New PSMA-specific binding proteins for cancer diagnosis and therapy

Abstract

The present invention relates to novel binding proteins that are specific for prostate specific membrane antigen (PSMA). The present invention further relates to PSMA binding proteins that further comprise a diagnostically or therapeutically active component. Further aspects of the present invention encompass the use of these PSMA binding proteins in medicine, such as in the diagnosis and treatment of cancers associated with PSMA expression.

Description

Technical Field

[0001] The present invention relates to novel binding proteins that are specific for prostate specific membrane antigen (PSMA). The invention further relates to PSMA binding proteins that further comprise diagnostically or therapeutically active components. Further aspects of the invention encompass the use of these PSMA binding proteins in medicine, such as in the diagnosis and treatment of cancers associated with PSMA expression. Background Art

[0002] Prostate-specific membrane antigen (PSMA) is expressed in tumors, especially in prostate cancer. Overexpression of PSMA has also been shown in the new vasculature of other solid tumors such as breast cancer, kidney cancer, lung cancer, ovarian cancer, colorectal cancer, bladder cancer, gastric cancer or brain cancer (e.g. glioblastoma) and multiple myeloma. It increases with the progression of cancer, especially at high levels in metastatic disease. The three-domain glycoprotein (extracellular, transmembrane and short intracellular domain) mediates tumor nutrition and cell proliferation. Targeted PSMA-specific monoclonal antibodies have been developed for the diagnosis and treatment of cancer, especially prostate cancer. To date, the only agent approved for diagnostic imaging and staining of newly diagnosed and recurrent prostate cancer patients is the radiolabeled mouse monoclonal antibody Capromab Pendetide. However, due to binding to the intracellular epitope of PSMA, the antibody has no therapeutic benefit. A second generation of monoclonal antibodies that bind to the extracellular epitope of PSMA has been developed, such as the mouse monoclonal antibody J591. J591 has been tested for in vivo imaging of progressive solid tumors or for capturing metastatic circulating tumor cells. Its therapeutic use is limited by toxic side effects and a short serum half-life.

[0003] PSMA-specific monoclonal antibodies have further major disadvantages as agents for diagnostic and therapeutic methods, such as for PSMA radioimmunotherapy and PSMA radioimaging. One is the complex molecular structure and correspondingly complex production process. Another is their large size, resulting in poor tissue penetration in vivo. Combining long circulation time antibody-based compounds for imaging applications may result in poor contrast due to high background signals.

[0004] Additionally, the frequent development of resistance to initially effective treatments constitutes a need for additional and improved therapeutics for prostate cancer and other cancers that overexpress PSMA.

[0005] Existing options fail to adequately address the diagnosis and treatment of PSMA-associated cancers, and therefore, many patients do not fully benefit from current strategies. Undoubtedly, new strategies are urgently needed to diagnose and treat PSMA-associated tumors.

[0006] One object of the present invention is to provide molecules for specifically targeting PSMA to allow targeted diagnostic and therapeutic options, including detection of PSMA-positive tumors. Targeting this tumor-associated protein may provide benefits to patients with unmet needs for new diagnostic and therapeutic approaches. Since the distribution of PSMA in normal tissues is low and restricted, targeting PSMA means potentially non-toxic diagnostic and therapeutic methods. Therefore, binding proteins specific for PSMA may be able to provide effective medical options for cancer and ultimately improve the quality of life of patients.

[0007] The present invention provides novel PSMA-binding molecules for use in new and improved strategies in the diagnosis and treatment of PSMA-associated cancers.

[0008] The above objects and advantages are achieved by the subject matter of the appended claims. The present invention meets the above needs by providing examples of PSMA binding proteins. The above summary does not necessarily describe all problems solved by the present invention. Summary of the invention

[0009] The present disclosure provides the following [1] to

[15] , but is not specifically limited thereto:

[0010] [1] A prostate-specific membrane antigen (PSMA) binding protein comprising at least one amino acid binding motif selected from GFAHR, GWAHR, SFAHR, SYAHR, GFAHL, WTTTF, WTPSI, WTETI, GHEYL, GDGDV, KHNTW, VAYRP, or WWNPN, or a motif having 80% identity thereto.

[0011] [2] The PSMA-binding protein according to [1], comprising one or more ubiquitin mutant proteins based on the ubiquitin according to SEQ ID NO: 1.

[0012] [3] The PSMA-binding protein according to [2], wherein the amino acid residues of the binding motif correspond to positions 62, 63, 64, 65, 66 of ubiquitin according to SEQ ID NO: 1, preferably the PSMA-binding protein comprises one or more ubiquitin mutants based on ubiquitin according to SEQ ID NO: 1 and contains the amino acid binding motif GFAHR or a motif having 80% identity thereto at the amino acid residues corresponding to positions 62, 63, 64, 65, 66 of ubiquitin according to SEQ ID NO: 1.

[0013] [4] The PSMA-binding protein according to [3], wherein additionally, the amino acids at positions 6 and 8 corresponding to ubiquitin of SEQ ID NO: 1 are substituted.

[0014] [5] The PSMA-binding protein according to [3], wherein additionally, the amino acids at positions 42, 44, 68, 70 and 72 corresponding to ubiquitin of SEQ ID NO: 1 are substituted.

[0015] [6] The PSMA-binding protein according to [3], wherein additionally, the amino acids corresponding to positions 6, 8, 9, 10, 12 and 46 of ubiquitin (SEQ ID NO: 1) are substituted, or wherein additionally, up to 6 amino acids are inserted between the amino acids corresponding to positions 9 and 10 of ubiquitin (SEQ ID NO: 1), or wherein additionally, the amino acids corresponding to positions 11, 33, 51, 48 and 74 of ubiquitin (SEQ ID NO: 1) are substituted.

[0016] [7] The PSMA-binding protein according to any one of [1]-[6], wherein the PSMA-binding protein is a multimer comprising a plurality of PSMA-binding proteins according to any one of [1]-[6], preferably a dimer of the PSMA-binding protein according to any one of [1]-[6].

[0017] [8] The PSMA-binding protein according to any one of [1] to [7], comprising or consisting of an amino acid sequence selected from the group of SEQ ID NOs: 3-55 or from amino acid sequences having at least 90% identity thereto.

[0018] [9] The PSMA-binding protein according to any one of [1] to [8], wherein the specific binding affinity of the PSMA-binding protein to the extracellular domain of PSMA is 500 nM or less.

[0019]

[10] The PSMA-binding protein according to any one of [1] to [9], further comprising one or more coupling sites for coupling a chemical moiety, preferably wherein the chemical moiety is selected from any of chelators, drugs, toxins, dyes and small molecules.

[0020]

[11] The PSMA-binding protein according to any one of [1] to

[10] , 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.

[0021]

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

[11] , further comprising at least one therapeutically active portion, which is optionally selected from a monoclonal antibody or a fragment thereof, a binding protein, a radionuclide, a cytotoxic compound, a cytokine, a chemokine, an enzyme or a derivative thereof, or any combination thereof.

[0022]

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

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

[0023]

[14] The PSMA-binding protein according to any one of [1] to

[13] , for use in diagnosing or treating PSMA-associated tumors, preferably for tumor imaging and radiotherapy treatment of PSMA-associated tumors.

[0024]

[15] A composition comprising a PSMA-binding protein according to any one of [1] to

[14] , for use in medicine, preferably for diagnosis or treatment of PSMA-associated tumors, preferably for tumor imaging and radiotherapy treatment of PSMA-associated tumors.

[0025]

[16] A method for producing a PSMA-binding protein according to any one of [1] to

[15] , comprising the steps of: a) culturing host cells under conditions suitable for obtaining the PSMA-binding protein, and b) isolating the produced PSMA-binding protein.

[0026] This summary does not necessarily describe all features of the invention. Other embodiments will become apparent from a review of the subsequent detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The attached figure shows:

[0028] Figure 1-3 Examples of PSMA-binding proteins are shown that have characteristic motifs in the amino acids corresponding to positions 62-66 of SEQ ID NO: 1 (ubiquitin). Figure 2 The numbers in the top row: 9a, 9b, 9c, 9d, 9e, 9f) corresponding amino acids show the structural features of such ubiquitin mutant proteins. Functional features are shown as affinity for PSMA determined by SPR (Biacore), thermal stability determined by DSF, and cell binding as described in the Examples.

[0029] Figure 1 PSMA binding proteins with the 5-amino acid motif WWNPN at positions 62-66 are shown. Amino acids corresponding to ubiquitin positions 6, 8, 9, 10, 12, 42, 44, 46, 62-66, 68, 70, 72 are substituted. In some binding proteins, further substitutions were found as shown in the column Further Substitutions.

