Novel FOLR1-specific binding proteins for cancer diagnosis and therapy

Abstract

The present invention relates to novel binding proteins specific for folate receptor alpha (FOLR1). The present invention also relates to FOLR1 binding proteins further comprising diagnostically or therapeutically active ingredients. Further aspects of the present invention relate to the use of these FOLR1 binding proteins in medicine, for example in the diagnosis and treatment of FOLR1-related cancers.

Description

Technical Field

[0001] The present invention relates to novel binding proteins specific for folate receptor alpha (FOLR1). The present invention also relates to FOLR1 binding proteins further comprising diagnostically or therapeutically active ingredients. Further aspects of the present invention relate to the use of these FOLR1 binding proteins in medicine, for example in the diagnosis and treatment of FOLR1-related cancers. Background Art

[0002] Folate receptor alpha (FOLR1) is a glycosylphosphatidylinositol-anchored membrane protein that has a high binding affinity for the active form of folate and coordinates its transport into the cell interior. FOLR1 is expressed only at low concentrations and with limited distribution in normal human tissues, such as the proximal tubule cells of the kidney.

[0003] FOLR1 is highly overexpressed in some solid tumors, especially in solid cancer types of epithelial origin. The cancer types with the highest frequency of FOLR1 expression are ovarian cancer, endometrial cancer, brain cancer, lung cancer, and kidney cancer. In addition, FOLR1 was found to be expressed at a moderate frequency in head and neck cancer, breast cancer, gastric cancer, and colorectal cancer.

[0004] Ovarian cancer has the highest mortality rate among all female cancers. FOLR1 levels have been reported to be elevated in almost all epithelial ovarian cancers and are associated with high levels of tumor aggressiveness, resulting in a lower disease-free interval and poor overall survival for patients. Despite initial response to chemotherapy, most patients experience disease recurrence due to tumor resistance to chemotherapy. Non-small cell lung cancer accounts for the majority of lung cancers, and these patients have a very poor prognosis with a low 5-year overall survival rate. High FOLR1 tissue expression has been observed in many estrogen receptor / progesterone receptor-negative breast cancers. In addition, overexpression of FOLR1 has been detected in patients with rare and aggressive forms of thoracic cancer and pleural mesothelioma.

[0005] Only a few targeted diagnostic or therapeutic approaches have been described for tumors with high FOLR1 levels. One example of a potential treatment for FOLR1-associated chemotherapy (platinum)-resistant ovarian cancer is the antibody-drug conjugate Mirvetuximabsoravtansine, which is currently in clinical testing. Another monoclonal antibody in clinical testing is Farletuzumab for ovarian and lung cancer.

[0006] Existing options fail to adequately address the diagnosis and treatment of FOLR1-associated cancers, and therefore, many patients fail to fully benefit from current strategies.

[0007] Undoubtedly, new strategies for the diagnosis and treatment of FOLR1-associated tumors are strongly needed.

[0008] One object of the present invention is to provide molecules for specific targeting of FOLR1 for targeted diagnostic and therapeutic options, including detection of FOLR1 positive tumors. Targeting this tumor-associated protein may provide benefits to patients with unmet needs for new diagnostic and therapeutic approaches. Due to the low and restricted distribution of FOLR1 in normal tissues, targeting FOLR1 suggests potential non-toxic diagnostic and therapeutic methods. Therefore, binding proteins specific for FOLR1 may provide effective medical options for cancer and ultimately improve the quality of life of patients.

[0009] The present invention provides novel FOLR1 binding molecules for new and improved strategies in the diagnosis and treatment of FOLR1-related cancers.

[0010] The above objects and advantages are achieved by the subject matter of the appended claims. The present invention satisfies the above stated needs by providing examples of FOLR1 binding proteins. The above summary does not necessarily describe all problems solved by the present invention. Summary of the invention

[0011] The present disclosure provides the following [1] to

[15] , but is not particularly limited thereto:

[0012] [1] A folate receptor alpha (FOLR1) binding protein comprising ubiquitin-derived amino acids according 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.

[0013] [2] The FOLR1 binding protein according to [1], wherein

[0014] The amino acid at position 9 corresponding to SEQ ID NO: 46 is selected from E, L, S or N, and

[0015] The amino acid corresponding to position 10 of SEQ ID NO: 46 is selected from E, Q, Y or I, and

[0016] The amino acid corresponding to position 12 of SEQ ID NO: 46 is selected from Y, E, W or D, and,

[0017] The amino acid corresponding to position 42 of SEQ ID NO: 46 is selected from E, K, Y, Q or M, and

[0018] The amino acid at position 44 corresponding to SEQ ID NO: 46 is selected from L, Y, V or F, and

[0019] The amino acid at position 46 corresponding to SEQ ID NO: 46 is selected from Y, D or S, and

[0020] The amino acid at position 62 corresponding to SEQ ID NO: 46 is selected from L, R, D or I, and

[0021] The amino acid at position 63 corresponding to SEQ ID NO: 46 is selected from G, F, L or A, and

[0022] The amino acid at position 64 corresponding to SEQ ID NO: 46 is selected from G, D or Y, and

[0023] The amino acid corresponding to position 65 of SEQ ID NO: 46 is selected from A, D, Y, G or M, and

[0024] The amino acid at position 66 corresponding to SEQ ID NO: 46 is selected from V, H, Y or T, and

[0025] The amino acid at position 68 corresponding to SEQ ID NO: 46 is selected from K, D, P or T, and

[0026] The amino acid at position 70 corresponding to SEQ ID NO: 46 is selected from Q, P, T, W or H, and

[0027] Optionally, one or more amino acids of SEQ ID NO: 46 are further modified.

[0028] [3] The FOLR1 binding protein according to [2], comprising amino acids having a sequence identity of 70% to 85% with SEQ ID NO: 46, preferably amino acids having a sequence identity of 75% to 83% with SEQ ID NO: 46, preferably amino acids having a sequence identity of 76% to 83% with SEQ ID NO: 46, and preferably amino acids having a sequence identity of 79% to 83% with SEQ ID NO: 46.

[0029] [4] The 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.

[0030] [5] The FOLR1 binding protein according to any one of [1] to [4], comprising

[0031] an amino acid residue selected from the group consisting of EEKY, EERY, EQKY, LYKE, SYKW or NIKD, corresponding to positions 9, 10, 11 and 12 of SEQ ID NO: 46, and

[0032] an amino acid residue selected from the group consisting of ELLWY, KLLWY, KLLRY, YLYWD, YLYGD, QLVWD or MLFWS corresponding to positions 42, 43, 44, 45 and 46 of SEQ ID NO: 46, and

[0033] An amino acid residue selected from LGGAVLKLQ, LGDAVLKLQ, LGGAVLKLP, RFGDHLDLT, RFGYHLDLT, DLGGYLPLW or IAYMTLTLH, which corresponds to positions 62, 63, 64, 65, 66, 67, 68, 69 and 70 of SEQ ID NO:46.

[0034] [6] The FOLR1 binding protein according to any one of [1] to [5], further comprising a substitution at 1, 2, 3, 4 or 5 amino acid positions, wherein the amino acid positions are selected from the positions corresponding to positions 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, preferably selected from the positions K6V, K6Q, L8R, L8E, L8Y, I13T, T14A, T14P, S20C, S20G, I23T, I23V, E23V Any of 24G, 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.

[0035] [7] FOLR1 according to any one of [1] to [6], wherein the FOLR1 binding protein is a multimer comprising a plurality of FOLR1 binding proteins according to any one of [1] to [6], preferably a dimer of the FOLR1 binding protein according to any one of [1] to [6], and more preferably a homodimer of the FOLR1 binding protein according to any one of [1] to [6].

[0036] [8] The FOLR1 binding protein according to any one of [1] to [7], which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-45 and SEQ ID NOs: 69-73, or an amino acid sequence having at least 90% identity thereto, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-45 and SEQ ID NOs: 69-73, or an amino acid sequence having at least 90% identity thereto.

