOLR1 expressed on the cell surface as measured by FACS and / or have a binding affinity for FOLR1 of 500 nM or less as measured by surface plasmon resonance assay.

[0052] In some embodiments, the FOLR1 binding proteins described herein have a binding affinity (K D). FOLR1 binding proteins bind to FOLR1 with a measurable binding affinity of less than 500nM, less than 200nM, less than 100nM, less than 50nM, less than 20nM, less than 10nM, less than 5nM, and more preferably less than 1nM. Suitable methods are known to the skilled person or described in the literature. Methods for measuring 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), kinetic exclusion analysis (KinExA assay), biolayer interferometry (BLI), flow cytometry, fluorescence spectroscopy techniques, 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 measured at 20°C, 25°C, or 30°C. Unless otherwise specifically indicated, the K D Values ​​are measured by SPR at 25°C. K D The smaller the value, the greater the binding affinity of the biomolecule for its binding partner. K D The higher the value, the less avidly the binding partners bind to each other. Examples of binding affinities of FOLR1-binding proteins to FOLR1 are shown in Table 4 (see Example 5).

[0053] In some embodiments, the FOLR1 binding proteins described herein have a specific binding affinity (K D) but not to FOLR2. In some embodiments, the FOLR1 (folate receptor alpha) binding proteins described herein specifically bind to FOLR1 but not detectably to FOLR2 (folate receptor beta), as measured by surface plasmon resonance assay (see FIG. 3), tested in FOLR2-expressing cell lines, and further described in the Examples (see Example 6). Thus, the binding of the FOLR1 binding proteins described herein is highly specific. FOLR1 and FOLR2 are folate receptor isoforms with low degrees of homology (less than 80%) and different expression patterns. FOLR1 is primarily expressed on the surface of malignant cancer cells, whereas FOLR2 is expressed on the surface of activated macrophages at sites of inflammation. Highly selective binding to FOLR1 may be important for targeted medical applications to cancers involving FOLR1 but not to inflammation, reducing potential toxic side effects.

[0054] In some embodiments, a FOLR1 binding protein described herein binds to FOLR1 but does not detectably bind to the Fc domain of human immunoglobulin IgG1, as measured by a surface plasmon resonance assay.

[0055] Half-maximal effective concentration EC 50 "refers to the FOLR1-binding protein concentration that induces a response halfway between baseline and the maximum after a specified exposure time, and thus represents the FOLR1-binding protein concentration at which 50% of its maximum effect is observed, in this case the half-maximal fluorescence intensity signal in a flow cytometric cell binding experiment. In some embodiments, the FOLR1-binding proteins described herein have an EC50 of less than 100 nM, less than 50 nM, less than 20 nM, less than 10 nM, less than 5 nM, and more preferably less than 1 nM for FOLR1-expressing cells. 50In some embodiments, the FOLR1 binding proteins described herein have an EC of less than 1 nM for FOLR1 after incubation for at least 24 hours at 37° C. in the presence of mouse serum. 50 Suitable methods are known to those skilled in the art. 50 The smaller the value, the stronger the binding of the FOLR1 binding protein to FOLR1. Examples of FOLR1 binding proteins that are stable even in the presence of serum are shown in Table 5 (see Example 9) and Table 6 (see Example 10).

[0056] In some embodiments, the FOLR1 binding proteins described herein are stable at elevated temperatures, preferably between 62° C. and 87° C. Stability analysis is known to those skilled in the art, for example, spectroscopic methods or methods utilizing fluorescence associated with chemical or physical unfolding. For example, the stability of a molecule can be measured using standard methods such as thermal melting (T m ) temperature, i.e., the temperature in degrees Celsius (°C) at which half of the molecules unfold. Typically, T m The higher the , the more stable the molecule. Temperature stability was measured by Differential Scanning Fluorometry (DSF), as described in more detail in Example 4 and Table 3.

[0057] Coupling site In some embodiments, the FOLR1 binding protein described herein further comprises one or more coupling sites for coupling a chemical moiety. The coupling sites can react with other chemical groups to couple the FOLR1 binding protein to the chemical moiety. Defining the number and position of the coupling sites allows for site-specific coupling of chemical moieties to the FOLR1 binding protein described herein. Thus, multiple chemical moieties can be attached to the FOLR1 binding protein as needed. The number of coupling sites can be adjusted to an optimal number for a particular application by one of skill in the art, and thus the amount of chemical moieties. In certain embodiments, the coupling sites may be selected from the group consisting of one or more amino acids that can be labeled with a particular chemical moiety, such as one or more cysteine ​​residues, one or more lysine residues, one or more tyrosine residues, one or more tryptophan residues, or one or more histidine residues. The FOLR1 binding protein may comprise 1 to 20 coupling sites, preferably 1 to 6 coupling sites, preferably 2 coupling sites, or preferably 1 coupling site.

[0058] Coupling Ring Domain One embodiment provides a FOLR1 binding protein comprising at least one coupling domain of 1-80 amino acids, comprising one or more coupling sites. In some embodiments, the coupling domain of 1-80 amino acids may comprise alanine, proline, or serine, and cysteine ​​as a coupling site. Examples of FOLR1 binding proteins with coupling domains are provided in SEQ ID NO: 44 and SEQ ID NO: 45 (coupling domain of three amino acids "SAC", coupling site is cysteine). In other embodiments, the coupling domain of 5-80 amino acids may comprise alanine, proline, serine, and cysteine ​​as a coupling site. In one embodiment, the coupling domain comprises 20-60% alanine, 20-40% proline, 10-60% serine, and one or more cysteines as coupling sites at the C-terminus or N-terminus of the FOLR1 binding protein described herein. In some embodiments, the amino acids alanine, proline, and serine are randomly distributed throughout the amino acid sequence of the coupling domain such that there are no more than 2, 3, 4, or 5 adjacent identical amino acid residues, preferably no more than 3 amino acids. The composition of the 1-20 coupling domains may be varied or identical.

[0059] In some embodiments, the chemical moiety is selected from any of a chelator, a drug, a toxin, a dye, and a small molecule. In some embodiments, at least one of the chemical moieties is a chelator designed as a complexing agent for coupling one or more other moieties to a compound targeted to the FOLR1 binding protein disclosed herein. One embodiment relates to a FOLR1 binding protein, as described in the Examples (Examples 7-10), in which the chelator is a complexing agent for coupling one or more radioisotopes or other detectable labels.

[0060] diagnostic part In various embodiments, the FOLR1 binding protein further comprises a diagnostic moiety. In other embodiments, the FOLR1 binding protein further comprises a plurality of diagnostic moieties. In some embodiments, such diagnostic moieties may be selected from radionuclides, fluorescent proteins, photosensitizers, dyes, or enzymes, or any combination of the above. In some embodiments, the FOLR1 binding protein comprising at least one diagnostic moiety can be used, for example, as an imaging agent, 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, imaging cancer cells associated with FOLR1 using radioimaging or photoluminescence or fluorescence.