[0030] Figure 2 PSMA binding proteins with the 5-amino acid motif KHNTW corresponding to positions 62-66 are shown. Amino acids corresponding to ubiquitin positions 42, 44, 62-66, 68, 70, 72 are substituted, and 6 amino acids are inserted between positions 9 and 10 of ubiquitin. In some binding proteins, further substitutions were found, as shown in the column "Further substitutions".

[0031] Figure 3 PSMA binding proteins with 5-amino acid motifs GFAHR or GWAHR (or similar) and / or 5-amino acids WTTTF, WTPSI, WTPTI or GDGDV are shown. Amino acids corresponding to ubiquitin positions 6, 8, 62-66 are substituted; two ubiquitin muteins are linked. In some PSMA binding proteins, further substitutions were found, for example, at ubiquitin positions 11, 33, 48, 51, 74, as shown in the column "Further Substitutions".

[0032] Figure 4 Functional characterization of PSMA binding proteins determined by flow cytometry. The histogram confirms that SEQ ID NO: 4 (referred to as 191871) binds to PSMA-overexpressing HEK293-cells or PSMA-expressing LNCaP-cells (grey peak); no binding to control cell lines HEK293-pEntry or PC3. Unmodified ubiquitin (bis-ubi; white peak) shows no binding to the cells. DETAILED DESCRIPTION

[0033] The present inventors have developed a solution to meet the strong need in the art to expand medical options for cancer diagnosis and treatment by providing novel PSMA-binding proteins. The functional characteristic of the PSMA-specific proteins as defined herein is that they have a high specific affinity for PSMA. In particular, the present invention provides ubiquitin mutant proteins (also known as The PSMA-binding proteins described herein provide a molecular format that has favorable physicochemical properties, high-level expression in bacteria, and allows the use of simple production methods. The novel PSMA-binding proteins may broaden the hitherto unmet medical strategies for the diagnosis and treatment of PSMA-related cancers. In particular, the PSMA-binding proteins may be used for diagnostic or imaging purposes, such as for the diagnosis or imaging of the presence of PSMA-expressing tumor cells, and for the treatment of PSMA-expressing tumors with radiotherapy.

[0034] Before describing the present invention in more detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as they may vary. It should also be understood that the terms used herein are only used for the purpose of describing specific aspects and embodiments, and are not intended to limit the scope of the present invention as reflected by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. This includes technicians working in the field of protein engineering and purification, as well as technicians working in the field of developing new target-specific binding molecules for technical applications and treatment and diagnosis.

[0035] Preferably, as in A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland), and the terms used herein are defined.

[0036] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" should be understood to imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps. The term "comprise(s)" or "comprising" may encompass limitations to "consists of" or "consisting of" if such limitations are necessary for any reason and to any extent.

[0037] Some documents may be cited in this specification (e.g., patents, patent applications, scientific publications, manufacturer's specifications, instructions for use, gene bank accession number sequence submissions, etc.). Nothing herein should be construed as an admission that the present invention is not entitled to predate such disclosures by virtue of prior invention. Certain documents cited herein may be characterized as "incorporated by reference." If there is a conflict between the definitions or teachings of such incorporated references and the definitions or teachings described in this specification, the text of this specification shall prevail.

[0038] All sequences mentioned herein are disclosed in the attached sequence listing, the entire contents and disclosure of which constitute part of the disclosure of this specification.

[0039] General Definition

[0040] The term "PSMA" refers to prostate-specific membrane antigen. Human PSMA is a glycoprotein of approximately 100 kD with a short intracellular domain (residues 1-19), a transmembrane domain (residues 20-43), and an extracellular domain (residues 44-750). PSMA is represented by UniProt ID Q04609 (version March 15, 2017); human PSMA mRNA is represented by NCBI reference sequence NM_004476.1. The term PSMA includes polypeptides that show at least 70%, 80%, 85%, 90%, 95%, or 100% sequence identity to Q04609.

[0041] The term "PSMA-binding protein" refers to a protein that binds to PSMA with high affinity.

[0042] The terms "protein" and "polypeptide" refer to any chain of two or more amino acids linked by peptide bonds, and do not refer to a specific length of the product. Therefore, peptides, proteins, amino acid chains, or any other terms used to refer to two or more amino acid chains are included in the definition of "polypeptide", and the term polypeptide can be used instead of or interchangeably with any of these terms. The term polypeptide is also intended to refer to the products of post-translational modifications of polypeptides that are well known in the art. The term "modification" or "amino acid modification" refers to the substitution, deletion or insertion of a reference amino acid at a specific position in the parent polypeptide sequence by another amino acid. Given the known genetic code and recombinant and synthetic DNA technology, skilled scientists can easily construct DNA encoding amino acid variants. The term "mutant protein" as used herein refers to a derivative of, for example, ubiquitin according to SEQ ID NO: 1 or diubiquitin according to SEQ ID NO: 2 or a similar protein, which differs from the amino acid sequence due to amino acid exchange, insertion, deletion or any combination thereof, provided that the mutant protein has a specific binding affinity for PSMA.

[0043] the term (Registered trademark of Navigo Proteins GmbH) refers to non-immunoglobulin derived binding proteins.

[0044] The term "substitution" is understood as the exchange of one amino acid by another. The term "insertion" encompasses the addition of amino acids to an original amino acid sequence.

[0045] The term ubiquitin refers to ubiquitin according to SEQ ID NO: 1, or to diubiquitin according to SEQ ID NO: 2, and to proteins with at least 95% identity, such as, for example, having point mutations at positions 45, 75, 76 that do not affect binding to the target (PSMA).

[0046] 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 expressed 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.

[0047] The term "fusion protein" refers to a protein comprising at least a first protein genetically linked to at least a second protein. A fusion protein is produced by linking two or more genes that originally encode different proteins. The fusion protein may further comprise additional domains that are not involved in binding to a target, such as, but not limited to, for example, a multimerization moiety, a polypeptide tag, a polypeptide linker, or a moiety that binds to a target other than PSMA.

[0048] The term "amino acid sequence identity" refers to a quantitative comparison of the identity (or difference) of the amino acid sequences of two or more proteins. "Percent (%) amino acid sequence identity 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 the sequences are compared and gaps are introduced if necessary to achieve the maximum percentage of sequence identity. To determine sequence identity, the sequence of the query protein is compared with the sequence of a reference protein or polypeptide. Methods for sequence alignment are well known in the art. For example, in order to determine the degree of amino acid sequence identity of any polypeptide relative to an amino acid sequence, the SIMLocal similarity program known in the art is preferably used. For multiple alignment analysis, as known to those skilled in the art, ClustalOmega is preferably used.

[0049] The term "multimeric binding molecule" refers to a binding protein comprising at least two, three, four, five or more binding proteins. The binding proteins may specifically bind to the same or overlapping epitopes on the target antigen, such as overlapping epitopes of PSMA, or they may bind to different epitopes on the target antigen, such as different epitopes of PSMA.

[0050] As used herein, the term "conjugate" refers to a protein comprising at least a first protein, eg, a PSMA-binding protein of the invention, chemically attached to another substance, such as to a non-protein (chemical) moiety or a second protein.

[0051] The term "epitope" includes any molecular determinant capable of being bound by a binding protein as defined herein, and is the region of the target antigen (ie, PSMA) that is bound by the binding protein, and may include specific amino acids that directly contact the binding protein.

[0052] Detailed description of embodiments of the present invention

[0053] Structural features of PSMA binding proteins. The prostate specific membrane antigen (PSMA) binding proteins defined herein comprise at least one amino acid binding motif selected from the group consisting of GFAHR, GWAHR, SFAHR, SYAHR, GFAHL, WTTTF, WTPSI, WTETI, GDGDV, KHNTW, VAYRP and WWNPN (SEQ ID Nos: 56-61 and 72-77), or amino acid motifs having at least 80% identity thereto, respectively. In various embodiments, the PSMA binding protein comprises a mutant protein of ubiquitin (SEQ ID NO: 1). In various embodiments, the PSMA binding protein comprises an amino acid binding motif selected from the group of GFAHR, GWAHR, SFAHR, SYAHR, GFAHL, WTTTF, WTPSI, WTETI, GDGDV, KHNTW, VAYRP and WWNPN or similar motifs, wherein the amino acid residues of the binding motif correspond to positions 62, 63, 64, 65, 66 of ubiquitin according to SEQ ID NO: 1. In various embodiments, the PSMA-binding protein comprises a mutein of ubiquitin according to SEQ ID NO: 1, wherein the ubiquitin mutein comprises at least one amino acid binding motif selected from GFAHR, GWAHR, SFAHR, SYAHR, GFAHL, WTTTF, WTPSI, WTETI, GDGDV, KHNTW, VAYRP or WWNPN or a similar motif at a position corresponding to position 62, 63, 64, 65, 66 of ubiquitin (SEQ ID NO: 1).