[0037] [9] The FOLR1 binding protein according to any one of [1] to [8], further comprising one or more coupling domains of 1 to 80 amino acids, which comprise one or more coupling sites for coupling a chemical moiety, preferably wherein the chemical moiety is selected from any one of a chelator, a drug, a toxin, a dye and a small molecule.

[0038]

[10] The FOLR1 binding protein according to any one of [1] to [9], further comprising at least one diagnostically active portion, which is optionally selected from a radionuclide, a fluorescent protein, a photosensitizer, a dye or an enzyme, or any combination thereof.

[0039]

[11] The FOLR1 binding protein according to any one of [1] to [9], further comprising at least one therapeutically active portion, which is optionally 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 thereof.

[0040]

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

[11] , further comprising at least one pharmacokinetic regulating moiety, which is optionally selected from polyethylene glycol, human serum albumin, an albumin binding protein or peptide, an immunoglobulin binding protein or peptide, or an immunoglobulin or an immunoglobulin fragment, a polysaccharide, or an unstructured amino acid sequence comprising the amino acids alanine, glycine, serine, or proline.

[0041]

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

[12] , which is used for diagnosis or treatment of FOLR1-related tumors, preferably for tumor imaging and radiotherapy treatment of FOLR1-related tumors.

[0042]

[14] A composition comprising the FOLR1 binding protein according to any one of [1] to

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

[0043]

[15] A method for producing the FOLR1 binding protein according to any one of [1] to

[13] , comprising the steps of: a) culturing a host cell under conditions suitable for obtaining the FOLR1 binding protein, and b) isolating the FOLR1 binding protein.

[0044] This summary does not necessarily describe all features of the invention. Other embodiments will become apparent by reading the detailed description that follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The attached figure shows:

[0046] Figure 1Selected amino acid sequences of the folate receptor alpha (FOLR1) binding protein are shown. Differences from ubiquitin (SEQ ID NO: 46) are shown in grey. The first row of numbers are the corresponding positions in SEQ ID NO: 46.

[0047] Figure 2 Affilin-199490 (homodimer of Affilin-197556b) binds to FOLR1 with high affinity. Analysis was performed by label-free interaction assay using SPR (Biacore). FOLR1 was immobilized on a CM-5 chip. The lines show different 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 K of Affilin-199490 was calculated. D The value is less than 0.5nM.

[0048] Figure 3 .Affilin-199490 does not bind to FOLR2 (folate receptor beta), but binds specifically to FOLR1 (folate receptor alpha).Analysis was performed by label-free interaction assay using SPR (Biacore). DETAILED DESCRIPTION

[0049] The present inventors have developed a solution to meet the strong ongoing need in the art for expanded medical options for cancer diagnosis and treatment by providing novel FOLR1 binding proteins. The functional characteristics of the FOLR1 specific proteins as defined herein are that they have a high specific affinity for FOLR1, but not for FOLR2. In particular, the present invention provides FOLR1 binding proteins (also referred to as ubiquitin mutant proteins) based on ubiquitin mutant proteins. The FOLR1 binding proteins as described herein provide a molecular format with favorable physicochemical properties, are expressed at high levels in bacteria, and allow for easy production methods. The novel FOLR1 binding proteins may broaden heretofore unmet medical strategies for the diagnosis and treatment of FOLR1-related cancers. In particular, the FOLR1 binding proteins may be used for imaging purposes, such as for the presence of tumor cells expressing FOLR1, and for radiotherapy treatment of tumors expressing FOLR1.

[0050] Before describing the present invention in more detail below, it should be understood that the present invention is not limited to the specific methods, schemes and reagents described herein, because they can be varied. It should also be understood that the terms used herein are only for the purpose of describing specific aspects and embodiments, and are not intended to limit the scope of the present invention reflected by the appended claims. Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. This includes technicians in the field of protein engineering and purification, as well as technicians in the field of novel target-specific binding molecules developed for technical applications and for treatment and diagnosis.

[0051] Preferably, the terms used herein are as in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and H. ed. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0052] Unless the context requires otherwise, throughout this specification and the claims that follow, the word "comprise" and variations such as "include" and "comprising" are understood to imply the inclusion of a specified integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps. The term "comprise" or "comprising" may encompass limitations to "consisting of," where such limitations are necessary for any reason and to any extent.

[0053] Throughout this specification, some documents (e.g., patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank accession number sequence submission documents, etc.) may be cited. Anything herein should not be construed as admitting that the present invention is not entitled to be earlier than such disclosure due to prior inventions. Certain documents cited herein may be described as "incorporated by reference." In the event of a conflict between the definition or teaching of these incorporated references and the definition or teaching cited in this specification, the text of this specification takes precedence.

[0054] All sequences referred to herein are disclosed in the attached sequence listing, the entire contents and disclosure of which constitute part of the disclosure of this specification.

[0055] General definitions of important terms used in this application

[0056] As used herein, the term "FOLR1" or "folate receptor alpha" refers to Uniprot Accession No. P15328, in particular residues 25-234. The term "FOLR1" includes all polypeptides that exhibit at least 80%, 85%, 90%, 95%, 96% or 97% or more or 100% sequence identity to FOLR1 of Uniprot Accession No. P15328 (human), in particular residues 25-234, or Uniprot Accession No. P35846 (mouse).

[0057] As used herein, the term "FOLR2" or "folate receptor beta" refers to Uniprot Accession No. P14207 or the corresponding protein in other species.

[0058] The term "FOLR1 binding protein" refers to a protein that binds to FOLR1 with high affinity.

[0059] The terms "protein" and "polypeptide" refer to any chain of two or more amino acids linked by peptide bonds, and do not refer to a specific length of the product. Thus, "peptide," "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids is included in the definition of "polypeptide," and the term "polypeptide" may be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to the products of post-translational modifications of a polypeptide, which are well known in the art.

[0060] The term "modification" or "amino acid modification" refers to the substitution, deletion or insertion of a reference amino acid by another amino acid at a specific position in the parent polypeptide sequence. Given the known genetic code, as well as recombinant and synthetic DNA technology, skilled scientists can easily construct DNA encoding amino acid variants.

[0061] The term "mutant protein" as used herein refers to a derivative or analogous protein of ubiquitin, such as that shown in SEQ ID NO: 46, which differs from the amino acid sequence by amino acid exchange, insertion, deletion or any combination thereof, provided that the mutant protein has specific binding affinity to FOLR1.

[0062] the term (Registered trademark of Navigo Proteins GmbH) refers to non-immunoglobulin derived binding proteins.

[0063] The term "substitution" is to be understood as the exchange of one amino acid by another. The term "insertion" includes the addition of amino acids to the original amino acid sequence.

[0064] The term "ubiquitin" refers to ubiquitin according to SEQ ID NO: 46, and refers to a protein with at least 95% identity, for example having point mutations at positions 45, 75, 76 that do not affect binding to the target (FOLR1).

[0065] The terms "binding affinity" and "binding activity" are used interchangeably herein and refer to the ability of a polypeptide to bind to another protein, peptide, or fragment or domain thereof. Binding affinity is typically measured by the equilibrium dissociation constant (K D ) is measured and reported, and this constant is used to evaluate and rank the strength of bimolecular interactions.

[0066] The term "fusion protein" relates to a protein comprising at least a first protein genetically linked to at least a second protein. A fusion protein is created by linking two or more genes that originally encode different proteins. The fusion protein may further comprise other domains that are not involved in target binding, such as, but not limited to, a multimerization moiety, a polypeptide tag, a polypeptide linker, or a moiety that binds to a target different from FOLR1.