[0061] therapeutic part In various embodiments, the FOLR1 binding protein further comprises a therapeutically effective moiety. In other embodiments, the FOLR1 binding protein further comprises a plurality of therapeutically effective moieties. In some embodiments, such therapeutically effective moieties can be selected from monoclonal antibodies or fragments thereof, extracellular domains of receptors or fragments thereof, radionuclides, cytotoxic compounds, cytokines, chemokines, enzymes, or derivatives thereof, or any combination of the above. In some embodiments, the FOLR1 binding protein comprising a therapeutically effective component can be used in targeted delivery of any of the above-listed components to FOLR1-expressing tumor cells, and can accumulate in the tumor cells, thereby resulting in low levels of toxicity to normal cells.

[0062] Radionuclides Suitable radionuclides for in vivo or in vitro imaging applications or radiotherapy include, but are not limited to, for example, the group of gamma-emitting isotopes, the group of positron emitters, the group of beta-emitters, and the group of alpha-emitters.In some embodiments, suitable conjugation partners include chelators such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA) or their activated derivatives, nanoparticles, and liposomes.As will be described in more detail in the examples, in various embodiments, DOTA can be suitable as a complexing agent for radioisotopes and other agents for imaging purposes.

[0063] Pharmacokinetic Modifier In some embodiments, the FOLR1 binding protein further comprises at least one pharmacokinetics modulating moiety, which is optionally selected from polyethylene glycol, human serum albumin, albumin-binding peptide, immunoglobulin-binding peptide or immunoglobulin or immunoglobulin fragment, or polysaccharide (e.g., hydroxyethyl starch), or a non-standard amino acid sequence that increases hydrodynamic radius, such as a multimer that contains the amino acids alanine, glycine, serine, and proline. In various embodiments, the moiety extends the half-life of the FOLR1 binding protein by at least 1.5 times. Several techniques for making FOLR1 binding protein with long half-life are known in the art, such as direct fusion or chemical coupling of the pharmacokinetics modulating moiety with the aforementioned FOLR1 binding protein. The pharmacokinetics modulating moiety can be linked to one or several sites of the FOLR1 binding protein, for example, via a peptide linker sequence or via the aforementioned coupling site.

[0064] The conjugation of proteinaceous or non-proteinaceous moieties to FOLR1 binding protein can be carried out by applying chemical methods well known in the art.In some embodiments, specific coupling chemical reactions can be applied to the derivatization of cysteine ​​or lysine residues.Chemical coupling can be carried out using chemical reactions well known to those skilled in the art, including but not limited to substitution, addition or cycloaddition, or oxidation chemical reactions (e.g., disulfide formation).

[0065] Molecules for purification / detection In some embodiments, additional amino acids may extend from either or both of the N-terminus or C-terminus of the FOLR1 binding protein. For example, the additional sequence may include a sequence introduced for purification or detection, etc. In one embodiment, the additional amino acid sequence includes one or more peptide sequences that confer affinity to certain chromatographic column materials. Exemplary examples of such sequences include, but are not limited to, Strep tags, oligohistidine tags, glutathione S-transferase, maltose binding protein, inteins, intein fragments, or albumin binding domains of protein G. An example of a FOLR1 binding protein with a Strep tag is provided in SEQ ID NO: 45.

[0066] Use in medicine Various embodiments relate to the FOLR1 binding protein disclosed herein for use in medicine.In one embodiment, the FOLR1 binding protein is used in medicine for diagnosing or treating cancer associated with FOLR1 expression.The FOLR1 binding protein disclosed herein allows selective diagnosis and treatment of cancer cells or tissues associated with FOLR1.FOLR1 is known to be upregulated in tumor cells, possibly resulting in uncontrolled tumor cell proliferation and metastasis.Examples of tumors associated with FOLR1 are ovarian cancer, endometrial cancer, brain cancer, lung cancer, kidney cancer, head and neck cancer, breast cancer, gastric cancer and colorectal cancer.

[0067] One embodiment is a method for diagnosing (including monitoring) a subject suffering from cancer related to FOLR1, comprising administering to the subject the above-mentioned FOLR1 binding protein, which is optionally conjugated to a radioactive molecule.In various embodiments, the FOLR1 binding protein disclosed herein can be used for the diagnosis of cancer related to FOLR1, and optionally, the FOLR1 binding protein is conjugated to a radioactive molecule.In some embodiments, the FOLR1 binding protein disclosed herein can be used as a biomarker.In some embodiments, imaging methods using FOLR1 binding protein with radioactive label or fluorescent label, etc., can be used to visualize FOLR1 on specific tissues or cells, for example, to evaluate the presence of tumor cells related to FOLR1, the distribution of tumors related to FOLR1, the recurrence of tumors related to FOLR1, and / or the response of patients to therapeutic treatment.

[0068] One embodiment is a method of treating a subject suffering from cancer associated with FOLR1, comprising administering to the subject the above-mentioned FOLR1 specific binding protein, optionally conjugated with a radioactive molecule and / or a cytotoxic substance.In various embodiments, the FOLR1 binding protein disclosed herein can be used for the treatment of cancer associated with FOLR1, where the FOLR1 binding protein is optionally conjugated with a cytotoxic substance and / or a radioactive molecule and / or expressed on the surface of target-specific CarT cells.Some embodiments relate to the use of FOLR1 binding protein labeled with suitable radioisotopes or cytotoxic compounds or the treatment of tumor cells associated with FOLR1, in particular to control or kill tumor cells, such as malignant cells, associated with FOLR1.In one embodiment, a curative dose of radiation is selectively delivered to tumor cells associated with FOLR1, but not to normal cells.

[0069] FOLR1 specific binding protein disclosed herein can be used for tumor targeting therapy or diagnosis, not recognizing inflamed tissue.Because it specifically binds to FOLR1 (not specifically binds to FOLR2), FOLR1 tumor targeting therapy may be beneficial in, for example, autoimmune patients or patients with inflammation.

[0070] composition Various aspects relate to compositions comprising FOLR1 binding protein disclosed herein.Compositions comprising FOLR1 binding protein as defined above for use in medicine, preferably for use in the diagnosis or treatment of various cancer tumors related to FOLR1, such as ovarian cancer, endometrial cancer, brain cancer, lung cancer, kidney cancer, head and neck cancer, breast cancer, gastric cancer, colorectal cancer, etc., preferably for use in the diagnosis or treatment of ovarian cancer, breast cancer and lung cancer.Compositions comprising FOLR1 binding protein as described above can be used for both diagnostic and therapeutic clinical applications.In particular, compositions comprising FOLR1 binding protein as described above can be used for clinical applications for imaging, monitoring, and eliminating or inactivating pathological cells expressing FOLR1.

[0071] Various embodiments relate to diagnostic compositions for diagnosing cancers related to FOLR1, comprising the FOLR1 binding protein defined herein and diagnostically acceptable carriers and / or diluents. These include, but are not limited to, stabilizers, surfactants, salts, buffers, colorants, etc. These compositions can be in the form of liquid formulations, lyophilized products, granules, emulsions or liposomal formulations.

[0072] Diagnostic compositions comprising the FOLR1 binding proteins described herein can be used for the diagnosis of cancers associated with FOLR1, as described above.