[0054] PSMA-binding protein with the motif WWNPN

[0055] In various embodiments, the PSMA binding protein has a characteristic amino acid motif WWNPN at positions 62, 63, 64, 65, 66 of ubiquitin (SEQ ID NO: 1). In various embodiments, the PSMA binding protein comprises a mutein of ubiquitin according to SEQ ID NO: 1, wherein the ubiquitin mutein comprises the amino acid binding motif WWNPN at positions corresponding to positions 62, 63, 64, 65, 66 of ubiquitin (SEQ ID NO: 1), and further substitutions at positions corresponding to positions 6, 8, 9, 10, 12, 42, 44, 46, 68, 70, 72 of SEQ ID NO: 1. In some embodiments, the PSMA-binding protein comprises or consists of at least one ubiquitin mutein modified by a further substitution selected from the group consisting of K6Y or K6W; L8R or L8A or L8K; T9A, T9V, T9E or T9Q; G10L or G10F; T12Q; R42M or R42K or R42T; I44F or I44K; A46K or A46R; H68S; V70N or V70D; R72D or R72N or R72G, in addition to Q62W, K63W, E64N, S65P and T66N. In some embodiments, the PSMA-binding protein comprises a ubiquitin mutein having an additional 1, 2 or 3 substitutions selected from the group consisting of, for example, but not limited to, E16A, E18G, I23V, K29R, Q31R, K33R, I36T, K48R, L71R, and L73R (see, e.g., SEQ ID NOs: 38, 39, 40, 41, 47, 48, 49, 50, 51).

[0056] In various embodiments, the PSMA-binding protein comprises or consists of a ubiquitin mutein having substitutions at amino acid positions 6, 8, 9, 10, 12, 42, 44, 46, 62, 63, 64, 65, 66, 68, 70, and 72 of SEQ ID NO: 1, wherein the ubiquitin mutein has the characteristic five-amino acid motif WWNPN at positions 62, 63, 64, 65, 66. Figure 1 Examples of PSMA-binding proteins having amino acid substitutions at positions 6, 8, 9, 10, 12, 42, 44, 46, 62, 63, 64, 65, 66, 68, 70, and 72 of SEQ ID NO: 1 are shown.

[0057] In one embodiment, the PSMA-binding protein comprises one or more amino acids selected from the group consisting of SEQ ID NOs:35-51, such as, but not limited to, SEQ ID NO:40 (Affilin-187191) (differences from SEQ ID NO: 1 that result in PSMA binding are underlined).

[0058] MQIFVYTRALKQITLEVEPSDTIENVKAKIQDKEGIPPDQQMLFWKGRQLEDGRTLSDYNIWWNPNLSLNLDLRAA.

[0059] PSMA-binding proteins with the motif KHNTW or VAYRP

[0060] In various embodiments, the PSMA binding protein has a characteristic amino acid motif KHNTW at positions 62, 63, 64, 65, 66 of ubiquitin (SEQ ID NO: 1). In various embodiments, the PSMA binding protein comprises or consists of a ubiquitin mutein modified by a substitution selected from R42H, I44Y, Q62K, K63H, E64N, S65T, T66W, H68E, V70M, and R72F of ubiquitin (SEQ ID NO: 1).

[0061] In various embodiments, the PSMA-binding protein has the characteristic amino acid motif VAYRP at positions 62, 63, 64, 65, 66 of ubiquitin (SEQ ID NO: 1). In other embodiments, the PSMA-binding protein comprises or consists of a ubiquitin mutein modified by a substitution selected from R42I, I44W, Q62V, K63A, E64Y, S65R, T66P, H68Y, V70T, and R72A of ubiquitin (SEQ ID NO: 1).

[0062] In some embodiments, the PSMA-binding protein comprises a ubiquitin mutein that is additionally modified by inserting 4-8 amino acids, preferably 6 amino acids, between amino acids T9 and G10 of ubiquitin (SEQ ID NO: 1). In various embodiments, the PSMA-binding protein has an inserted 6 amino acid motif between positions 9 and 10, wherein the amino acid at position 6 is preferably an aromatic amino acid, or such an inserted M. The amino acid motif may include, but is not limited to: FEHXSF, wherein X is selected from any amino acid, preferably P, H, K or N; PQPPEX, wherein X is selected from W, F or Y; PPFAFW, PIPPDW or DMYRFM.

[0063] In various embodiments, the PSMA-binding protein comprises or consists of a mutant protein of ubiquitin according to SEQ ID NO: 1, wherein the ubiquitin mutant protein comprises at least one amino acid binding motif selected from KHNTW or VAYRP at positions corresponding to positions 62, 63, 64, 65, 66 of ubiquitin (SEQ ID NO: 1), and comprises further substitutions at positions corresponding to positions 42, 44, 68, 70, 72 of ubiquitin (SEQ ID NO: 1) and 4-8 amino acids inserted between amino acids T9 and G10 of ubiquitin (SEQ ID NO: 1).

[0064] In various embodiments, the PSMA-binding protein comprises or consists of a ubiquitin mutein having substitutions at amino acids corresponding to positions 42, 44, 62, 63, 64, 65, 66, 68, 70, and 72 of SEQ ID NO: 1 and 6 amino acids inserted at position 9 of SEQ ID NO: 1, and wherein the ubiquitin mutein has a characteristic five amino acid motif KHNTW or VAYRP corresponding to positions 62, 63, 64, 65, 66. An additional 1, 2, or 3 positions may be substituted, such as, but not limited to, A46V of ubiquitin (see SEQ ID NO: 29).

[0065] Figure 2 The specific amino acids at positions 42, 44, 62, 63, 64, 65, 66 of SEQ ID NO: 1 and the 6 amino acids inserted between positions 9 and 10 of SEQ ID NO: 1 (eg Figure 2 Examples shown in 9a, 9b, 9c, 9d, 9e, and 9f.

[0066] In one embodiment, the PSMA binding protein comprises an amino acid selected from the group consisting of SEQ ID NOs: 25-34, such as SEQ ID NO: 25 (Affilin-164667)

[0067] MQIFVKTLTFEHPSFGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQHLYWAGKQLEDGRTLSDYNIKHNTWLELMLFLRAA (differences from SEQ ID NO: 1 that result in PSMA binding are underlined).

[0068] PSMA binding protein with motif GWAHR / GFAHR or similar and or WTTTF or similar

[0069] In some embodiments, the PSMA binding protein comprises a ubiquitin mutein having a characteristic five amino acid motif GWAHR or GFAHR or SFAHR or SYAHR or GFAHL or variants thereof at positions 62, 63, 64, 65, 66 of ubiquitin. In some embodiments, the PSMA binding protein comprises one or more ubiquitin muteins based on ubiquitin according to SEQ ID NO: 1, and the one or more ubiquitin muteins comprise the amino acid binding motif GFAHR or a motif with 80% identity thereof at amino acid residues corresponding to positions 62, 63, 64, 65, 66 of ubiquitin according to SEQ ID NO: 1. In some embodiments, the PSMA binding protein comprises one or more ubiquitin muteins based on ubiquitin according to SEQ ID NO: 1, and the one or more ubiquitin muteins comprise the amino acid binding motif X1X2AHX3 at amino acid residues corresponding to positions 62, 63, 64, 65, 66 of ubiquitin according to SEQ ID NO: 1, wherein X1 is selected from G or S, X2 is selected from aromatic amino acids, preferably W or F, and X3 is selected from R or L. In some embodiments, the PSMA-binding protein comprises one or more ubiquitin muteins based on ubiquitin according to SEQ ID NO: 1, and the one or more ubiquitin muteins comprise the amino acid binding motif G(X)AHR, wherein X is an aromatic amino acid residue, preferably wherein X is selected from F or W. In some embodiments, the PSMA-binding protein comprises one or more ubiquitin muteins based on ubiquitin according to SEQ ID NO: 1, and the one or more ubiquitin muteins comprise the amino acid binding motif GFAHR, GWAHR, SFAHR, or SYAHR or GFAHL at the amino acid residues corresponding to positions 62, 63, 64, 65, 66 of ubiquitin according to SEQ ID NO: 1.