[0067] The term "amino acid sequence identity" refers to a quantitative comparison of the identity (or differences) of the amino acid sequences of two or more proteins. The "percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the 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 percentage of sequence identity. To determine sequence identity, the sequence of the query protein is aligned with the sequence of a reference protein or reference polypeptide. Methods of sequence alignment are well known in the art. For example, to determine the degree of amino acid sequence identity of any polypeptide relative 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, as known to those skilled in the art, Clustal Omega is preferably used.

[0068] DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0069] Structural characterization of FOLR1 binding proteins. FOLR1 binding proteins as defined herein include mutant proteins of ubiquitin. FOLR1 binding proteins include amino acids based on ubiquitin according to SEQ ID NO: 46, having substitutions at least at positions 9, 10, 12, 42, 44, 46, 62, 63, 64, 65, 66, 68 and 70.

[0070] In some embodiments, a FOLR1 binding protein as 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.

[0071] In various embodiments, the FOLR1 binding protein as disclosed herein has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 46. The FOLR1 binding protein as disclosed herein has at least 81%, 82%, 83%, 84% or 85% sequence identity to the amino acid sequence of SEQ ID NO: 46.

[0072] In preferred embodiments, the FOLR1 binding proteins as disclosed herein may have any amino acid identity between 70% identity and 85% identity with the amino acid sequence of SEQ ID NO: 46. In even more preferred embodiments, the FOLR1 binding proteins as disclosed herein may have any amino acid identity between 73% identity and 83% identity with the amino acid sequence of SEQ ID NO: 46. In even more preferred embodiments, the FOLR1 binding proteins as disclosed herein may have any amino acid identity between 75% identity and 83% identity with the amino acid sequence of SEQ ID NO: 46. In even more preferred embodiments, the FOLR1 binding proteins as disclosed herein may have any amino acid identity between 76% identity and 83% identity with the amino acid sequence of SEQ ID NO: 46. In even more preferred embodiments, the FOLR1 binding proteins as disclosed herein may have any amino acid identity between 79% identity and 83% identity with the amino acid sequence of SEQ ID NO: 46.

[0073] The FOLR1 binding protein as defined herein comprises a mutant protein of SEQ ID NO: 46, i.e., a mutant protein comprising ubiquitin. The FOLR1 binding protein comprises amino acids based on SEQ ID NO: 46 having substitutions at least at positions 9, 10, 12, 42, 44, 46, 62, 63, 64, 65, 66, 68 and 70. In the 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, the amino acid corresponding to position 64 of SEQ ID NO:46 is G, D or Y, 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 The amino acid at position 66 of SEQ ID NO: 46 is V, H, Y or T, the amino acid at position 68 of SEQ ID NO: 46 is K, D, P or T, the amino acid at position 70 of SEQ ID NO: 46 is Q, P, T, W or H, and optionally, one or more, preferably 1, 2, 3, 4 or 5 amino acids of SEQ ID NO: 46 are substituted. In some embodiments, the amino acid at position 11 of SEQ ID NO: 46 is K or R, and the amino acid at position 45 of SEQ ID NO: 46 is W, R or G.

[0074] Structural characterization of 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, wherein the amino acid residue motif is selected from the group of EEKY, EERY, EQKY, LYKE, SYKW or NIKD (SEQ ID NOs: 61-66).

[0075] In one embodiment, the FOLR1 binding protein comprises an amino acid residue motif at positions corresponding to positions 42, 43, 44, 45 and 46 of SEQ ID NO: 46, wherein the amino acid residue motif is selected from the group of ELLWY, KLLWY, KLLRY, YLYWD, YLYGD, QLVWD or MLFWS (SEQ ID NOs: 54-60).

[0076] 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).

[0077] 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, wherein the amino acid residue motif is selected from the group of EEKY, EERY, EQKY, LYKE, SYKW or NIKD; an amino acid residue motif at positions corresponding to positions 42, 43, 44, 45 and 46 of SEQ ID NO:46, wherein the amino acid residue motif is selected from the group of ELLWY, KLLWY, KLLRY, YLYWD, YLYGD, QLVWD or MLFWS; 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 the group of LGGAVLKLQ, LGDAVLKLQ, LGGAVLKLP, RFGDHLDLT, RFGYHLDLT, DLGGYLPLW or IAYMTLTLH.

[0078] In one embodiment, the FOLR1 binding protein comprises an amino acid motif at positions 9-12, 42-46, and 62-70 corresponding to ubiquitin as defined in SEQ ID NO:46, the amino acid motif being selected from the group consisting of 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, as shown in Table 1.

[0079] Table 1. Amino acids at selected positions in FOLR1 binding proteins

[0080] The numbers in the first row are the corresponding positions in SEQ ID NO:46.

[0081]

[0082] In one embodiment, the FOLR1 binding protein comprises a further substitution at 1, 2, 3, 4, 5 amino acid positions selected from the group consisting of positions 6, 8, 13, 14, 20, 23, 24, 25, 29, 30, 31, 32, 33, 34, 48, 49, 51, 52, 58, 59, 60, 71, and 72 corresponding to SEQ ID NO:46. In some embodiments, the substitution is 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, preferably wherein the FOLR1 binding protein comprises amino acids having a sequence identity of between 70% and 85% to SEQ ID NO: 46, preferably to SEQ ID NO: 47. NO:46 has a sequence identity of between 75% and 83% amino acids, preferably a sequence identity of between 76% and 83% amino acids to SEQ ID NO:46. In some embodiments, the further substitution is selected from Y59H (e.g., see SEQ ID NO:7). In some embodiments, the further substitution is selected from E24A (e.g., see SEQ ID NO:1; has a sequence identity of 81.6% to SEQ ID NO:46; 14 of the 76 amino acids are modified). In some embodiments, the further substitution is selected from any one of the group of K29R and K48E (e.g., see SEQ ID NO:4; has a sequence identity of 80.3% 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 of T14A, I30V and K48E (e.g., see SEQ ID NO: 2) (78.9% sequence identity to SEQ ID: 46; 16 amino acids are modified; see, e.g., the dimer of SEQ ID NO: 2 as shown in SEQ ID NO: 30). In some embodiments, the further substitution is selected from any one of the group of K6V, L8R and R72G (e.g., see SEQ ID NO: 24; 78.9% sequence identity to SEQ ID: 46; 16 amino acids are modified). In some embodiments, the further substitution is selected from any one of the group of K6V, L8R, W45G, R72G (e.g., see SEQ ID NO: 25; 77.6% sequence identity to SEQ ID: 46; 17 amino acids are modified).In some embodiments, the further substitution is selected from any one of the group consisting of K6V, L8R, I13T, T14A, R72G (e.g., see SEQ ID NO: 27; 76.3% sequence identity to SEQ ID: 46; 18 amino acids modified).

[0083] exist Figure 1 Other examples are provided in .

[0084] Structural characterization of non-substituted positions in SEQ ID NO: 46. In various embodiments, the FOLR1 binding proteins as disclosed herein are further structurally characterized in that certain residues are not mutated, for example, 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, 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, 73-76 correspond to the amino acids set forth in SEQ ID NO:46.

[0085] Multimers. In preferred embodiments, the FOLR1 binding protein is a multimer comprising a plurality of FOLR1 binding proteins as defined herein. The multimer may comprise two, three, four or more FOLR1 binding proteins. In one embodiment, the FOLR1 binding protein comprises 2, 3, 4 or more FOLR1 binding proteins linked to each other, i.e., the FOLR1 binding protein may be a dimer, trimer or tetramer, etc. In some embodiments, the multimer is a dimer of the FOLR1 binding protein 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 may be produced by fusing two identical proteins of any one of the groups of SEQ ID NOs: 1-29 or any one of the amino acid sequences having at least 90% identity therewith. For example, the FOLR1 binding protein of SEQ ID NO: 33 is a homodimer 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.

[0086] Table 2. Homodimeric FOLR1 binding proteins

[0087]

[0088]

[0089] In other embodiments, the multimer is a heterodimer, for example, the two amino acid sequences of the FOLR1-specific Affilin protein have different amino acid sequences.