[0073] Various embodiments relate to pharmaceutical (e.g., therapeutic) compositions for treating disease, comprising the FOLR1 binding protein disclosed herein and pharma- ceutically (e.g., therapeutically) acceptable carriers and / or diluents.Pharmaceutical (e.g., therapeutic) compositions may optionally comprise additional auxiliary agents and excipients known per se.These include, but are not limited to, for example, stabilizers, surfactants, salts, buffers, coloring agents, etc.

[0074] Pharmaceutical compositions comprising a FOLR1 binding protein as defined herein can be used for the treatment of diseases, as described above.

[0075] The composition comprises an effective amount of FOLR1 binding protein as defined herein.The dosage of protein varies according to organism, disease type, age and weight of patient, and other factors known per se.Depending on each galenic preparation, these compositions can be administered parenterally, systemically, intraperitoneally, intramuscularly, subcutaneously, transdermally, or by other application methods conventionally used.

[0076] These compositions can be in the form of liquid preparations, lyophilisates, creams, lotions for topical application, sprays; powders, granules; emulsions or liposomal preparations. The type of formulation varies 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 field.

[0077] The various components of the composition may be packaged as a kit together with instructions for use.

[0078] Preparation of FOLR1-binding protein The FOLR1 binding protein described herein can be prepared by any of many conventional and well-known techniques, such as simple organic synthesis strategy, solid-phase-assisted synthesis technique, fragment ligation technique, or by commercially available automated synthesis equipment.On the other hand, they can also be prepared by conventional recombinant technology alone or by conventional recombinant technology combined with conventional synthetic technology.In addition, they can be prepared by cell-free in vitro transcription / translation.

[0079] Various embodiments relate to polynucleotides encoding the FOLR1 binding proteins disclosed herein. One embodiment further provides an expression vector comprising the polynucleotide, and a host cell comprising the isolated polynucleotide or the expression vector.

[0080] Various embodiments relate to methods for producing the FOLR1-binding proteins disclosed herein, comprising culturing host cells under appropriate conditions that allow expression of the FOLR1-binding protein, and optionally isolating the FOLR1-binding protein.

[0081] For example, one or more polynucleotides encoding FOLR1 binding protein may be expressed in a suitable host, and the produced FOLR1 binding protein may be isolated. The host cell comprises said nucleic acid molecule or vector. Suitable host cells include prokaryotes or eukaryotes. Vector refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) that can be used to transfer protein coding information into a host cell. Various cell culture systems, including but not limited to mammalian, yeast, plant, or insect, can also be used to express recombinant protein. Suitable conditions for culturing prokaryotic or eukaryotic host cells are well 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 volume shake flasks to large fermenters, applying techniques well known to those skilled in the art.

[0082] One embodiment is directed to a method for preparing the binding protein detailed above, comprising the steps of: (a) preparing a nucleic acid encoding a FOLR1-binding protein defined herein; (b) introducing the nucleic acid into an expression vector; (c) introducing the expression vector into a host cell; (d) culturing the host cell; (e) subjecting the host cell to culture conditions in which the FOLR1-binding protein is expressed, thereby producing the FOLR1-binding protein defined herein; (f) optionally isolating the FOLR1-binding protein produced in step (e); and (g) optionally conjugating the FOLR1-binding protein to another functional moiety defined herein.

[0083] Generally, the isolation of purified FOLR1 binding protein from the culture mixture can be carried out by applying conventional methods and techniques well known in the art, such as centrifugation, precipitation, coagulation, various forms of chromatography, filtration, dialysis, concentration, and combinations thereof. Chromatographic methods are well known in the art, and include, but are not limited to, ion exchange chromatography, gel filtration chromatography (size exclusion chromatography), hydrophobic interaction chromatography, or affinity chromatography.

[0084] To facilitate purification, FOLR1 binding protein can be fused to other peptide sequences that enhance affinity to separation materials.Preferably, such fusions are selected so that they do not adversely affect the functionality of FOLR1 binding protein or can be separated after purification by introducing a specific protease cleavage site.Such methods are also known to those skilled in the art. EXAMPLES

[0085] The following examples are provided to further illustrate the present invention. A specific example of the present invention is a specific modification of ubiquitin (SEQ ID NO: 46) that results in binding to FOLR1. 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.

[0086] Example 1. Identification of FOLR1-binding proteins Library construction and library cloning. A library containing randomized amino acid positions was synthesized in-house with randomized oligonucleotides generated using synthetic trinucleotide phosphoramidites (ELLA Biotech) to achieve a balanced amino acid distribution with other amino acid residues at the randomized positions in parallel with the elimination of cysteines. The sequence of ubiquitin (SEQ ID NO: 46): TIFF0007608538000003.tif10163.

[0087] Amino acids 6, 8, 9, 10, 12, 42, 44, 46, 62, 63, 64, 65, 66, 68, 70, 72 of SEQ ID NO: 46 were randomized. The corresponding cDNA library was amplified by PCR and ligated with modified pCD87SA phagemid (herein referred to as pCD12) using standard methods known to those skilled in the art. The pCD12 phagemid contains a modified torA leader sequence (deletion of amino acid sequence QPAMA) to achieve protein processing without additional amino acids at the N-terminus. An aliquot of the ligation mixture was used for electroporation of E. coli ER2738 (Lucigen). Established recombinant gene methods were used unless otherwise specified.

[0088] Example 2. Identification of FOLR1-binding proteins Targets. A DNA sequence encoding human FOLR1 (Uniprot accession number P15328; residues 25-234) was genetically fused to either the Fc fragment of human IgG1 followed by a C-terminal His tag or an Avi tag followed by a C-terminal His tag. Full-length cDNA with human codon usage was provided by OriGene Technologies, cloned into the mammalian expression vector pCEP4, and expressed in mammalian Expi293F cells at various scales ranging from 400 ml up to 2 l in shake flasks. Expression cultures containing the pCEP4-FOLR1-Avi-His construct were further co-transfected with pCEP4-BirA to lead to site-specific biotin labeling of the target protein. Expression cultures were analyzed by SDS-PAGE and by immunoblot analysis using antibodies against FOLR1, the Fc portion of human IgG1, or biotin.

[0089] The cell culture supernatant of FOLR1-Fc-His expression was centrifuged and filtered for affinity chromatography using a 1 mL HisTrap excel column (GE Healthcare). The target protein was eluted by injecting an imidazole-containing buffer and loaded onto a Superdex 200 XK 16 / 600 gel filtration column. The cell culture supernatant of FOLR1-Avi-His expression was centrifuged, filtered, and desalted for affinity chromatography using a streptavidin mutein matrix (Roche). The target protein was eluted by injecting a biotin-containing buffer and loaded onto a Superdex 200 10 / 300 gel filtration column. The purity of the recovered target protein was analyzed and confirmed by SDS-PAGE and SE-HPLC. The biological activity of the target protein against the substrate folic acid was confirmed by concentration-dependent ELISA.