[0070] In other embodiments, the PSMA-binding protein comprises a ubiquitin mutein having the characteristic five amino acid motif WTTTF (SEQ ID NO:58) or WTPSI (SEQ ID NO:75) or WTETI (SEQ ID NO:76) or GDGDV (SEQ ID NO:77) or variants thereof at positions 62, 63, 64, 65, 66 of ubiquitin. In some embodiments, the PSMA-binding protein comprises or consists of two ubiquitin moieties, wherein the first ubiquitin moiety has the motif GFAHR (SEQ ID NO:56) or GWAHR (SEQ ID NO:57) or SFAHR (SEQ ID NO:72) or SYAHR (SEQ ID NO:73) or GFAHL (SEQ ID NO:74) or a similar motif, or the second ubiquitin moiety has the motif WTTTF or WTPSI or WTETI or GDGDV or a similar motif. In various embodiments, the PSMA-binding protein comprises or consists of two ubiquitin moieties, wherein the first ubiquitin moiety has the motif GFAHR or GWAHR or SFAHR or SYAHR or GFAHL or a similar motif, and the second ubiquitin moiety has the motif WTTTF or WTPSI or WTETI or GDGDV or a similar motif. Figure 3 Examples of specific amino acids at positions 6, 8, 62, 63, 64, 65, 66 in a PSMA-binding protein composed of two ubiquitin moieties are shown. In various embodiments, the PSMA-binding protein comprises or consists of two ubiquitin muteins fused to each other, wherein the two ubiquitin muteins have substitutions at the amino acids corresponding to positions 6, 8, 62, 63, 64, 65, 66 of SEQ ID NO: 1, wherein the N-terminally positioned ubiquitin mutein has a characteristic five-amino acid motif GFAHR or GWAHR or SFAHR or SYAHR or GFAHL corresponding to positions 62, 63, 64, 65, 66, and the C-terminally positioned ubiquitin mutein has a characteristic five-amino acid motif WTTTF or WTPSI or WTETI or GDGDV corresponding to positions 62, 63, 64, 65, 66, such as Figure 3 shown.

[0071] In some embodiments, the ubiquitin mutein has a substitution selected from K6R or K6W; L8M, L8P, L8Q, L8W, L8D, L8G, L8H or L8I; Q62G, Q62S or Q62W; K63F, K63W, K63G, K63P or K63Y; E64A or E64G; S65H or S65D; and T66R, T66Q or T66L. In some embodiments, the ubiquitin mutein has a substitution selected from K6M, K6L, K6A or K6H; L8Q, L8R, L8F, L8N or L8H; Q62W or Q62G; K63T, K63H or K63D; E64T, E64P, E64E or E64G; S65T, S65Y or S65D and T66F, T66I, T66L or T66V.

[0072] In some embodiments, the PSMA-binding protein comprises a ubiquitin mutein additionally modified by further substitutions, such as but not limited to positions 10, 11, 33, 48, 48, 51, 74 of ubiquitin, and in specific embodiments, such as selected from the group of G10Q, K11Q, K33T, K48T, E51A, R74C of ubiquitin (e.g., see SEQ ID NOs: 11, 12, 17, 19).

[0073] In some embodiments, the PSMA-binding protein comprises two ubiquitin muteins linked to each other, ie, the PSMA-binding protein comprises a mutein of diubiquitin (SEQ ID NO: 2).

[0074] In one embodiment, for example, the PSMA-binding protein comprises one or more amino acids selected from the group consisting of SEQ ID NOs: 3-24 (differences from SEQ ID NO: 1 or SEQ ID NO: 2 that result in PSMA binding are underlined).

[0075] SEQ ID NO:3 (Affilin-162462)

[0076] MQIFVRTPTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIGFAHRLHLVLRLRAAMQIFVMTQTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIWTTTFLHLVLRLRAA;

[0077] SEQ ID NO:4 (Affilin-191871)

[0078] MQIFVRTQTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIGWAHRLHLVLRLRAAMQIFVHTNTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIWTETILHLVLRLRAA.

[0079] The PSMA-binding protein of the present invention comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-55. In some embodiments, the PSMA-binding protein comprises an amino acid sequence that exhibits at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NOs: 3-55. For example, but not limited to, Figure 1 , Figure 2 and Figure 3 The amino acid sequences of selected PSMA-binding proteins are shown in FIG.

[0080] In some embodiments, the PSMA-binding proteins disclosed herein have at least 74%, 75%, 76%, 77%, 78%, or 79% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In various embodiments, the PSMA-binding proteins disclosed herein have at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The PSMA-binding proteins disclosed herein have at least 81%, 82%, 83%, 84%, or 85% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the PSMA-binding proteins disclosed herein may have any amino acid identity between 74% and 90% identity to the amino acid sequence of ubiquitin (SEQ ID NO: 1) or diubiquitin (SEQ ID NO: 2).

[0081] In some embodiments, the PSMA-binding proteins disclosed herein may have at least 90% identity to any amino acid sequence of SEQ ID NO:3. In some embodiments, the PSMA-binding proteins disclosed herein may have at least 90% identity to any amino acid sequence of SEQ ID NO:4. In some embodiments, the PSMA-binding proteins disclosed herein may have at least 90% identity to any amino acid sequence of SEQ ID NO:25. In some embodiments, the PSMA-binding proteins disclosed herein may have at least 90% identity to any amino acid sequence of SEQ ID NO:26. In some embodiments, the PSMA-binding proteins disclosed herein may have at least 90% identity to any amino acid sequence of SEQ ID NO:40.

[0082] Multimer. In a preferred embodiment, the PSMA-binding protein is a multimer comprising a plurality of PSMA-binding proteins as defined herein. The multimer may comprise two, three, four or more PSMA-binding proteins. In one embodiment, the PSMA-binding protein comprises 2, 3, 4 or more PSMA-binding proteins linked to each other, i.e., the PSMA-binding protein may be a dimer, trimer or tetramer, etc. In some embodiments, the multimer is a dimer of the PSMA-binding protein defined above. In various embodiments, the PSMA-binding protein is a homodimer. A homodimeric PSMA-binding protein as understood herein is a protein in which two PSMA-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: 3-51 or any amino acid sequence having at least 90% identity therewith. For example, but not limited to, homodimers are exemplified in SEQ ID NO: 52 (a dimer of SEQ ID NO: 3) and SEQ ID NO: 53 (a dimer of SEQ ID NO: 25).

[0083] In other embodiments, the multimer is a heterodimer, for example, the two amino acid sequences of the PSMA binding protein are not identical. For example, heterodimers such as SEQ ID NO: 54 (a dimer of SEQ ID NO: 25 and SEQ ID NO: 3, from N- to C-terminus) and SEQ ID NO: 55 (a dimer of SEQ ID NO: 3 and SEQ ID NO: 25, from N- to C-terminus) are produced.

[0084] In some embodiments, two or more PSMA-binding proteins are directly linked. In some embodiments, two or more PSMA-binding proteins are linked via a peptide linker. In various embodiments, two or more PSMA-binding proteins are linked via a peptide linker of up to 30 amino acids. In other embodiments, two or more PSMA-binding proteins are linked via a peptide linker of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids. In one embodiment, two PSMA-binding proteins are linked via 16 amino acids.

[0085] One embodiment relates to a linker comprising at least two or more amino acids selected from the group of glycine, serine, alanine or proline. In some embodiments, two or more PSMA-binding proteins are linked via a peptide linker having an amino acid sequence according to any one of SAPAASPSPAAPAPSPASPAPASPASAPSAPASAPPAASA (SEQ ID NO: 62) or PAAPAPSPASPAPASPASAPS (SEQ ID NO: 63), or a peptide linker having at least 90% identity thereto. Other linkers for fusion proteins are known in the art and can be used.

[0086] Functional characteristics. In some embodiments, the PSMA-binding proteins described herein bind to PSMA expressed on cells as determined by FACS and / or have a binding affinity for PSMA of 500 nM or less as determined by surface plasmon resonance assay.