[0090] 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.

[0091] One embodiment relates to a linker composed of an amino acid such as glycine, serine, alanine or proline. The linker may be composed of glycine and serine and may be glycine-rich (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 according to any of SEQ ID NO: 67 or a peptide linker having 90% identity thereto. Other linkers for protein fusion are known in the art and may be used.

[0092] The FOLR1 binding protein as described herein comprises, consists essentially of, or consists of an amino acid sequence selected from the group of SEQ ID NOs: 1-45 and SEQ ID NOs: 69-74, respectively, or selected from an amino acid sequence having at least 90% identity to SEQ ID NOs: 1-45 and SEQ ID NOs: 69-74, 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 have at least 90% identity to SEQ ID NO: 2. In some other 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 to the amino acid sequence of SEQ ID NO: 28 (Affilin-187770). For example, but not limited to, the selected FOLR1 binding protein is shown in Figure 1 .

[0093] Functional Characteristics. In some embodiments, a FOLR1 binding protein as described herein binds to FOLR1 expressed on a cell (as determined by FACS) and / or binds to FOLR1 with an affinity of 500 nM or less (as determined by surface plasmon resonance assay).

[0094] In some embodiments, the binding affinity (K) of a FOLR1 binding protein as described herein for FOLR1 is D) is less than 500 nM. The FOLR1 binding protein binds to FOLR1 with a measurable binding affinity of less than 500 nM, less than 200 nM, 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. Suitable methods are known to those skilled in the art or described in the literature. Methods for determining binding affinity are known per se and may be selected from, for example, the following methods known in the art: enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), kinetic exclusion analysis (KinExA assay), biolayer interferometry (BLI), flow cytometry, fluorescence spectroscopy, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA) and enhanced chemiluminescence (ECL). Some methods are described in the examples below. In general, the dissociation constant K D is determined at 20°C, 25°C or 30°C. If not otherwise specified, K D The values ​​were determined by SPR at 25 °C. K D The lower the value, the greater the binding affinity of the biomolecule for its binding partner. D The higher the value, the weaker the binding between the binding partners. Examples of binding affinities of FOLR1 binding proteins to FOLR1 are provided in Table 4 (see Example 5).

[0095] In some embodiments, the specific binding affinity (K) of a FOLR1 binding protein as described herein for FOLR1 is D ) is less than 500 nM, but not for FOLR2. In some embodiments, as measured by surface plasmon resonance (see Figure 3 ), as determined by , as tested on cell lines expressing FOLR2, and as further described in the Examples (see Example 6), the FOLR1 (folate receptor alpha) binding protein as described herein specifically binds to FOLR1, but no binding to FOLR2 (folate receptor beta) is detected. Therefore, the binding of the FOLR1 binding protein as described herein is highly specific. FOLR1 and FOLR2 are folate receptor subtypes with low homology (less than 80%) and different expression patterns. FOLR1 is mainly expressed on malignant cancer cells, while FOLR2 is expressed on activated macrophages at sites of inflammation. Highly selective binding to FOLR1 may be important for targeted medical applications of FOLR1-related cancers, but may not be important for inflammation and may have reduced potential toxic side effects.

[0096] In some embodiments, a FOLR1 binding protein as described herein binds to FOLR1 but has no detectable binding to the human Fc domain of immunoglobulin IgG1 as determined by surface plasmon resonance assay.

[0097] EC50 50 The concentration of FOLR1 binding protein that elicits a response halfway between baseline and maximum after a specified exposure time, and therefore represents the concentration of FOLR1 binding protein at which 50% of the maximum effect is observed, in this case, the fluorescence intensity signal reaches half of the maximum in a cell-bound, flow cytometry experiment. In some embodiments, the EC of a FOLR1 binding protein as described herein on cells expressing FOLR1 is 50 Less than 100 nM, less than 50 nM, less than 20 nM, less than 10 nM, less than 5 nM, more preferably less than 1 nM. In some embodiments, after incubation at 37° C. for at least 24 hours in the presence of mouse serum, the EC of the FOLR1 binding protein as described herein for FOLR1 is less than 100 nM, less than 50 nM, less than 20 nM, less than 10 nM, less than 5 nM, more preferably less than 1 nM. 50 Less than 1 nM. Suitable methods are known to those skilled in the art. 50 The lower the value, the stronger the FOLR1 binding protein binds to FOLR1. Table 5 (see Example 9) and Table 6 (see Example 10) provide examples of FOLR1 binding proteins that are stable even in the presence of serum.

[0098] In some embodiments, the FOLR1 binding proteins described herein are stable at elevated temperatures, preferably between 62°C and 87°C. For stability analysis, spectroscopic or fluorescence-based methods, such as those associated with chemical or physical unfolding, are known to those skilled in the art. For example, standard methods can be used to measure thermal melting (T m ) temperature (in degrees Celsius, °C) at which half of the molecules unfold. Typically, T m The higher the , the more stable the molecule. As described in further detail in Example 4 and Table 3, temperature stability was determined by differential scanning fluorimetry (DSF).

[0099] Coupling site. In some embodiments, the FOLR1 binding protein as described herein further comprises one or more coupling sites for coupling of a chemical moiety. The coupling site is capable of reacting with other chemical groups to couple the FOLR1 binding protein to the chemical moiety. The defined number and defined position of the coupling site enables the chemical moiety to be site-specifically coupled to the FOLR1 binding protein as described herein. Thus, if desired, a large number of chemical moieties can be bound to the FOLR1 binding protein. One skilled in the art can adjust the number of coupling sites to the optimal number for certain applications, thereby adjusting the amount of the chemical moiety accordingly. In selected embodiments, the coupling site can be selected from a group of one or more amino acids that can be labeled with a specific chemical method, 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 can comprise 1 to 20 coupling sites, preferably 1 to 6 coupling sites, preferably 2 coupling sites, or preferably one coupling site.

[0100] Coupling domain. One embodiment provides a FOLR1 binding protein comprising at least one 1 to 80 amino acid coupling domain comprising one or more coupling sites. In some embodiments, the 1 to 80 amino acid coupling domain 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 NOs: 44 and 45 (coupling domain of 3 amino acids "SAC", coupling site is cysteine). In other embodiments, the 5 to 80 amino acid coupling domain may consist of alanine, proline, serine, and cysteine ​​as a coupling site. In one embodiment, the coupling domain consists of 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 as described herein. In some embodiments, the amino acids alanine, proline and serine are randomly distributed throughout the coupling domain amino acid sequence such that no more than 2, 3, 4 or 5 identical amino acid residues are adjacent, preferably a maximum of 3 amino acids. The composition of the 1 to 20 coupling domains may be different or identical.

[0101] In some embodiments, the chemical moiety is selected from any of a chelator, a drug, a toxin, a dye, and a small molecule.

[0102] In some embodiments, at least one of the chemical moieties is a chelator designed as a complexing agent for coupling to one or more other moieties to couple the target compound to the FOLR1 binding protein as disclosed herein. One embodiment relates to a FOLR1 binding protein, wherein the chelator is a complexing agent for coupling to one or more radioisotopes or other detectable labels, as described in the Examples (Examples 7-10).

[0103] Diagnostic moiety. In various embodiments, the FOLR1 binding protein further comprises a diagnostic moiety. In other embodiments, the FOLR1 binding protein further comprises more than one diagnostic moiety. In some embodiments, such diagnostic moieties can be selected from radionuclides, fluorescent proteins, photosensitizers, dyes, or enzymes, or any combination thereof. 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 or metastasis of tumor cells, tumor distribution, and / or recurrence of tumors. Methods for detecting or monitoring cancer cells relate to imaging methods. Such methods involve imaging FOLR1-associated cancer cells by, for example, radioimaging or photoluminescence or fluorescence.