[0090] Primary selection by TAT phage display method The naive library was enriched for FOLR1 using phage display as a selection system. The library-bearing phagemid pCD12 was used to transform competent bacterial cells ER2738 (Lucigene), after which phage amplification and purification were performed using standard methods known to those skilled in the art. For selection, the target proteins were immobilized on magnetic beads. Therefor, the target proteins fused to IgG1-Fc were immobilized on Protein A or Protein G Dynabeads®. Site-specific biotin-labeled target proteins fused to Avi-tags, or additionally randomly biotin-labeled target proteins fused to IgG1-Fc, were immobilized on Streptavidin or Neutravidin SpeedBeads™. The FOLR1 concentration during the phage incubation period was reduced from 200 nM (1st round) to 100 nM (2nd round), 50 nM (3rd round), and 25 nM (4th round). In the first selection, a preselection with biotin-labeled Fc fragment of IgG1 was performed starting from the second round. In the second selection, phage particles were preincubated with mouse serum during the last three rounds (3 h in the second round, 23 h in the third and fourth rounds, respectively) and a preselection with biotin-labeled Fc fragment of IgG1 was also performed. FOLR1-phage complexes were magnetically separated from the supernatant and washed several times. FOLR1-bound phages were eluted by trypsin. To identify target-specific phage pools, eluted and re-amplified phages from each selection round were analyzed by phage pool ELISA. Wells of a medium-binding capacity microtiter plate (Greiner Bio-One) were coated with FOLR1 (2.5 μg / ml). Bound phages were detected using α-M13 HRP-conjugated antibody (GE Healthcare).

[0091] Cloning of target-binding phage pools into expression vectors Selected pools showing specific binding to FOLR1 in the phage pool ELISA were amplified by PCR according to methods known in the art, cut with the appropriate restriction nuclease, and ligated into a derivative of the expression vector pET-28a (Merck, Germany) containing Strep-Tag II (IBA GmbH).

[0092] Single colony hit analysis After transformation of BL21(DE3) cells (Merck, Germany), kanamycin-resistant single colonies were grown. Expression of FOLR1-binding proteins was achieved by culturing in 384-well plates (Greiner Bio-One) using autoinduction medium (Studier, 2005, Protein Expr. Purif. 41(1):207-234). Cells were harvested and subsequently lysed chemically or enzymatically by BugBuster reagent (Novagen) and mechanically by freeze / thaw cycles, respectively. After centrifugation, the resulting supernatants were screened by ELISA using targets immobilized on High Bind 384 ELISA microtiter plates (Greiner Bio-One). Detection of proteins bound to FOLR1 was achieved using Strep-Tactin® HRP conjugate (IBA GmbH) in combination with TMB-Plus substrate (Biotrend, Germany). The reaction was stopped by adding 0.2M H2SO4 solution and measured using a plate reader at 450 nm and 620 nm as a reference.

[0093] Construction of a mature library To mature the selected mutants, error-prone PCR was performed using the dNTP analogs dPTP and 8-oxo-dGTP (Jena Bioscience). The resulting mature library cDNA was ligated with pCD12 as described above, and test transformations in E. coli SS320 were performed.

[0094] Maturity Selection and Analysis For affinity maturation, two rounds of panning were performed. The first maturation selection was performed with random biotin-labeled target proteins fused to IgG1-Fc at concentrations of 80 nM and 8 nM in the first and second rounds, respectively. In both rounds, phage particles were preincubated with mouse serum for 3 hours in the first round and 23 hours in the second round, respectively, and preselection with biotin-labeled Fc fragment of IgG1 was also performed. The second maturation selection was performed with site-specific biotin-labeled target proteins fused to Avi-tag at concentrations of 50 nM and 1 nM in the first and second rounds, respectively. In both rounds, phage particles were preincubated with mouse serum for 23 hours in the first and second rounds. To analyze the matured and selected pools for specific target binding, phage pool ELISA was performed, followed by cloning of positive pools into expression vector pET-28a and hit ELISA as described above.

[0095] Example 3. Expression and purification of FOLR1 binding proteins FOLR1 binding proteins were cloned into expression vectors using standard methods known to those skilled in the art, and purified and analyzed as described below. All FOLR1 specific proteins were expressed and highly purified by affinity chromatography and gel filtration. After affinity chromatography purification, size exclusion chromatography (SE HPLC or SEC) was performed using an Akta system and a Superdex™ 200 HiLoad 16 / 600 column (GE Healthcare). The column had a volume of 120 ml and was equilibrated with 2 column volumes (CV). Samples were loaded at a flow rate of 1 ml / min of purification buffer. Fraction collection was started when the signal intensity reached 10 mAU. After SDS-PAGE analysis, positive fractions were collected and their protein concentrations were measured.

[0096] Further analysis included SDS-PAGE, SE-HPLC, and RP-HPLC. Protein concentration was determined based on absorbance measurements at 280 nm using molar extinction coefficient. RP chromatography (RP HPLC) was performed using a Dionex HPLC system and a Vydac 214MS54 C4 (4.6×250 mm, 5 μm, 300 Å) column (GE Healthcare). For example, affilin 202521 (SEQ ID NO: 30 with C-terminal coupling sequence "SAC") has a purity of about 100% by SE-HPLC and RP-HLCP, respectively.

[0097] Example 4. FOLR1 binding proteins are stable at high temperatures The thermal stability of FOLR1 specific proteins was measured by differential scanning fluorimetry (DSF). Each probe at a concentration of 0.1 μg / μL was transferred to a MicroAmp Optical 384-well plate and SYPRO Orange dye was added at an appropriate dilution. A temperature gradient from 25°C to 95°C was programmed with a heating rate of 1°C / min (V2A-7 Applied Biosystems). Fluorescence was constantly measured at excitation wavelengths of 520 nm and emission wavelengths of 623 nm (V2A-7, Applied Biosystems). The midpoint of the transition to thermal unfolding (Tm, melting point) was measured and is shown in Table 3 for selected FOLR1 binding proteins.

[0098] Table 3. Temperature stability of FOLR1 binding protein TIFF0007608538000004.tif118128

[0099] Example 5. Analysis of FOLR1-binding proteins (surface plasmon resonance, SPR) A CM5 sensor chip (GE Healthcare) was equilibrated with SPR running buffer. The carboxyl groups exposed on the surface were activated to give reactive ester groups by passing a mixture of EDC and NHS through the chip. 700–1500 RU of FOLR1 (ligand-bound) was immobilized on the flow cell. A flow cell without ligand was used as a reference. An injection of ethanolamine after ligand immobilization was used to block unreacted NHS groups. Upon ligand binding, protein analytes accumulated on the surface, increasing the refractive index. This change in refractive index was measured in real time and plotted as response units or resonance units (RU) versus time. Serial dilutions of analytes were added to the chip at a flow rate of 30 μl / min. Binding was performed for 30 s and dissociation for 60 s. After each run, the chip surface was regenerated with 30 μl of regeneration buffer and equilibrated with running buffer. Control samples were added to the matrix at a flow rate of 30 μl / min, except that they were bound for 60 s and dissociated for 120 s. Renaturation and re-equilibration were performed as previously described. Binding studies were performed using a Biacore 3000 (GE Healthcare) and data evaluation was processed by the software BIAevaluation 3.0 provided by the manufacturer using the Langmuir 1:1 model (RI=0).

[0100] FIG. 2, FIG. 3, and Table 4 show the binding affinity of FOLR1 binding proteins to FOLR1-Fc.