[0087] In some embodiments, the binding affinity (K) of a PSMA-binding protein as described herein for PSMA is D ) is less than 500 nM. The PSMA binding protein binds to PSMA 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 less than 1 nM. Suitable methods are known to those skilled in the art or described in the literature. Methods for determining binding affinity are known per se and can be selected 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 of these methods are described in the examples below. In general, the dissociation constant K DDetermined in the temperature range between 20°C and 30°C, for example at 20°C, 25°C or 30°C. Unless otherwise specified, K D The values ​​were all measured by SPR at 25°C. 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. Figure 1 , Figure 2 , Figure 3 Examples are provided in the and Examples. The binding of the PSMA-binding proteins described herein is highly specific for PSMA. Ubiquitin (SEQ ID NO: 1) does not bind to PSMA. The PSMA-binding proteins described herein bind to PSMA, but do not detectably bind to the Fc domain of human immunoglobulin IgG1, as determined by surface plasmon resonance assays. D PSMA binding of less than 500 nM may be important for targeted medical applications in PSMA-associated cancers. D Proteins less than 500 nM may have reduced potential toxic side effects. In some embodiments, a PSMA-binding protein comprises a ubiquitin mutant protein having: (a) a five amino acid residue motif selected from GFAHR, GWAHR, SFAHR, SYAHR, GFAHL, WTTTF, WTPSI, WTETI, GHEYL, GDGDV, KHNTW, VAYRP, and WWNPN at positions corresponding to positions 62, 63, 64, 65, and 66 of ubiquitin (SEQ ID NO: 1), or a motif having 80% identity thereto; (b) a sequence identity of 80% to 93% to SEQ ID NO: 1; and (b) a binding affinity (K) for PSMA of D ) is less than 500nM. The half maximum effective concentration EC 50 The concentration of PSMA-binding protein that induces a response halfway between baseline and maximum after a specified exposure time, and therefore represents the concentration of PSMA-binding protein at which 50% of its maximal effect is observed, in this case, the half-maximal fluorescence intensity signal in a cell binding flow cytometric assay. In some embodiments, the PSMA-binding proteins described herein have an effect on the EC of PSMA-expressing cells. 50 Less than 100 nM, less than 50 nM, less than 20 nM, less than 10 nM, less than 5 nM, and less than 1 nM. In some embodiments, the PSMA-binding proteins described herein have an effect on the EC of PSMA-expressing cells after incubation at 37° C. in the presence of mouse serum for at least 24 hours. 50 Less than 1 nM. Suitable methods are known to those skilled in the art. 50The lower the value, the greater the binding of the PSMA-binding protein to PSMA. Table 4 provides examples of PSMA-binding proteins that are stable even in the presence of serum.

[0088] In some embodiments, the PSMA-binding proteins described herein are stable at elevated temperatures up to 85° 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, the stability of a molecule can be measured by thermal melting (T m ) temperature, which is the temperature at which half of the molecules unfold, in degrees Celsius (℃). Usually, T m The higher the value, the more stable the molecule. The temperature stability was determined by differential scanning fluorimetry (DSF), as described in Examples and Figure 1 , Figure 2 , Figure 3 As described in further detail in .

[0089] Competition binding experiments comparing PSMA-binding proteins show that PSMA-binding proteins with different motifs can bind to different or non-overlapping epitopes (see Examples). For example, SEQ ID NO: 25 with the motif KHNTW binds to a different epitope than SEQ ID NO: 15 with the motifs GFAHR and WTTTF. Thus, certain PSMA-binding proteins do not compete for PSMA binding and are particularly useful in certain diagnostic or therapeutic applications.

[0090] Additional functional characterization was performed by cellular PSMA binding assays using PSMA overexpressing cells. Immunofluorescence microscopy and flow cytometry analysis confirmed the specific binding of the PSMA-binding proteins described herein to PSMA-positive tumor cell lines from human origin and to PSMA on live cells (see Examples).

[0091] Coupling Sites. In some embodiments, the PSMA-binding proteins described herein further comprise one or more coupling sites for coupling chemical moieties. Coupling sites are capable of reacting with other chemical groups to couple the PSMA-binding protein to the chemical moiety. The determined number and determined position of the coupling sites enable site-specific coupling of the chemical moiety to the PSMA-binding proteins described herein. Thus, if desired, a large number of chemical moieties can be coupled to the PSMA-binding protein. One skilled in the art can adjust the number of coupling sites to the optimal number for a certain application, thereby adjusting the number of chemical moieties accordingly. In selected embodiments, the coupling sites can be selected from a group of one or more amino acids that can be labeled with a specific chemical, 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 PSMA-binding protein can comprise 1 to 20 coupling sites, such as 1 to 6 coupling sites, such as 2 coupling sites, or one coupling site.

[0092] Coupling Domain. One embodiment provides a PSMA-binding protein comprising: at least one coupling domain of 1 to 80 amino acids comprising one or more coupling sites. In some embodiments, the coupling domain of 1 to 80 amino acids may comprise alanine, proline or serine and cysteine ​​as a coupling site. An example of a PSMA-binding protein having an amino acid SAC coupling domain is provided in SEQ ID NO:5 (Affilin-191871 with c-terminal amino acid SAC). In other embodiments, the coupling domain of 1 to 80 amino acid residues 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 cysteine ​​residues as a coupling site at the C- or N-terminus of a PSMA-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 so that no more than 2, 3, 4 or 5 identical amino acid residues are adjacent, preferably 3 amino acids at most. The composition of 1 to 20 coupling domains can be different or identical. The amino acid composition of selected examples of coupling domains with coupling sites (cysteine) is shown in Table 1.

[0093] Table 1. Amino acid composition of examples of coupling domains

[0094]

[0095] In some embodiments, the chemical moiety is selected from any one of a chelator, a drug, a toxin, a dye, and a small molecule. In some embodiments, at least one chemical moiety is a chelator designed as a complexing agent for coupling one or more other moieties and a target compound to a PSMA-binding protein disclosed herein. One embodiment relates to a PSMA-binding protein wherein the chelator is a complexing agent for coupling one or more radioisotopes or other detectable labels, as described in the Examples.

[0096] Diagnostic moiety. In various embodiments, the PSMA-binding protein further comprises a diagnostic moiety. In other embodiments, the PSMA-binding protein further comprises more than one diagnostic moiety. In some embodiments, such diagnostic moieties may be selected from radionuclides, fluorescent proteins, photosensitizers, dyes or enzymes, or any combination of the foregoing. In some embodiments, PSMA-binding proteins comprising at least one diagnostic moiety may be employed, for example, as imaging agents, for example, to assess the presence of tumor cells or metastases, tumor distribution and / or tumor recurrence. Methods of detecting or monitoring cancer cells involve imaging methods. Such methods involve imaging PSMA-associated cancer cells by, for example, radioimaging or photoluminescence or fluorescence.

[0097] Therapeutic moiety. In various embodiments, the PSMA-binding protein further comprises a therapeutically active moiety. In other embodiments, the PSMA-binding protein further comprises more than one therapeutically active moiety. In some embodiments, such therapeutically active moieties may be selected from monoclonal antibodies or fragments thereof, binding proteins such as ubiquitin muteins (Affilins), extracellular domains of receptors or fragments thereof, radionuclides, cytotoxic compounds, cytokines, chemokines, enzymes or derivatives thereof, or any combination thereof. In some embodiments, the PSMA-binding protein comprising a therapeutically active component may be used to target delivery of any of the above-listed components to PSMA-expressing tumor cells and accumulate therein, thereby producing a lower level of toxicity to normal cells.

[0098] Radionuclides. Radionuclides suitable for in vivo or in vitro imaging or radiotherapy include, for example, but not limited to, the group of gamma-emitting isotopes, the group of positron emitters, the group of beta emitters, and the group of alpha emitters. In some embodiments, suitable conjugation partners include chelating agents such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA) or their activated derivatives, nanoparticles, and liposomes. In various embodiments, DOTA may be suitable as a complexing agent for radioisotopes and other imaging agents, as described in further detail in the Examples.

[0099] Pharmacokinetic Modulating Moieties. In some embodiments, the PSMA-binding protein further comprises at least one pharmacokinetic modulating moiety, which is optionally selected from polyethylene glycol, human serum albumin, an albumin binding protein, an immunoglobulin binding protein or an immunoglobulin or immunoglobulin fragment, a polysaccharide (e.g., hydroxyethyl starch), or an unstructured amino acid sequence that increases the hydrodynamic radius, such as a polymer comprising the amino acids alanine, glycine, serine, proline. In various embodiments, the moiety increases the half-life of the PSMA-binding protein by at least 1.5 times. Various techniques for producing PSMA-binding proteins with extended half-lives are known in the art, such as direct fusion or chemical coupling methods of pharmacokinetic modulating moieties to the above-described PSMA-binding proteins. The pharmacokinetic modulating moiety can be attached, for example, at one or more sites of the PSMA-binding protein via a peptide linker sequence or via a coupling site as described above.