[0104] Therapeutic moiety. In various embodiments, the FOLR1 binding protein further comprises a therapeutically active moiety. In other embodiments, the FOLR1 binding protein further comprises more than one therapeutically active moiety. In some embodiments, such therapeutically active 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 thereof. In some embodiments, the FOLR1 binding protein comprising a therapeutically active component can be used to target delivery of any of the above listed components to tumor cells expressing FOLR1 and accumulate therein, thereby resulting in low-level toxicity to normal cells.

[0105] Radionuclides. Radionuclides suitable for in vivo or in vitro imaging applications or radiotherapy include, for example, but not limited to, a group of gamma-emitting isotopes, a group of positron emitters, a group of beta emitters, and a group of alpha emitters. In some embodiments, suitable conjugation partners include chelating agents, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA) or activated derivatives thereof, nanoparticles and liposomes. In various embodiments, DOTA may be suitable as a complexing agent for radioisotopes and other agents used for imaging, as described in further detail in the Examples.

[0106] Moieties that modulate pharmacokinetics. In some embodiments, the FOLR1 binding protein further comprises at least one moiety that modulates pharmacokinetics, which is optionally selected from polyethylene glycol, human serum albumin, an albumin binding peptide, an immunoglobulin binding peptide, or an immunoglobulin or immunoglobulin fragment, or a polysaccharide (e.g., hydroxyethyl starch), or an unstructured amino acid sequence that increases the hydrodynamic radius (e.g., a polymer comprising the amino acids alanine, glycine, serine, proline). In various embodiments, the moiety extends the half-life of the FOLR1 binding protein by at least 1.5 times. Various techniques for producing FOLR1 binding proteins with extended half-lives are known in the art, for example, by directly fusing the moiety that modulates pharmacokinetics to the FOLR1 binding protein as described above or by chemical coupling methods. The moiety that modulates pharmacokinetics can be attached to, for example, one or more sites of the FOLR1 binding protein via a peptide linker sequence or via a coupling site as described above.

[0107] Conjugation of a proteinaceous or non-proteinaceous moiety to a FOLR1 binding protein can be performed using chemical methods well known in the art. In some embodiments, a coupling chemistry specific for derivatization of cysteine ​​or lysine residues can be used. Chemical coupling can be performed by chemical methods well known to those skilled in the art, including but not limited to substitution, addition or cycloaddition or oxidation chemistry (e.g., disulfide formation).

[0108] Molecules for purification / detection. In some embodiments, additional amino acids may be extended at the N-terminus or C-terminus or both of the FOLR1 binding protein. Additional sequences may include, for example, sequences introduced for purification or detection. In one embodiment, the additional amino acid sequences include one or more peptide sequences that confer affinity for certain chromatographic column materials. Typical examples of such sequences include, but are not limited to, Strep-tags, oligohistidine tags, glutathione S-transferase, maltose binding protein, inteins, intein fragments, or the albumin binding domain of protein G. SEQ ID NO: 45 provides an example of a FOLR1 binding protein with a Strep-tag.

[0109] Medical Uses. Various embodiments relate to FOLR1 binding proteins as disclosed herein for use in medicine. In one embodiment, the FOLR1 binding protein is used in medicine to diagnose or treat cancer associated with FOLR1 expression. The FOLR1 binding protein as disclosed herein allows for the selective diagnosis and treatment of FOLR1 associated cancer cells or cancer tissues. FOLR1 is known to be upregulated in tumor cells, which may lead to uncontrolled growth of tumor cells and the formation of metastases. Examples of FOLR1 associated tumors are ovarian cancer, endometrial cancer, brain cancer, lung cancer, kidney cancer, head and neck cancer, breast cancer, gastric cancer and colorectal cancer.

[0110] One embodiment is a method of diagnosing (including monitoring) a subject with a FOLR1-associated cancer, the diagnostic (monitoring) method comprising administering to the subject a FOLR1-binding protein as described, optionally conjugated to a radioactive molecule. In various embodiments, a FOLR1-binding protein as disclosed herein can be used to diagnose a FOLR1-associated cancer, optionally wherein the FOLR1-binding protein is conjugated to a radioactive molecule. In some embodiments, a FOLR1-binding protein as disclosed herein can be used as a biomarker. In some embodiments, an imaging method using a labeled (e.g., radiolabeled or fluorescently labeled) FOLR1-binding protein can be used to visualize FOLR1 on a specific tissue or cell, for example, to assess the presence of FOLR1-associated tumor cells, the distribution of FOLR1-associated tumors, the recurrence of FOLR1-associated tumors, and / or to assess a patient's response to treatment.

[0111] One embodiment is a method for treating a subject with a FOLR1-associated cancer, the method comprising administering to the subject a FOLR1-specific binding protein, which is optionally conjugated to a radioactive molecule and / or a cytotoxic agent. In various embodiments, the FOLR1 binding protein as disclosed herein can be used to treat FOLR1-associated cancer, optionally wherein the FOLR1 binding protein is conjugated to a cytotoxic agent and / or a radioactive molecule and / or expressed on the surface of a target-specific CarT cell. Some embodiments relate to the use of a FOLR1 binding protein labeled with a suitable radioisotope or cytotoxic compound in the treatment of FOLR1-associated tumor cells, in particular for controlling or killing FOLR1-associated tumor cells (e.g., malignant cells). In one embodiment, a therapeutic dose of radiation is selectively delivered to FOLR1-associated tumor cells but not to normal cells.

[0112] FOLR1 specific binding proteins as disclosed herein can be used for tumor targeted therapy or diagnosis without recognizing inflammatory tissue. Due to the specific binding to FOLR1 (but not to FOLR2), FOLR1 tumor targeted therapy may be beneficial, for example, in autoimmune patients or patients with inflammation.

[0113] Compositions. Various embodiments relate to compositions comprising a FOLR1 binding protein as disclosed herein. Compositions comprising a FOLR1 binding protein as defined above are used in medicine, preferably for the diagnosis or treatment of various FOLR1-related cancer tumors, such as ovarian cancer, endometrial cancer, brain cancer, lung cancer, kidney cancer, head and neck cancer, breast cancer, gastric cancer, colorectal cancer, etc., preferably ovarian cancer, breast cancer and lung cancer. Compositions comprising a FOLR1 binding protein as described above can be used in clinical applications for diagnostic and therapeutic purposes. In particular, compositions comprising a FOLR1 binding protein as described above can be used in clinical applications for imaging, monitoring, eliminating or inactivating pathological cells expressing FOLR1.

[0114] Various embodiments relate to a diagnostic composition for diagnosing FOLR1-related cancers, comprising a FOLR1 binding protein as defined herein and a diagnostically acceptable carrier and / or diluent. These include, for example, but not limited to, stabilizers, surfactants, salts, buffers, colorants, etc. The composition may be in the form of a liquid formulation, a lyophilized formulation, a granule, an emulsion, or a liposomal formulation.

[0115] As mentioned above, a diagnostic composition comprising a FOLR1 binding protein as described herein can be used to diagnose a FOLR1-associated cancer.

[0116] Various embodiments relate to pharmaceutical (e.g., therapeutic) compositions for treating a disease, comprising a FOLR1 binding protein as disclosed herein, and a pharmaceutically (e.g., therapeutically) acceptable carrier and / or diluent. The pharmaceutical (e.g., therapeutic) composition may optionally comprise other adjuvants and excipients known per se. These include, but are not limited to, stabilizers, surfactants, salts, buffers, colorants, and the like.

[0117] As mentioned above, pharmaceutical compositions comprising a FOLR1 binding protein as defined herein may be used to treat a disease.

[0118] The composition contains an effective dose of a FOLR1 binding protein as defined herein. The amount of protein administered depends on the organism, the type of disease, the age and weight of the patient and other factors known per se. According to the galenic formulation, these compositions can be administered parenterally by injection or infusion, systemically, intraperitoneally, intramuscularly, subcutaneously, transdermally or by other customary application methods.