[0101] Table 4. Binding affinity (K) of FOLR1 binding protein to FOLR1-Fc measured by SPR assay (Biacore) D ) TIFF0007608538000005.tif80128

[0102] Example 6. Functional characterization: Specific binding to cell surface expressed FOLR1 but not FOLR2 (flow cytometry) Flow cytometry is used to analyze the specific interaction of FOLR1-binding proteins with surface-exposed FOLR1, but not with FOLR2. Transfected HEK293-FOLR1, HEK293-FOLR2, and control empty vector HEK293-pEntry cells were trypsinized, resuspended in medium containing FCS, washed, and stained in pre-chilled FACS blocking buffer. 1 × 10 cells were used for cell staining. 6 Cell concentrations of 100 μl / well were prepared in triplicate for each cell line in a 96-well plate (Greiner). 50 nM of affilin protein or anti-human FOLR1 monoclonal antibody (clone LK26; Biolegend, 908301) or anti-human FOLR2 antibody (clone EM35; Sysmex, BD029864) as positive control were added to FOLR-overexpressing and control cells. After 45 min, the supernatant was removed and 100 μl / well of rabbit anti-Strep tag antibody (GenScript; A00626) diluted 1:300 in FACS blocking buffer was added. Anti-FOLR1 and anti-FOLR2 antibodies were detected with anti-mouse IgG-Alexa488 (Invitrogen; A-10680) diluted 1:1000. After removal of the primary antibody, goat anti-rabbit IgG Alexa Fluor 488 antibody (Invitrogen; A11008) was added at a dilution of 1:1000. Flow cytometry was performed using a Guava easyCyte 5HT instrument (Merck-Millipore) at an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

[0103] All FOLR1 binding proteins showed specific binding to HEK293-FOLR1 overexpressing cells, but no binding to FOLR1 negative cell lines (e.g. HEK293-pEntry). None of the FOLR1 binding proteins tested for binding to FOLR2 showed binding to FOLR2. Controls: anti-FOLR1 antibody as positive control; ubiquitin (HEK293-FOLR1 cells) as negative control.

[0104] Example 7. Labeling of fusion proteins with DOTA FOLR1-bound proteins were incubated with a 20-fold excess of maleimide-DOTA (2,2',2''-(10-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, CheMatech) dissolved in 50 mM HEPES, 150 mM sodium chloride, 5 mM EDTA, pH 7.0 for 3 h at room temperature. To reduce metal ions that may interact with DOTA molecules, all columns and AKTA instruments (GE Healthcare) were incubated with 0.1 M EDTA solution for 30 min. Only metal-free or metal-reduced components were used when preparing solutions. After incubation, samples were separated from unbound DOTA molecules by gel filtration (Superdex S200, GE Healthcare) in 100 mM sodium acetate, pH 5.0-5.8. Protein samples were also incubated with 5 mM iron(II) chloride at room temperature for 1 h to verify the availability of DOTA molecules for coupling with radioisotopes. After incubation, unbound iron was removed using a HiTrap desalting column (GE Healthcare). The extent of labeling was measured using MALDI-TOF analysis. In a separate experiment, FOLR1 protein was coupled with DOTA to obtain Lu 3+ Labeled with Lu 3+ The loading was performed at c=0.2 mg / ml in 100 nM Na-acetate, pH 5.8, with a maximum labeling temperature of 70° C. Molecules were analyzed by CD spectroscopy at 207 nm.

[0105] Example 8. Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOF) Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOF MS) was performed as follows: FOLR1-binding proteins were purified and concentrated using C18-P10-ZipTips (Millipore; catalog number ZTC18S096). The tips were washed with 0.1% (v / v) trifluoroacetic acid (TFA) in water and eluted with 50% (v / v) acetonitrile / 0.1% TFA. Samples were treated with 2% (v / v) TFA in water and embedded in 2,5-dihydroxyacetophenone (DHAP) matrix (Bruker, catalog number 8231829). The mass of FOLR1-binding proteins was measured by autoflex™ speed mass spectrometer (Bruker). Protein calibration standards (Bruker, part number 8206355 and part number 8207234) were used for the calibration of the autoflex speed mass spectrometer.

[0106] The FOLR1-binding protein with and without DOTA labeling was analyzed based on MALDI-TOF mass spectrum and the peaks were compared. The MALDI-TOF analysis showed that the DOTA molecule attached as a label to the FOLR1-binding protein was available for coupling with iron(II) chloride molecules.

[0107] After labeling of FOLR1-binding proteins, K D Although the affinity of the FOLR1-binding protein for the target was slightly changed, the labeling did not significantly affect the affinity of the FOLR1-binding protein for the target. The results are summarized in Tables 5 and 6.

[0108] Example 9. Serum stability of FOLR1 binding proteins (flow cytometry) The stability of FOLR1 binding proteins in the presence of serum was analyzed. The binding proteins were incubated with a dilution series of 1 μM (Affilin-199479, Affilin-199487, Affilin-197556a, Affilin-199488, Affilin-199488-Dota, Affilin-199490, and Affilin-199490-Dota) and 200 nM (Affilin-199489) dissolved in 100% mouse serum for 0 or 24 hours at 37°C. 100 μl of the Affilin-serum solution was used to analyze serum stability in HEK293-FOLR1 cells by FACS as previously described (Example 6). FACS analysis confirmed that Affilin proteins were bound to FOLR1 even after 24 hours of incubation in mouse serum (see Table 5). Alternative FOLR1 proteins were tested (results not shown) and confirmed binding to FOLR1 even in the presence of serum.

[0109] Table 5. Binding of FOLR1 binding proteins in the presence of serum (flow cytometry) TIFF0007608538000006.tif64164

[0110] Example 10. Serum stability of FOLR1 binding proteins (ELISA) High-binding capacity plates (Greiner, 781061) were immobilized with 0.1-2.5 μg / ml FOLR1-Fc overnight at 4° C. 197556a (SEQ ID NO: 1 with C-terminal sequence SAWSHPQFEK), 202521 (SEQ ID NO: 30 with C-terminal coupling sequence "SAC"; see SEQ ID NO: 44), and 202521-Dota-Lu (with C-terminal coupling sequence "SAC" and lutetium Lu 3+A dilution series of FOLR1 (SEQ ID NO: 30) with Dota labeled with K was incubated in 100% mouse serum overnight at 37°C. The ELISA plate was washed with 1x PBS and blocked with 3% BSA / 0.5% Tween / PBS for 2 hours at room temperature. The dilution series after 0 and 24 hours of incubation in the presence of serum was incubated on the ELISA plate for 1 hour at room temperature. After washing with PBST, the wells were incubated with biotin-labeled anti-ubiquitin antibody (1:1000) for 1 hour at room temperature. Binding was visualized using streptavidin-HRP (1:10,000). FOLR1-bound proteins were still K-labeled even after 24 hours of incubation in serum. D Thus, ELISA analysis confirmed that affilin proteins bound to FOLR1 with intact high affinity even after 24 hours of incubation in mouse serum (see Table 6).