[0100] Conjugation of protein or non-protein moieties to PSMA-binding proteins can be performed using chemistries well known in the art. In some embodiments, coupling chemistries specific for derivatization of cysteine ​​or lysine residues can be used. Chemical coupling can be performed by chemistry well known to those skilled in the art, including but not limited to substitution, addition or cycloaddition or oxidation chemistry (e.g., disulfide formation).

[0101] Molecules for purification / detection. In some embodiments, additional amino acids may be extended at the N-terminus or C-terminus or both of the PSMA-binding protein. The 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 to certain chromatographic column materials. Typical examples of such sequences include, but are not limited to, Strep-tag (see, e.g., SEQ ID NO: 71), oligohistidine-tag, glutathione S-transferase, maltose-binding protein, intein, intein fragments, or the albumin binding domain of protein G.

[0102] Use in medicine. Various embodiments relate to the use of PSMA-binding proteins as disclosed herein in medicine. In one embodiment, the PSMA-binding proteins are used in medicine to diagnose or treat cancers associated with PSMA expression. The PSMA-binding proteins as disclosed herein allow for the selective diagnosis and treatment of PSMA-associated cancer cells or cancer tissues. PSMA is known to be upregulated in tumor cells. PSMA is highly expressed in prostate cancer and its metastases (e.g., selected from, but not limited to, liver, thyroid, B-cell follicular lymphoma, lymph node and bone metastases) and other solid tumors (preferably selected from breast cancer, kidney cancer / renal cancer, multiple myeloma, brain tumors, lung cancer, ovarian cancer, colorectal cancer, bladder cancer and gastric cancer).

[0103] One embodiment is a method of diagnosing (including monitoring) a subject with a PSMA-associated cancer, the method comprising administering to the subject a PSMA-binding protein as described, optionally conjugated to a radioactive molecule. In various embodiments, a PSMA-binding protein as disclosed herein can be used to diagnose a PSMA-associated cancer, optionally wherein the PSMA-binding protein is conjugated to a radioactive molecule. In some embodiments, imaging methods using a PSMA-binding protein with a label (such as a radioactive or fluorescent label) can be used to visualize PSMA on specific tissues or cells, for example to assess the presence of PSMA-associated tumor cells, PSMA-associated tumor distribution, recurrence of PSMA-associated tumors, and / or to assess a patient's response to therapeutic treatment.

[0104] One embodiment is a method of treating a subject with a PSMA-associated cancer, the method comprising administering to the subject a PSMA-specific binding protein, optionally conjugated to a radioactive molecule and / or a cytotoxic agent. In various embodiments, the PSMA-binding protein as disclosed herein can be used to treat PSMA-associated cancer, optionally wherein the PSMA-binding protein is conjugated to a cytotoxic agent and / or a radioactive molecule. Some embodiments relate to the use of a PSMA-binding protein labeled with a suitable radioisotope or cytotoxic compound for treating PSMA-associated tumor cells, particularly for controlling or killing PSMA-associated tumor cells, such as malignant cells. In one embodiment, a therapeutic dose of radiation is selectively delivered to PSMA-associated tumor cells, rather than normal cells.

[0105] Compositions. Various embodiments relate to a composition comprising a PSMA-binding protein disclosed herein. The composition comprising the PSMA-binding protein defined above is used in medicine, preferably for the diagnosis or treatment of various PSMA-related cancer tumors, such as prostate cancer and its metastasis, liver, thyroid, B-cell follicular lymphoma, lymph node and bone metastasis, kidney cancer / renal cancer, multiple myeloma, brain tumors, lung cancer, ovarian cancer, colorectal cancer, bladder cancer, gastric cancer, etc. The composition comprising the above PSMA-binding protein can be used in clinical applications for diagnostic and therapeutic purposes. In particular, the composition comprising the above PSMA-binding protein can be used in clinical applications for imaging, monitoring and elimination or inactivation of pathological cells expressing PSMA. Various embodiments relate to a diagnostic composition for diagnosing PSMA-related cancers, comprising a PSMA-binding protein as defined herein and a diagnostically acceptable carrier and / or diluent. These include, for example, but are not limited to, stabilizers, surfactants, salts, buffers, colorants, etc. The composition can be in the form of a liquid preparation, a lyophilized agent, a granule, an emulsion or a liposomal preparation.

[0106] Diagnostic compositions comprising a PSMA-binding protein described herein can be used to diagnose PSMA-associated cancers, as described above.

[0107] Various embodiments relate to a pharmaceutical (e.g., therapeutic) composition for treating a disease, comprising a PSMA-binding protein as disclosed herein and a pharmaceutically (e.g., therapeutically) acceptable carrier and / or diluent. The pharmaceutical (e.g., therapeutic) composition may optionally contain other adjuvants and excipients known per se. These include, for example, but are not limited to, stabilizers, surfactants, salts, buffers, colorants, etc.

[0108] Pharmaceutical compositions comprising a PSMA-binding protein as defined herein can be used to treat a disease, as described above.

[0109] The compositions contain an effective dose of a PSMA-binding protein as defined herein. The amount of protein to be administered depends on the organism, the type of disease, the age and weight of the patient, and other factors known per se. Depending on the galenic preparation, these compositions can be administered parenterally by injection or infusion, systemically, intraperitoneally, intramuscularly, subcutaneously, transdermally, or by other conventionally used application methods.

[0110] 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.

[0111] The various components of the composition may be packaged as a kit with instructions for use.

[0112] Preparation of PSMA-binding proteins. The PSMA-binding proteins 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. Various embodiments relate to a polynucleotide encoding a PSMA-binding protein as disclosed herein. One embodiment further provides an expression vector comprising the polynucleotide, and a host cell comprising the isolated polynucleotide or expression vector.

[0113] Various embodiments are directed to a method of producing a PSMA-binding protein disclosed herein, comprising culturing a host cell under suitable conditions that allow expression of the PSMA-binding protein and optionally isolating the PSMA-binding protein.

[0114] For example, one or more polynucleotides encoding PSMA-binding proteins can be expressed in a suitable host, and the produced PSMA-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., a nucleic acid, plasmid, phage, or virus) that can be used to transfer protein coding information into a host cell. Various cell culture systems, such as, but not limited to, mammals, yeast, plants, or insects, can also be used to express recombinant proteins. Suitable conditions for culturing prokaryotic or eukaryotic host cells are well known to those skilled in the art. Cell culture and protein expression to produce proteins can be performed at any scale, from small volume shake flasks to large fermenters, using techniques well known to those skilled in the art.

[0115] One embodiment relates to a method for preparing a binding protein as detailed above, the method comprising the following steps: (a) preparing a nucleic acid encoding a PSMA-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 for expressing the PSMA-binding protein, thereby producing a PSMA-binding protein as defined herein; (f) optionally isolating the PSMA-binding protein produced in step (e); and (g) optionally conjugating the PSMA-binding protein to other functional moieties as defined herein.

[0116] In general, the purified PSMA-binding protein can be isolated from the culture mixture using conventional methods and techniques well known in the art, such as centrifugation, precipitation, flocculation, chromatography in various embodiments, filtration, dialysis, concentration, and combinations thereof, etc. Chromatographic methods are well known in the art and include, but are not limited to, ion exchange chromatography, gel filtration chromatography (size exclusion chromatography), or affinity chromatography.

[0117] To simplify purification, the PSMA-binding protein can be fused to other peptide sequences that have increased affinity for the separation material. Preferably, such fusions are selected that do not adversely affect the function of the PSMA-binding protein or that can be separated after purification due to the introduction of specific protease cleavage sites. Such methods are also known to those skilled in the art.

[0118] Example

[0119] The following examples are provided to further illustrate the present invention. The present invention is particularly illustrated by specific modifications of ubiquitin (SEQ ID NO: 1 or SEQ ID NO: 2), resulting in binding to PSMA. However, the present invention is not limited thereto, and the following examples merely illustrate the utility of the present invention on the basis of the above description.

[0120] Example 1. Identification of PSMA-binding proteins

[0121] Library construction and cloning. Ubiquitin libraries containing randomized amino acid positions were synthesized either by Triplex technology (MorphoSys Slonomics, Germany) or in-house from randomized oligonucleotides generated by synthetic trinucleotide phosphoramidites (ELLA Biotech) to achieve a balanced amino acid distribution while excluding cysteine ​​and other amino acid residues at random positions.