[0119] The composition can be in the form of a liquid preparation, a lyophilized preparation, a cream, a topical lotion, an aerosol, a powder, a granule, an emulsion or a liposome preparation. The type of preparation depends 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.

[0120] The various components of the composition may be packaged into a kit together with instructions for use.

[0121] Preparation of FOLR1 binding proteins. FOLR1 binding proteins as described herein can be prepared by any of a number of conventional and well-known techniques, such as common organic synthesis strategies, solid phase assisted synthesis techniques, fragment ligation techniques, or by commercially available automated synthesizers. On the other hand, they can also be prepared by conventional recombinant techniques alone or in combination with conventional synthetic techniques. In addition, they can also be prepared by cell-free in vitro transcription / translation.

[0122] Various embodiments relate to polynucleotides encoding FOLR1 binding proteins as disclosed herein. One embodiment further provides an expression vector comprising the polynucleotide, and a host cell comprising the isolated polynucleotide or expression vector.

[0123] Various embodiments relate to a method of producing a FOLR1 binding protein as disclosed herein, the method comprising culturing a host cell under suitable conditions allowing expression of the FOLR1 binding protein, and optionally isolating the FOLR1 binding protein.

[0124] For example, one or more polynucleotides encoding FOLR1 binding proteins can be expressed in a suitable host, and the resulting FOLR1 binding proteins can be isolated. The host cell comprises the nucleic acid molecule or vector. Suitable host cells include prokaryotes or eukaryotes. A vector refers to any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) that can be used to transfer protein coding information into a host cell. A variety of cell culture systems can also be used to express recombinant proteins, such as, but not limited to, mammals, yeast, plants, or insects. 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 purposes can be performed on any scale (from small-volume shake flasks to large fermenters) using techniques well known to any person skilled in the art.

[0125] One embodiment relates to a method for preparing a binding protein as described above in detail, the method comprising the following steps: (a) preparing a nucleic acid encoding a FOLR1 binding protein as 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 capable of expressing the FOLR1 binding protein, thereby producing the FOLR1 binding protein as defined herein; (f) optionally isolating the FOLR1 binding protein produced in step (e); and (g) optionally conjugating the FOLR1 binding protein to other functional parts as defined herein.

[0126] Generally, purified FOLR1 binding protein can be isolated from the culture mixture using conventional methods and techniques well known in the art, such as centrifugation, precipitation, flocculation, various embodiments of chromatography, filtration, dialysis, concentration, and combinations thereof, etc. Chromatography is well known in the art and includes, but is not limited to, ion exchange chromatography, gel filtration chromatography (size exclusion chromatography), hydrophobic interaction chromatography, or affinity chromatography.

[0127] To simplify purification, the FOLR1 binding protein can be fused to other peptide sequences that have increased affinity for the separation material. Preferably, a fusion is selected that does not have a deleterious effect on the function of the FOLR1 binding protein, or that can be separated after purification due to the introduction of a specific protease cleavage site. Such methods are also known to those skilled in the art.

[0128] Example

[0129] The following examples are provided to further illustrate the present invention. The present invention is particularly exemplified by specific modifications to ubiquitin (SEQ ID NO: 46) that result in binding to FOLR1. However, the present invention is not limited thereto, and the following examples merely demonstrate the utility of the present invention based on the above description.

[0130] Example 1. Identification of FOLR1 binding proteins

[0131] Library construction and library cloning.

[0132] Libraries containing randomized amino acid positions were synthesized in-house by randomized oligonucleotides generated from synthetic trinucleotide phosphoramidites (ELLA Biotech) to achieve a well-balanced amino acid distribution while excluding cysteine ​​and other amino acid residues at randomized positions.

[0133] Sequence of ubiquitin (SEQ ID NO: 46):

[0134] MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRAA

[0135] SEQ ID NO: 46 is randomized at amino acid positions 6, 8, 9, 10, 12, 42, 44, 46, 62, 63, 64, 65, 66, 68, 70, 72. The corresponding cDNA library was amplified by PCR and ligated to a modified pCD87SA phagemid (referred to herein as pCD12) using standard methods known to the skilled person. The pCD12 phagemid contains a modified torA leader sequence (missing the amino acid sequence QPAMA) to enable protein processing, while no amino acids are added at the N-terminus. An aliquot of the ligation mixture was used for electroporation of E. coli ER2738 (Lucigen). Unless otherwise stated, established recombinant genetic methods were used.

[0136] Example 2. Identification of FOLR1 binding proteins

[0137] Target. The DNA sequence encoding human FOLR1 (uniprot accession number P15328; residues 25-234) was genetically fused to the Fc fragment of human IgG1 (with a His-tag attached to the C-terminus) or to the Avi-tag (with a His-tag attached to the C-terminus). The full-length cDNA using human codons was provided by OriGene Technologies, cloned into the mammalian expression vector pCEP4, and expressed in mammalian Expi293F cells at different scales from 400 ml to 2 L in shake flasks. Expression cultures with the pCEP4-FOLR1-Avi-His construct were then co-transfected with pCEP4-BirA to induce site-specific biotinylation of the target protein. Expression cultures were analyzed by SDS-PAGE and immunoblot analysis with antibodies against FOLR1, the Fc part of human IgG1, or biotin.

[0138] The cell culture supernatant expressed by FOLR1-Fc-His was centrifuged and filtered for affinity chromatography on a HisTrap excel1mL column (GE Healthcare). The target protein was eluted by injecting a buffer containing imidazole and applied to a Superdex 200XK 16 / 600 gel filtration column. The cell culture supernatant expressed by FOLR1-Avi-His was centrifuged, filtered and desalted to be applied to affinity chromatography on a streptavidin mutant protein matrix (Roche). The target protein was eluted by injecting a buffer containing biotin and applied to a Superdex 200 10 / 300 gel filtration column. The purity of the target protein recovered was analyzed and confirmed by SDS-PAGE and SE-HPLC. The biological activity of the target protein to the substrate folic acid was confirmed by concentration-dependent ELISA.

[0139] Preliminary screening was performed by TAT phage display. Using phage display as a screening system, a natural library was enriched for FOLR1. After transforming competent bacteria ER2738 cells (Lucigene) with phagemid pCD12 carrying the library, phage amplification and purification were performed using standard methods known to technicians. The target protein was fixed on magnetic beads for screening. The target protein fused to IgG1-Fc was fixed on Protein A or Protein G. The site-specific biotinylated target protein fused to the Avi-tag or the target protein fused to IgG1-Fc (additionally randomly biotinylated) was immobilized on Streptavidin or Neutravidin SpeedBeads TM On. The FOLR1 concentration during phage incubation was reduced from 200 nM (first round) to 100 nM (second round), to 50 nM (third round), and to 25 nM (fourth round). In the first screening, prescreening was performed using the Fc fragment of biotinylated IgG1, starting from the second round. In the second screening, phage particles were preincubated with mouse serum for the last three rounds, 3 h in the second round, 23 h in the third and fourth rounds, and prescreened with the Fc fragment of biotinylated IgG1. The FOLR1-phage complex was magnetically separated from the supernatant and washed several times. Phages bound to FOLR1 were eluted with trypsin. To identify the target-specific phage library, the eluted and re-amplified phages from each round of screening were analyzed by phage library ELISA. The wells of a culture medium-bound 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).

[0140] The phage pools binding to the target were cloned into an expression vector. The screening pools showing specific binding to FOLR1 in the phage pool ELISA were amplified by PCR according to methods known in the art, cut with appropriate restriction nucleases, and ligated into a derivative of the expression vector pET-28a (Merck, Germany) containing Strep-Tag II (IBA GmbH).