[0111] Table 6. Binding of FOLR1 binding protein in the presence of serum (ELISA) TIFF0007608538000007.tif28163

[0112] Sequence information SEQUENCE LISTING <110> Navigo Proteins GmbH <120> Novel FOLR1 specific binding proteins for cancer diagnosis and treatment <150> EP 18213661.4 <151> 2018-12-18 <150> EP 19160572.4 <151> 2019-03-04 <160> 73 <170> PatentIn version 3.5 <210> 1 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197556a <400> 1 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Ala Leu Glu 1 5 10 15 Val Glu Pro Cys Asp Thr Ile Glu Asn Val Lys Ala Lys Val Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Glu Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 2 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197556b <400> 2 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Ala Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Val Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Glu Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 3 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197251 <400> 3 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Val Gln Gly 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Glu Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Gly Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 4 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 196962 <400> 4 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Thr Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Arg Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 5 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197525 <400> 5 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Arg Val Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 6 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 187731 <400> 6 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 7 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197132 <400> 7 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp His Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 8 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197340 <400> 8 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Arg Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Arg Leu Glu Asp Gly Arg Thr Leu Ser Asn Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 9 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 196920 <400> 9 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Arg Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Gly 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Lys Asp Gly Arg Thr Leu Ser Asp Tyr Ser Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 10 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197103 <400> 10 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Thr Glu Asp Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Pro Arg Leu Arg Ala Ala 65 70 75 <210> 11 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197032 <400> 11 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Arg Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Pro Arg Leu Arg Ala Ala 65 70 75 <210> 12 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 196964 <400> 12 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Leu Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Thr Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 13 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197312 <400> 13 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Pro Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Thr Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Asp Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 14 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 196868 <400> 14 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Thr Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 15 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197044 <400> 15 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Gly Asp Thr Ile Glu Asn Val Lys Ala Glu Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Pro Arg Leu Arg Ala Ala 65 70 75 <210> 16 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197230 <400> 16 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Glu Ile Gln Gly 20 25 30 Arg Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 17 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197383 <400> 17 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asn 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Asp 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 18 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 196934 <400> 18 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Arg Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Gly Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Asp 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 19 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197126 <400> 19 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Ala Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Pro Leu Arg Leu Arg Ala Ala 65 70 75 <210> 20 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197014 <400> 20 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Pro Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Pro Leu Arg Leu Arg Ala Ala 65 70 75 <210> 21 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 196888 <400> 21 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Pro Leu Arg Leu Arg Ala Ala 65 70 75 <210> 22 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197106 <400> 22 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Gln Ala Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Pro Leu Arg Leu Arg Ala Ala 65 70 75 <210> 23 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 197252 <400> 23 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Val Glu Asn Val Lys Ala Lys Ile Arg Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Gly Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Gly Leu Arg Ala Ala 65 70 75 <210> 24 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 189864 <400> 24 Met Gln Ile Phe Val Val Thr Arg Leu Tyr Lys Glu Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Tyr Leu Tyr Trp Asp Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Arg Phe Gly 50 55 60 Asp His Leu Asp Leu Thr Leu Gly Leu Arg Ala Ala 65 70 75 <210> 25 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 189872 <400> 25 Met Gln Ile Phe Val Val Thr Arg Leu Tyr Lys Glu Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Tyr Leu Tyr Gly Asp Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Arg Phe Gly 50 55 60 Asp His Leu Asp Leu Thr Leu Gly Leu Arg Ala Ala 65 70 75 <210> 26 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 181919 <400> 26 Met Gln Ile Phe Val Val Thr Arg Leu Tyr Lys Glu Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Tyr Leu Tyr Trp Asp Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Arg Phe Gly 50 55 60 Tyr His Leu Asp Leu Thr Leu Gly Leu Arg Ala Ala 65 70 75 <210> 27 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 189853 <400> 27 Met Gln Ile Phe Val Val Thr Arg Leu Tyr Lys Glu Thr Ala Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Tyr Leu Tyr Trp Asp Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Arg Phe Gly 50 55 60 Tyr His Leu Asp Leu Thr Leu Gly Leu Arg Ala Ala 65 70 75 <210> 28 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 187770 <400> 28 Met Gln Ile Phe Val Val Thr Glu Ser Tyr Lys Trp Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Gln Leu Val Trp Asp Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Asp Leu Gly 50 55 60 Gly Tyr Leu Pro Leu Trp Leu Tyr Leu Arg Ala Ala 65 70 75 <210> 29 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> 187803 <400> 29 Met Gln Ile Phe Val Gln Thr Tyr Asn Ile Lys Asp Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Met Leu Phe Trp Ser Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Ile Ala Tyr 50 55 60 Met Thr Leu Thr Leu His Leu Lys Leu Arg Ala Ala 65 70 75 <210> 30 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> 199490 <400> 30 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Ala Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Val Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Glu Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala Gly Gly Ser Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Met Gln Ile Phe 85 90 95 Val Lys Thr Leu Glu Glu Lys Tyr Ile Ala Leu Glu Val Glu Pro Ser 100 105 110 Asp Thr Ile Glu Asn Val Lys Ala Lys Val Gln Asp Lys Glu Gly Ile 115 120 125 Pro Pro Asp Gln Gln Glu Leu Leu Trp Tyr Gly Glu Gln Leu Glu Asp 130 135 140 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly Ala Val Leu Lys 145 150 155 160 Leu Gln Leu Arg Leu Arg Ala Ala 165 <210> 31 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> 199489 dimer of SEQ ID NO: 2 <400> 31 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Ala Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Val Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Glu Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Lys Thr Leu Glu Glu Lys Tyr Ile Ala Leu Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys Val Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Glu Leu Leu Trp Tyr Gly Glu Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly Ala Val Leu Lys 130 135 140 Leu Gln Leu Arg Leu Arg Ala Ala 145 150 <210> 32 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> 199484 homodimer of SEQ ID NO 4 <400> 32 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Thr Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Arg Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala Gly Gly Ser Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Met Gln Ile Phe 85 90 95 Val Lys Thr Leu Glu Glu Lys Tyr Thr Thr Leu Glu Val Glu Pro Ser 100 105 110 Asp Thr Ile Glu Asn Val Lys Ala Arg Ile Gln Asp Lys Glu Gly Ile 115 120 125 Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu Gln Leu Glu Asp 130 135 140 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly Ala Val Leu Lys 145 150 155 160 Leu Gln Leu Arg Leu Arg Ala Ala 165 <210> 33 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> 199483 homodimer of SEQ ID NO: 4 <400> 33 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Thr Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Arg Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Lys Thr Leu Glu Glu Lys Tyr Thr Thr Leu Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Arg Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly Ala Val Leu Lys 130 135 140 Leu Gln Leu Arg Leu Arg Ala Ala 145 150 <210> 34 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> 199488 homo-dimer of SEQ ID NO: 5 <400> 34 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Arg Val Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala Gly Gly Ser Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Met Gln Ile Phe 85 90 95 Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu Val Glu Pro Ser 100 105 110 Asp Thr Ile Glu Asn Val Lys Ala Arg Val Gln Asp Lys Glu Gly Ile 115 120 125 Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu Gln Leu Glu Asp 130 135 140 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly Ala Val Leu Lys 145 150 155 160 Leu Gln Leu Arg Leu Arg Ala Ala 165 <210> 35 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> 199487 homo-dimer of SEQ ID NO: 5 <400> 35 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Arg Val Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Arg Val Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly Ala Val Leu Lys 130 135 140 Leu Gln Leu Arg Leu Arg Ala Ala 