[0122] Using multiple libraries to identify PSMA-binding proteins

[0123] Library SPV2: Ubiquitin randomized at amino acid positions 6, 8, 9, 10, 12, 42, 44, 46, 62, 63, 64, 65, 66, 68, 70 and 72 (SEQ ID NO: 1).

[0124] Library SPL27: Ubiquitin (SEQ ID NO: 1) with randomized amino acids corresponding to positions 62, 63, 64, 65, 66, 68, 70 and 72 and an insertion of six random amino acids introduced between T9 and G10 of ubiquitin. The presence of amino acid residues Cys, Ile, Leu, Val and Phe were omitted from the insertion.

[0125] Library SPVF19: diubiquitin (SEQ ID NO: 2) randomized at amino acid positions 6, 8, 62, 63, 64, 65, 66, 68, 70, 72, 82, 84, 138, 139, 140, 141, 142 (this corresponds to randomization of positions 6, 8, 62, 63, 64, 65, 66 in the two ubiquitin moieties; in library SPVF19, the two ubiquitin moieties are directly linked).

[0126] 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 artisan. The pCD12 phagemid contains a modified torA leader sequence to enable protein processing without the need to add additional amino acids at the N-terminus. An aliquot of the ligation mixture was used for electroporation of E. coli ER2738 (Lucigen). Unless otherwise indicated, established recombinant genetic methods were used, such as described in Sambrook, J & Russel, DW

[2001] (Cold Spring Harbor Laboratory, NY).

[0127] Target expression, purification and analysis. The DNA sequence encoding the extracellular domain of human PSMA (uniprot accession number Q04609; residues 45-750) was genetically fused to the Fc region of human IgG1, followed by the addition of a His-tag at the N-terminus. The full-length cDNA using human codons was provided by GeneArt (Thermo Scientific), cloned into the mammalian expression vector pCEP4, and expressed in mammalian Expi293F cells at a scale of 250 ml in shake flasks. Expression was analyzed by SDS-PAGE and by immunoblot analysis using antibodies against PSMA and the Fc-portion of human IgG1. 130 ml of the large-scale expressed cell culture supernatant was centrifuged and filtered for application to affinity chromatography on a protein A HP 1 mL column (GE Healthcare). The target protein was eluted by a gentle pH shift (pH 4) and applied to a Superdex XK16 / 600 gel filtration column. 9 mg of His-Fc-PSMA could be recovered; SDS-PAGE analysis and SE-HPLC analysis confirmed the purity of the target protein. The enzyme activity of the target protein towards the substrate N-acetyl-L-aspartyl-L-glutamate (NAAG) was confirmed.

[0128] Primary selection by TAT phage display. The naive library was enriched for the target PSMA using TAT phage display as a selection system. After transformation of competent bacteria ER2738 cells (Lucigene) with phagemid pCD12 carrying the library, phage amplification and purification were performed using standard methods known to the skilled person. For selection, Protein A or The target protein was immobilized as an Fc fusion of the extracellular domain of human PSMA on protein G. The target concentration during phage incubation was reduced from 200nM (first round) to 100nM (second round) and 50nM (third round) and 25nM (fourth round). In some selection rounds, mouse serum was added to select molecules with increased serum stability. The target phage complex was magnetically separated from the supernatant and washed several times. The target-bound phage was eluted by trypsin. In order to deplete the phage library of Fc-binding variants, a pre-selection of phages with an Fc fragment of immobilized IgG1 was performed before the second and third rounds (Athens Research & Technology). In order to identify target-specific phage pools, the eluted and re-amplified phages of each round of selection were analyzed by phage library ELISA. Wells of a medium binding microtiter plate (Greiner Bio-One) were coated with PSMA-Fc (2.5 μg / ml) and the Fc-fragment of IgG1 (2.5 μg / ml), respectively. Bound phages were detected using an α-M13 HRP-conjugated antibody (GE Healthcare).

[0129] The target binding phage pool was cloned into an expression vector. The selected pool showing specific binding to the target in the phage pool ELISA was 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 a Strep tag II (IBA GmbH).

[0130] Single colony hit analysis. After transformation of BL21 (DE3) cells (Merck, Germany), kanamycin-resistant single colonies were grown. Expression of the targeted binding modified ubiquitin variants was achieved by culturing in 384-well plates (Greiner Bio-One) using auto-induction medium. Cells were harvested and subsequently lysed chemically or enzymatically by BugBuster reagent (Novagen), and mechanically by freeze / thaw cycles. After centrifugation, the supernatants were screened by ELISA with the immobilized target on high-binding 384 ELISA microtiter plates (Greiner Bio-One). The bound proteins were detected by combining HRP conjugate (IBAGmbH) with TMB-Plus substrate (Biotrend, Germany). The reaction was terminated by adding 0.2 M H2SO4 solution and measured at 450 nm and 620 nm in a microplate reader.

[0131] Construction of mature libraries. To mature each selected variant, a modular shuffling approach was used, in which the binding molecule was divided into two modules. For the ubiquitin mutant protein based on SEQ ID NO: 1, the first module contained amino acids 1-40 and the second module contained amino acids 32-76. For the diubiquitin mutant protein based on SEQ ID NO: 2, the first module contained amino acids 1-77 and the second module contained amino acids 71-152. For the cloning of the modular shuffling mature library, the module of one of the variants remained unchanged and fused to the native second module of the original library, and vice versa. The fusion of the two modules was achieved by overlap extension PCR. The cDNA of the mature library obtained was connected to pCD12 as described above. Alternatively, an error-prone PCR method was used, in which additional mutations were induced in a predefined molecule or library.

[0132] Maturation selection and analysis. For affinity maturation, two rounds of panning were performed. For both rounds, preselection was performed using the Fc fragment of IgG1. In some selection rounds, mouse serum was added to select molecules with increased serum stability. To analyze the specific target binding of the matured and selected pools, phage pool ELISA was performed, and then the positive pools were cloned into the expression vector pET-28a and hit ELISA was performed as described above.

[0133] Example 2. Expression and purification of PSMA-binding protein

[0134] PSMA-binding molecules were cloned into expression vectors using standard methods known to those of skill in the art, purified, and analyzed as described below.

[0135] All constructs were expressed in E. coli BL21 (DE3) using a low copy plasmid system under the control of the T7 promoter. After induction with lactose contained in the culture medium (autoinduction medium), the protein was produced in the cytoplasm in a mostly soluble form. After fresh transformation with a defined plasmid, all overnight cultures were inoculated from a single colony. PSMA binding proteins were produced in ZYM5052 autoinduction medium according to Studieret al. (2005). Overnight cultures were grown to saturation in 2xYT medium in a shake flask at a volume of 20-100 mL. The main culture was inoculated to an OD600 of 0.05 to 0.1 and incubated at 30°C for up to 24 hours in a shake flask with or without baffles at a speed of 200 rpm in ZYM5052 with 50 μg / mL kanamycin. Depending on the expression level, either in 1 L shake flasks (350 mL of medium per flask) or in 5 L flasks (1 L of medium per flask).

[0136] The affinity-tagged Affilin protein was purified by affinity chromatography and gel filtration. System and Superdex TM Size exclusion chromatography (SEHPLC or SEC) was performed on a 200HiLoad 16 / 600 column (GE Healthcare). The column volume was 120 ml and balanced with 2CV. The sample was applied 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.

[0137] The dimeric PSMA binding protein without the affinity tag was purified using cation exchange chromatography (SP Sepharose HP, GE Healthcare) followed by anion exchange chromatography (Q Sepharose HP, GE Healthcare) to reduce the amount of endotoxin.

[0138] Finally, size exclusion chromatography (Sephacryl S200HR, GE Healthcare) was performed. Further analysis included SDS-PAGE, SE-HPLC, and RP-HPLC. Protein concentration was determined by absorbance measurement at 280 nm using the molar absorptivity. For example, the purity of SEQ ID NO:5 was 98% according to SE-HPLC. The protein was purified using a Dionex HPLC system and PLRP-S (5 μm, ) column (Agilents) was used for RP chromatography (RP-HPLC).

[0139] Example 3. PSMA-binding protein is stable at high temperature

[0140] The thermal stability of the binding proteins of the present invention was determined by differential scanning fluorimetry (DSF). Each sample was transferred to 480 multiwell plates 96 (Roche) and SYPRO orange dye was added at appropriate dilutions. A temperature ramp from 20 to 90°C was programmed at a heating rate of 1°C per minute ( 480, Roche). The fluorescence was continuously measured at an excitation wavelength of 465 nm and an emission wavelength of 580 nm ( 480,Roche). Figure 1 , Figure 2 and Figure 3 The transition midpoints (Tm, melting points) of thermal unfolding of the selected mutant proteins are shown. The PSMA-binding proteins of the present invention have a melting temperature as high as 85°C. m The T of SEQ ID NO: 6 is 69.3°C. mThe temperature stability of the selected dimeric PSMA binding proteins is shown in Table 2.