[0141] Single colony hit analysis. After transformation of BL21 (DE3) cells (Merck, Germany), single colonies with kanamycin resistance were grown. Expression of FOLR1 binding protein was achieved by cultivation in 384-well plates (Greiner Bio-One) using autoinduction medium (Studier, 2005, Protein Expr. Purif. 41 (1): 207-234). Cells were harvested and then chemically lysed or enzymatically lysed by BugBuster reagent (Novagen) and mechanically lysed by freeze / thaw cycles. After centrifugation, the resulting supernatant was screened by ELISA using targets immobilized on High Bind 384 ELISA microtiter plates (Greiner Bio-One). Strep- Proteins bound to FOLR1 were detected by combining HRP conjugate (IBA GmbH) with TMB-Plus substrate (Biotrend, Germany). The reaction was terminated by adding 0.2 M H2SO4 solution and measured in a microplate reader at 450 nm versus 620 nm.

[0142] Construction of mature library. To mature the selected variants, error-prone PCR was performed using the dNTP analogs dPTP and 8-oxo-dGTP (Jena Bioscience). The cDNA of the mature library obtained was ligated with pCD12 as described above and tested for transformation in E. coli SS320.

[0143] Maturation screening and analysis. In order to mature affinity, two rounds of panning were performed. The first maturation screening was performed with random biotinylated target proteins fused to IgG1-Fc at concentrations of 80nM and 8nM, respectively, in the first and second rounds. For these two rounds, the phage particles were pre-incubated with mouse serum for 3 hours and 23 hours, respectively, in the first and second rounds, and pre-screened with the Fc fragment of biotinylated IgG1. The second maturation screening was performed with a site-directed biotinylated target protein fused to an Avi-tag at a concentration of 50nM and 1nM, respectively, in the first and second rounds. For these two rounds, the phage particles were pre-incubated with mouse serum for 23 hours in the first and second rounds. In order to analyze the mature screening library for specific target binding, a phage library ELISA was performed, and then the positive library was cloned into the expression vector pET-28a, and the hit ELISA was performed as described above.

[0144] Example 3. Expression and purification of FOLR1 binding protein

[0145] The FOLR1 binding proteins were cloned into expression vectors using standard methods known to the skilled artisan, 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, the proteins were purified using System and Superdex TM Size exclusion chromatography (SE HPLC or SEC) was performed on a 200 HiLoad 16 / 600 column (GE Healthcare). The volume of the column was 120 ml and balanced with 2CV. The sample was subjected to purification buffer at a flow rate of 1 ml / min. Fraction collection began when the signal intensity reached 10 mAU. After SDS-PAGE analysis, the positive fractions were combined and their protein concentrations were measured.

[0146] Further analysis included SDS-PAGE, SE-HPLC, and RP-HPLC. Protein concentration was determined by absorbance measurement at 280 nm using the molar absorption coefficient. Proteins were purified using a Dionex HPLC system and a Vydac 214MS54 C4 (4.6×250 mm, 5 μm, ) column (GE Healthcare) for RP chromatography (RP HPLC). For example, Affilin 202521 (SEQ ID NO: 30 with a C-terminal conjugated sequence "SAC") has a purity of about 100% according to SE-HPLC and RP-HLCP, respectively.

[0147] Example 4. FOLR1 binding protein is stable at high temperature

[0148] The thermal stability of FOLR1-specific proteins was determined by differential scanning fluorimetry (DSF). Each probe was transferred to a MicroAmp Optical 384-well plate at a concentration of 0.1 μg / μL, and SYPRO Orange dye was added at appropriate dilutions. The temperature ramp was set from 25°C to 95°C with a heating rate of 1°C per minute (V2A-7 Applied Biosystems). Fluorescence was continuously measured at an excitation wavelength of 520nm and an emission wavelength of 623nm (V2A-7, Applied Biosystems). The midpoint of the transition (Tm, melting point) of thermal unfolding was determined and is shown in Table 3 for selected FOLR1-binding proteins.

[0149] Table 3. Temperature stability of FOLR1 binding proteins

[0150] Affilin SEQ ID DSF,℃ 197556a 1 80 199490 30 85 199489 31 79 197251 3 80 199484 32 71 199483 33 62 197525 5 82 199487 35 64 199488 34 70 187731 6 87 199477 37 79 199478 36 85 197340 8 72 196920 9 73 196964 12 74 196868 14 79 197383 17 80 196934 18 81 199479 38 65 199480 39 70 199481 40 71 199482 41 72 197126 19 79 199485 43 76 199486 42 72 196888 21 76 181919 26 63 187803 29 62

[0151] Example 5. Analysis of FOLR1 binding proteins (surface plasmon resonance, SPR)

[0152] The CM5 sensor chip (GE Healthcare) was balanced with SPR running buffer. A mixture of EDC and NHS was activated by activating the carboxyl groups exposed to the surface to produce reactive ester groups. 700-1500RU FOLR1 (on the ligand) was fixed on the flow cell. A flow cell without a ligand was used as a reference. Ethanolamine was injected after ligand immobilization to block unreacted NHS groups. After ligand binding, protein analytes accumulated on the surface, thereby increasing the refractive index. This change in refractive index was measured in real time and plotted as a response or resonance unit (RU) versus time. The analyte was diluted continuously onto the chip at a flow rate of 30 μl / min. Association was performed for 30 seconds and dissociation for 60 seconds. After each run, the chip surface was regenerated with 30 μl regeneration buffer and balanced with running buffer. The control sample was applied to the matrix at a flow rate of 30 μl / min, while association was performed for 60 seconds and dissociation was performed for 120 seconds. Regeneration and rebalancing were performed as described above. Binding studies were performed by using Biacore 3000 (GE Healthcare); data evaluation was performed by BIAevaluation 3.0 software provided by the manufacturer using the Langmuir 1:1 model (RI=0).

[0153] Figure 2 , Figure 3 and Table 4 show the binding affinity of FOLR1 binding proteins to FOLR1-Fc.

[0154] Table 4. Binding affinity (K) of FOLR1 binding proteins to FOLR1-Fc determined by SPR assay (Biacore) D ).

[0155] Affilin SEQ ID Biacore, M 197556a 1 4.11E-10 199490 30 1.19E-10 199489 31 2.06E-10 199487 35 1.51E-10 199488 34 8.49E-11 199477 37 1.87E-11 199478 36 2.07E-10 199479 38 4.74E-11 199480 39 3.09E-11 199481 40 3.12E-11 199482 41 2.61E-11 197126 19 2.15E-09 189864 24 2.91E-11 189872 25 4.17E-12 181919 26 1.68E-08 189853 27 7.42E-12 187803 29 3.88E-07

[0156] Example 6. Functional characterization: specific binding to FOLR1 expressed on the cell surface, but not to FOLR2 (flow cytometry)

[0157] Flow cytometry was used to analyze the specific interaction of FOLR1 binding proteins with surface-exposed FOLR1 but not with FOLR2. Transfected HEK293-FOLR1-cells, HEK293-FOLR2-cells and empty vector control HEK293-pEntry-cells were trypsinized and resuspended in medium containing FCS, washed and stained in pre-chilled FACS blocking buffer. 1×10 6Cells were plated at a concentration of 10 cells / ml for cell staining and each cell line was added to a 96-well plate (Greiner) in triplicate at 100 μl / well. 50 nM of Affilin protein or monoclonal anti-human FOLR1 antibody (clone LK26; BioLegend, 908301) as a positive control or anti-human FOLR2 antibody (clone EM35; Sysmex, BD029864) was added to FOLR-overexpressing cells and control cells. After 45 minutes, the supernatant was removed and rabbit anti-Strep-tag antibody (GenScript; A00626) was added at 100 μl / well (diluted 1:300 in FACS blocking buffer). Anti-FOLR1 antibody and anti-FOLR2 antibody were detected with anti-mouse-IgG-Alexa 488 (Invitrogen; A-10680) diluted 1:1000. After removal of the primary antibody, goat anti-rabbit IgG Alexa Fluor 488 antibody (Invitrogen; A11008) was applied at a dilution of 1: 1000. Flow cytometry measurements were performed on a Guava easyCyte 5HT apparatus (Merck-Millipore) with an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

[0158] All FOLR1 binding proteins showed specific binding to HEK293-FOLR1 overexpressing cells, but no binding to FOLR1 negative cell lines (e.g. HEK293-pEntry). All FOLR1 binding proteins tested for binding to FOLR2 showed no binding to FOLR2. Controls: Anti-FOLR1 antibody as positive control; Ubiquitin as negative control (HEK293-FOLR1 cells).