145 150 <210> 36 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> 199478 homodimer of SEQ ID NO: 6 <400> 36 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala Gly Gly Ser Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Met Gln Ile Phe 85 90 95 Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu Val Glu Pro Ser 100 105 110 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 115 120 125 Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys Gln Leu Glu Asp 130 135 140 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly Ala Val Leu Lys 145 150 155 160 Leu Gln Leu Arg Leu Arg Ala Ala 165 <210> 37 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> 199477 homodimer of SEQ ID NO: 6 <400> 37 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Lys Thr Leu Glu Glu Lys Tyr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly Ala Val Leu Lys 130 135 140 Leu Gln Leu Arg Leu Arg Ala Ala 145 150 <210> 38 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> 199479 homodimer of SEQ ID NO 18 <400> 38 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Arg Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Gly Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Asp 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Lys Thr Leu Glu Glu Arg Tyr Ile Thr Leu Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Gly Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Asp Ala Val Leu Lys 130 135 140 Leu Gln Leu Arg Leu Arg Ala Ala 145 150 <210> 39 <211> 162 <212> PRT <213> Artificial Sequence <220> <223> 199480 homodimer of SEQ ID NO. 18 <400> 39 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Arg Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Gly Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Asp 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala Gly Gly Ser Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Met Gln Ile Phe Val Lys Thr Leu Glu Glu 85 90 95 Arg Tyr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Gly Asn Val 100 105 110 Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys 115 120 125 Leu Leu Trp Tyr Gly Glu Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp 130 135 140 Tyr Asn Ile Leu Gly Asp Ala Val Leu Lys Leu Gln Leu Arg Leu Arg 145 150 155 160 Ala Ala <210> 40 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> 199481 homodimer of SEQ ID NO 18 <400> 40 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Arg Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Gly Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Asp 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala Gly Gly Ser Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Met Gln Ile Phe 85 90 95 Val Lys Thr Leu Glu Glu Arg Tyr Ile Thr Leu Glu Val Glu Pro Ser 100 105 110 Asp Thr Ile Gly Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 115 120 125 Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu Gln Leu Glu Asp 130 135 140 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Asp Ala Val Leu Lys 145 150 155 160 Leu Gln Leu Arg Leu Arg Ala Ala 165 <210> 41 <211> 172 <212> PRT <213> Artificial Sequence <220> <223> 199482 homodimer of SEQ ID NO 18 <400> 41 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Arg Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Gly Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Asp 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala Gly Gly Ser Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 85 90 95 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Arg Tyr Ile Thr Leu Glu 100 105 110 Val Glu Pro Ser Asp Thr Ile Gly Asn Val Lys Ala Lys Ile Gln Asp 115 120 125 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 130 135 140 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Asp 145 150 155 160 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 165 170 <210> 42 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> 199486 homodimer of SEQ ID NO 20 <400> 42 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Pro Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Pro Leu Arg Leu Arg Ala Ala Gly Gly Ser Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Met Gln Ile Phe 85 90 95 Val Lys Thr Leu Glu Glu Lys Tyr Ile Pro Leu Glu Val Glu Pro Ser 100 105 110 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 115 120 125 Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys Gln Leu Glu Asp 130 135 140 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly Ala Val Leu Lys 145 150 155 160 Leu Pro Leu Arg Leu Arg Ala Ala 165 <210> 43 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> 199485 homodimer of SEQ ID NO: 20 <400> 43 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Pro Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Pro Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Lys Thr Leu Glu Glu Lys Tyr Ile Pro Leu Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly Ala Val Leu Lys 130 135 140 Leu Pro Leu Arg Leu Arg Ala Ala 145 150 <210> 44 <211> 171 <212> PRT <213> Artificial Sequence <220> <223> 202521 (199490) homodimer of SEQ ID NO 2 with C-terminal SAC but no Streptag <400> 44 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Ala Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Val Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Glu Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala Gly Gly Ser Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Met Gln Ile Phe 85 90 95 Val Lys Thr Leu Glu Glu Lys Tyr Ile Ala Leu Glu Val Glu Pro Ser 100 105 110 Asp Thr Ile Glu Asn Val Lys Ala Lys Val Gln Asp Lys Glu Gly Ile 115 120 125 Pro Pro Asp Gln Gln Glu Leu Leu Trp Tyr Gly Glu Gln Leu Glu Asp 130 135 140 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly Ala Val Leu Lys 145 150 155 160 Leu Gln Leu Arg Leu Arg Ala Ala Ser Ala Cys 165 170 <210> 45 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> 199490 (homodimer of SEQ ID NO 2) with c-terminal SAC and Streptag <400> 45 Met Gln Ile Phe Val Lys Thr Leu Glu Glu Lys Tyr Ile Ala Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Val Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Glu Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala Gly Gly Ser Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Met Gln Ile Phe 85 90 95 Val Lys Thr Leu Glu Glu Lys Tyr Ile Ala Leu Glu Val Glu Pro Ser 100 105 110 Asp Thr Ile Glu Asn Val Lys Ala Lys Val Gln Asp Lys Glu Gly Ile 115 120 125 Pro Pro Asp Gln Gln Glu Leu Leu Trp Tyr Gly Glu Gln Leu Glu Asp 130 135 140 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly Ala Val Leu Lys 145 150 155 160 Leu Gln Leu Arg Leu Arg Ala Ala Ser Ala Cys Ala Ser Trp Ser His 165 170 175 Pro Gln Phe Glu Lys 180 <210> 46 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> ubiquitin <400> 46 Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Ala Ala 65 70 75 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> motif 62-70 <400> 47 Leu Gly Gly Ala Val Leu Lys Leu Gln 1 5 <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> motif 62-70 <400> 48 Leu Gly Asp Ala Val Leu Lys Leu Gln 1 5 <210> 49 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> motif 62-70 <400> 49 Leu Gly Gly Ala Val Leu Lys Leu Pro 1 5 <210> 50 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> motif 62-70 <400> 50 Arg Phe Gly Asp His Leu Asp Leu Thr 1 5 <210> 51 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> motif 62-70 <400> 51 Arg Phe Gly Tyr His Leu Asp Leu Thr 1 5 <210> 52 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> motif 62-70 <400> 52 Asp Leu Gly Gly Tyr Leu Pro Leu Trp 1 5 <210> 53 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> motif 62-70 <400> 53 Ile Ala Tyr Met Thr Leu Thr Leu His 1 5 <210> 54 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> motif 42-46 <400> 54 Glu Leu Leu Trp Tyr 1 5 <210> 55 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> motif 42-46 <400> 55 Lys Leu Leu Trp Tyr 1 5 <210> 56 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> motif 42-46 <400> 56 Lys Leu Leu Arg Tyr 1 5 <210> 57 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> motif 42-46 <400> 57 Tyr Leu Tyr Trp Asp 1 5 <210> 58 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> motif 42-46 <400> 58 Tyr Leu Tyr Gly Asp 1 5 <210> 59 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> motif 42-46 <400> 59 Gln Leu Val Trp Asp 1 5 <210> 60 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> motif 42-46 <400> 60 Met Leu Phe Trp Ser 1 5 <210> 61 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> motif 9-12 <400> 61 Glu Glu Lys Tyr 1 <210> 62 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> motif 9-12 <400> 62 Glu Glu Arg Tyr 1 <210> 63 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> motif 9-12 <400> 63 Glu Gln Lys Tyr 1 <210> 64 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> motif 9-12 <400> 64 Leu Tyr Lys Glu 1 <210> 65 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> motif 9-12 <400> 65 Ser Tyr Lys Trp 1 <210> 66 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> motif 9-12 <400> 66 Asn Ile Lys Asp 1 <210> 67 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> linker <400> 67 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 1 5 10 15 <210> 68 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> c-terminal sequence (StrepTaq) <400> 68 Ala Ser Trp Ser His Pro Gln Phe Glu Lys 1 5 10 <210> 69 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> Affilin 197103 E10Q <400> 69 Met Gln Ile Phe Val Lys Thr Leu Glu Gln Lys Tyr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Thr Glu Asp Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Glu 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Pro Arg Leu Arg Ala Ala 65 70 75 <210> 70 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> Affilin 197312 E10Q <400> 70 Met Gln Ile Phe Val Lys Thr Leu Glu Gln Lys Tyr Ile Pro Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Thr Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Asp Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Gln Leu Arg Leu Arg Ala Ala 65 70 75 <210> 71 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> Affilin 197014 E10Q <400> 71 Met Gln Ile Phe Val Lys Thr Leu Glu Gln Lys Tyr Ile Pro Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Pro Leu Arg Leu Arg Ala Ala 65 70 75 <210> 72 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> Affilin 199486 E10Q <400> 72 Met Gln Ile Phe Val Lys Thr Leu Glu Gln Lys Tyr Ile Pro Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Pro Leu Arg Leu Arg Ala Ala Gly Gly Ser Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Met Gln Ile Phe 85 90 95 Val Lys Thr Leu Glu Gln Lys Tyr Ile Pro Leu Glu Val Glu Pro Ser 100 105 110 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 115 120 125 Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys Gln Leu Glu Asp 130 135 140 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly Ala Val Leu Lys 145 150 155 160 Leu Pro Leu Arg Leu Arg Ala Ala 165 <210> 73 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 199485 E10Q <400> 73 Met Gln Ile Phe Val Lys Thr Leu Glu Gln Lys Tyr Ile Pro Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly 50 55 60 Ala Val Leu Lys Leu Pro Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Lys Thr Leu Glu Gln Lys Tyr Ile Pro Leu Glu Val Glu Pro Ser 85 90 95 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 100 105 110 Pro Pro Asp Gln Gln Lys Leu Leu Trp Tyr Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Leu Gly Gly Ala Val Leu Lys 130 135 140 Leu Pro Leu Arg Leu Arg Ala Ala 145 150