[0159] Example 7. Labeling of fusion protein with DOTA

[0160] FOLR1 binding protein was incubated with 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) in 50 mM HEPES, 150 mM NaCl, 5 mM EDTA pH 7.0 at room temperature for 3 h. To reduce metal ions that may interact with DOTA molecules, all columns and The device (GE Healthcare) was incubated with 0.1 M EDTA solution for 30 minutes. For the preparation of the solution, only metal-free or metal-reduced components were used. After incubation, the samples were separated from unbound DOTA molecules by gel filtration (Superdex S200, GE Healthcare) in 100 mM sodium acetate pH 5.0-5.8. The protein samples were also incubated with 5 mM iron (II) chloride at room temperature for 1 hour to demonstrate the effectiveness of coupling the DOTA molecules to the radioisotope. After incubation, unbound iron was removed using a HiTrap desalting column (GE Healthcare). The degree of labeling was determined by MALDI-TOF analysis. In further experiments, FOLR1 protein was coupled to DOTA and labeled with Lu 3+ In 100 nM sodium acetate at pH 5.8, Lu 3+ The loading amount was c = 0.2 mg / ml and the labeling temperature was up to 70° C. The molecules were analyzed by CD spectroscopy at 207 nm.

[0161] Example 8. Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOF)

[0162] Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOF MS) was performed as follows: FOLR1 binding protein was purified and concentrated using C18-P10-ZipTips (Millipore; catalog number ZTC18S096). The tip was washed with 0.1% (v / v) trifluoroacid (TFA) in water and eluted with 50% (v / v) acetonitrile / 0.1% TFA. The sample was treated with 2% (v / v) TFA in water and embedded in 2,5-dihydroxyacetophenone (2,5-dihydroxyacetophenone, DHAP) matrix (Bruker, catalog number 8231829). The mass of FOLR1 binding protein was measured on an autoflexTM speed mass spectrometer (Bruker). Protein calibration standards (Bruker, part number 8206355 and part number 8207234) were used to tune the autoflex speed mass spectrometer.

[0163] The FOLR1 binding protein with and without DOTA labeling was analyzed by MALDI-TOF mass spectrometry and their peaks were compared. MALDI-TOF analysis showed that the DOTA molecules labeled on the FOLR1 binding protein can be used to couple with the iron (II) chloride molecules.

[0164] Although after labeling FOLR1 binding proteins, K D There were slight changes, but the labeling did not significantly affect the affinity of FOLR1 binding protein to the target. The results are summarized in Tables 5 and 6.

[0165] Example 9. Serum stability of FOLR1 binding protein (flow cytometry)

[0166] The stability of the FOLR1 binding proteins in the presence of serum was analyzed. The binding proteins were incubated at 37°C for 0 h or 24 h with a dilution series starting from 1 μM (Affilin-199479, Affilin-199487, Affilin-197556a, Affilin-199488, Affilin-199488-Dota, Affilin-199490 and Affilin-199490-Dota) and 200 nM (Affilin-199489) in 100% mouse serum. 100 μl of the Affilin-serum solution was used to analyze serum stability on HEK293-FOLR1-cells by FACS as described above (Example 6). FACS analysis confirmed that the Affilin protein binds to FOLR1 even after 24 hours of incubation in mouse serum (see Table 5). The FOLR1 protein was further tested (results not shown) and demonstrated binding to FOLR1 even in the presence of serum.

[0167] Table 5. Binding of FOLR1 binding proteins in the presence of serum (flow cytometry)

[0168]

[0169] Example 10. Serum stability of FOLR1 binding protein (ELISA)

[0170] High binding plates (Greiner, 781061) were fixed with 0.1 μg / ml-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 Lu 3+A dilution series of lutetium-labeled Dota (SEQ ID NO: 30) was incubated in 100% mouse serum at 37°C overnight. The ELISA plate was washed with 1× PBS and blocked with 3% BSA / 0.5% Tween / PBS for 2 hours at room temperature. The dilution series was incubated in the presence of serum for 0 hours or 24 hours and then incubated on the ELISA plate for 1 hour at room temperature. After washing with PBST, the wells were incubated with biotinylated anti-ubiquitin antibody (1:1000) for 1 hour at room temperature. Binding was visualized with streptavidin-HRP (1:10.000). FOLR1 binding protein did not show K after 24h serum incubation. D Therefore, ELISA analysis confirmed that the high affinity binding of Affilin protein to FOLR1 remained unchanged even after 24 hours of incubation in mouse serum (see Table 6).

[0171] Table 6. Binding of FOLR1 binding proteins in the presence of serum (ELISA)

[0172]

[0173] Sequence Listing <110> Navig Protein Ltd. <120> Novel FOLR1-specific binding proteins for cancer diagnosis and therapy <130> FSP1V211426ZX <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> Dimer of 199489 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> Homodimer of 199484 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> Homodimer of 199483 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> 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> Homodimer of 199487 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> Homodimer of 199478 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> Homodimer of SEQ ID NO: 6, 199477 <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> 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> Homodimer of 199480 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> Homodimer of 199481 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> Homodimer of SEQ ID NO 18, 199482 <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> 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> Homodimer of 199485 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 Homodimer of SEQ ID NO: 2 (199490) with C-terminal SAC but without Strep tag <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 Strep tags <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> Connectors <400> 67 Gly Gly Ser Gly 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. A folate receptor alpha (FOLR1) binding protein consisting of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 30 or SEQ ID NO:

31.

2. The FOLR1 binding protein according to claim 1, further comprising a coupling domain of three amino acids "SAC" for chemical moiety coupling.

3. The FOLR1 binding protein according to claim 2, wherein the chemical moiety is selected from any one of a chelator, a drug, a toxin and a dye.

4. The FOLR1 binding protein according to claim 1, further comprising at least one diagnostically active portion, which is optionally selected from a radionuclide, a fluorescent protein, a photosensitizer, a dye or an enzyme, or any combination thereof.

5. The FOLR1 binding protein according to claim 1, further comprising at least one therapeutically active moiety, which is optionally selected from a monoclonal antibody, a radionuclide, a cytotoxic compound, a cytokine, a chemokine, an enzyme, or any combination thereof.

6. The FOLR1 binding protein according to any one of claims 1 to 5, further comprising at least one pharmacokinetics-modulating moiety, optionally selected from polyethylene glycol, human serum albumin, the albumin binding domain of protein G, an immunoglobulin or an immunoglobulin fragment, or a polysaccharide.

7. The FOLR1 binding protein according to any one of claims 1 to 5, for use in the diagnosis or treatment of FOLR1-positive tumors.

8. The FOLR1 binding protein according to claim 7, for use in imaging of FOLR1 positive tumors and radiotherapy treatment of FOLR1 positive tumors.

9. A composition comprising the FOLR1 binding protein according to any one of claims 1 to 6, for use in the diagnosis or treatment of FOLR1-positive tumors.

10. The composition according to claim 9, for use in imaging of FOLR1 positive tumors and in radiotherapy treatment of FOLR1 positive tumors.

11. A method for producing the FOLR1 binding protein according to any one of claims 1 to 6, comprising the following steps: a) culturing a host cell comprising a nucleic acid sequence encoding the FOLR1 binding protein according to claim 1 under conditions suitable for obtaining the FOLR1 binding protein, and b) isolating the produced FOLR1 binding protein.

12. A composition comprising the folate receptor alpha (FOLR1) binding protein according to any one of claims 1 to 6.

Images