Claims

1. 1. A folate receptor alpha (FOLR1) binding protein comprising an amino acid sequence selected from the group of SEQ ID NOs: 2, 26, 28, 29, or an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 2, 26, 28, or 29, respectively, having a specific binding affinity of less than 500 nM to FOLR1 but no binding to FOLR2 (folate receptor beta), which is: - in an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, the amino acid residues corresponding to positions 9, 10, 11, and 12 are EEKY, EERY, or EQKY, the amino acid residues corresponding to positions 42, 43, 44, 45, and 46 are ELLWY, KLLWY, or KLLRY, and the amino acid residues corresponding to positions 62, 63, 64, 65, 66, 67, 68, 69, and 70 are LGGAVLKLQ, LGDAVLKLQ, or LGGAVLKLP; an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 26, in which the amino acid residues corresponding to positions 9, 10, 11, and 12 are LYKE, the amino acid residues corresponding to positions 42, 43, 44, 45, and 46 are YLYWD or YLYGD, and the amino acid residues corresponding to positions 62, 63, 64, 65, 66, 67, 68, 69, and 70 are RFGDHLDLT or RFGYHLDLT; In an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 28, the amino acid residues corresponding to positions 9, 10, 11, and 12 are SYKW, the amino acid residues corresponding to positions 42, 43, 44, 45, and 46 are QLVWD, and the amino acid residues corresponding to positions 62, 63, 64, 65, 66, 67, 68, 69, and 70 are DLGGYLPLW; and In an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 29, the amino acid residues corresponding to positions 9, 10, 11, and 12 are NIKD, the amino acid residues corresponding to positions 42, 43, 44, 45, and 46 are MLFWS, and the amino acid residues corresponding to positions 62, 63, 64, 65, 66, 67, 68, 69, and 70 are IAYMTLTLH; The FOLR1 binding protein.

2. The folate receptor alpha (FOLR1) binding protein of claim 1, comprising an amino acid sequence selected from any one of the group consisting of SEQ ID NOs: 1-43.

3. The FOLR1-binding protein of claim 1 or 2, which is a multimer comprising multiple FOLR1-binding proteins of claim 1 or 2.

4. The FOLR1 binding protein of any one of claims 1 to 3, further comprising one or more coupling domains of 1 to 80 amino acids comprising one or more coupling sites for coupling a chemical moiety, wherein the chemical moiety is selected from any of a chelator, a drug, a toxin, a dye, and a small molecule.

5. The FOLR1 binding protein of any one of claims 1 to 4, further comprising at least one diagnostically effective moiety, optionally selected from a radionuclide, a fluorescent protein, a photosensitizer, a dye, or an enzyme, or any combination of the foregoing.

6. The FOLR1 binding protein of any one of claims 1 to 4, further comprising at least one therapeutically effective moiety optionally selected from a monoclonal antibody or fragment thereof, a radionuclide, a cytotoxic compound, a cytokine, a chemokine, an enzyme, or a derivative thereof, or any combination of the foregoing.

7. The FOLR1 binding protein of any one of claims 1 to 6, further comprising at least one pharmacokinetic modulating moiety selected from polyethylene glycol, human serum albumin, an albumin-binding peptide, an immunoglobulin-binding peptide, or an immunoglobulin or immunoglobulin fragment, a polysaccharide, or an unstructured amino acid sequence comprising the amino acids alanine, glycine, serine, and proline.

8. 8. A FOLR1 binding protein according to any one of claims 1 to 7 for use in the diagnosis or treatment of a tumor associated with FOLR1.

9. The FOLR1 binding protein of claim 8 for imaging of FOLR1-associated tumors or for radiotherapy of FOLR1-associated tumors.

10. 10. A composition comprising a FOLR1 binding protein according to any one of claims 1 to 9 for use in medicine.

11. A composition comprising a FOLR1 binding protein according to any one of claims 1 to 10 for use in the diagnosis or treatment of a tumor associated with FOLR1.

12. The composition of claim 11 for imaging of FOLR1-associated tumors or for radiotherapy of FOLR1-associated tumors.

13. (a) culturing a host cell under conditions suitable for obtaining said FOLR1 binding protein; and (b) isolating the produced FOLR1-binding protein. A method for producing a FOLR1 binding protein according to any one of claims 1 to 12, comprising:

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