[0141] Example 4. Analysis of PSMA-binding proteins (surface plasmon resonance, SPR)

[0142] The CM5 sensor chip (GE Healthcare) was balanced with SPR running buffer. The surface-exposed carboxyl groups were activated by passing a mixture of EDC and NHS to produce reactive ester groups. 700-1500RU PSMA-Fc (on the ligand) was fixed on the flow cell, and IgG-Fc (detached from the ligand) was fixed on another flow cell, with a ratio of 1:3 (hIgG-Fc: target) to the target. Ethanolamine was injected after ligand fixation to block unreacted NHS groups. After ligand binding, protein analytes accumulated on the surface, increasing the refractive index. This change in refractive index is measured in real time and plotted as a response or resonance unit (RU) versus time curve. The analyte was applied to the chip in serial dilutions at a flow rate of 30 μl / min. Binding was performed for 120 seconds, and dissociation was performed for 360 seconds. After each run, the chip surface was regenerated with 30 μl regeneration buffer and balanced with running buffer. The dilution series was used as a positive control, while the dilution series of unmodified ubiquitin represented a negative control. Control samples were applied to the matrix at a flow rate of 30 μl / min, while they bound for 60 seconds and dissociated for 120 seconds. Regeneration and re-equilibration were performed as described previously. Binding studies were performed using Biacore 3000 (GE Healthcare); data evaluation was run using the BIAevaluation 3.0 software provided by the manufacturer using a Langmuir 1:1 model (RI=0). After fitting the data using a 1:1 Langmuir model, K was calculated, for example. D The values ​​are shown in Figure 1 , Figure 2 , Figure 3 and Table 2. The estimated dissociation constants (K D ) were normalized for off-target and are shown. For example, the K of SEQ ID NO: 5 relative to hPSMA-Fc (1100RU immobilized by protein A) D is 1.8 nM, K of SEQ ID NO:6 D The K of SEQ ID NO:22 is 113 nM. D The K of SEQ ID NO:45 is 484 nM. D is 33 nM and the K of SEQ ID NO: 26 D Table 2 shows the binding affinity determined by SPR and the temperature stability of the dimeric PSMA binding protein (see Example 3).

[0143] Table 2. Binding affinity and temperature stability of dimeric PSMA binding proteins

[0144]

[0145] Example 5. Functional characterization: Binding to PSMA expressed on the cell surface (flow cytometry)

[0146] Flow cytometry was used to analyze the binding of PSMA binding proteins to PSMA exposed on the cell surface. Human prostate cancer cell line LNCaP overexpressing PSMA, HEK293-PSMA cells overexpressing PSMA, PC3 cells not expressing PSMA, and empty vector control HEK293-pEntry cells were used. Cells were trypsinized and resuspended in medium containing FCS, washed and stained in pre-cooled FACS blocking buffer. Cells were prepared at a concentration of 1x10 6 Cells / ml were used for cell staining, and 100 μl was filled into the wells of a 96-well plate (Greiner) in triplicate for each cell line. In multiple experiments, different concentrations, such as 50 nM of PSMA binding protein or 0.5 μg / ml of monoclonal anti-human-PSMA antibody (clone LNI-17; Biolegend; 342502) were added to PSMA overexpression and control cells as positive controls. The PSMA binding protein includes a C-terminal streptococcal tag for purification and detection purposes (see, e.g., SEQ ID NO: 71). After 45 minutes, the supernatant was removed and 100 μl / well rabbit anti-streptococcal tag antibody (obtained from GenScript; A00626) diluted 1:300 in FACS blocking buffer was added. Anti-PSMA antibodies were detected in positive control wells with anti-mouse-IgG-Alexa 488 (Invitrogen; A-10680) diluted 1:1000. After removal of the anti-streptococcal tag antibody from the other wells, goat anti-rabbit IgG Alexa Fluor 488 antibody (obtained from Invitrogen; A11008) was applied at a dilution of 1:1000. Flow cytometry measurements were performed on a Guava easyCyte 5HT apparatus from Merck-Millipore at an excitation wavelength of 488 nm and an emission wavelength of 525 / 30 nm. All PSMA-binding proteins of the invention (including dimers) showed binding to PSMA expressed on the surface of LNCaP-cells and HEK293-PSMA-cells (see Figure 1 , Figure 2 , Figure 3; binding is shown as "yes" in the figure), and no binding on the PSMA-negative cell lines HEK293-pEntry or PC3 cells. Ubiquitin showed no binding on LNCaP-cells and HEK293-PSMA-cells. Positive binding of anti-PSMA antibodies to PSMA-expressing cells was also observed. For example, SEQ ID NO: 4 showed strong cell binding to HEK-PSMA and LNCaP cells.

[0147] Example 6. Binding to PSMA expressed on the cell surface (immunocytochemistry and fluorescence microscopy)

[0148] A concentration of 50 nM was tested on PSMA expressing LNCaP-cells and the control cell line PC3 (no PSMA expression). Diubiquitin was used as a control for non-PSMA-binding proteins and 1 μg / ml anti-PSMA-ab was used as a positive control for PSMA binding. Cells were plated at 1×10 5 The concentration of cells / ml was inoculated on poly-D-lysin-coated Chamber slides (Sigma-Aldrich). After 72 hours of culture, cells were fixed with methanol (5min, -20°C), followed by blocking (5% fetal horse serum in PBS, 1h) and incubated with 50nM PSMA binding protein for 45 minutes at room temperature. PSMA binding was detected by incubation with rabbit anti-streptococcal tag antibody (1:500) for 1 hour, followed by incubation with anti-rabbit-IgG-Alexa488-antibody (1:1000) for 1 hour. Cell nuclei were stained with 4μg / ml DAPI. All incubation steps were performed at room temperature. PSMA binding of dimer SEQID NO:54, dimer SEQ ID NO:55 and monomer SEQ ID NO:25 on LNCaP cells was confirmed, while binding to PC3 cells could not be observed.

[0149] Example 7. Functional Characterization: Binding of PSMA-binding Protein to PSMA Expressed on Tumor Tissue (Immunohistochemistry)

[0150] The binding proteins of the present invention were analyzed using tissue sections of frozen LNCaP-xenograft-tumor and F9-isograft-tumor sections. The tissue sections were fixed with ice-cold acetone for 10 minutes. The dimer of the PSMA binding protein includes a C-terminal streptococcal tag (see, e.g., SEQ ID NO:71) for purification and detection purposes. After blocking and incubation with 50nM and 10nM dimers of SEQ ID NO:54, 100nM and 10nM dimers of SEQ ID NO:52 and SEQ ID NO:53, and 100nM control protein unmodified diubiquitin, the sections were incubated with rabbit anti-streptococcal tag antibody (1:500) for 1 hour. The sections were then treated with NovolinkTM polymer (Leica, RE7290-CE). The sections were incubated with AEC solution (DAKO) for 1 minute to visualize the binding of the protein. Nuclei were stained with Mayer's hematoxylin alum solution (Merck Millipore, cat. no. 109249). All incubation steps were performed at room temperature. 2 μg / ml anti-PSMA-ab GCP-04 (Novus Biologicals) and 14 μg / ml GCP-05 (Thermo Scientific) were used as positive controls. Strong PSMA binding of 50 nM heterodimer (SEQ ID NO: 54) and 100 nM homodimer (SEQ ID NO: 52 and SEQ ID NO: 53) on LNCaP-tumor tissue was confirmed. Anti-PSMA-antibodies GCP-04 and GCP-05 showed the same staining pattern, while unmodified ubiquitin showed no staining. No nonspecific staining on F9-tumor tissue was observed.

[0151] Example 8. Competition analysis of PSMA binding proteins

[0152] To investigate whether the isolated PSMA-Affilin proteins bind to the same or different PSMA epitopes, the following assays were performed: PSMA-Fc fusion proteins (60 nM) were immobilized on a CM5 Biacore chip coupled to recombinant protein A using NHS / EDC chemistry, yielding 1000 response units (RU). In the first experiment, SEQ ID NO: 25 (motif KHNTW) and SEQ ID NO: 15 (motifs GFAHR and WTTTF) were injected at a defined concentration (0.5 μM) at a flow rate of 30 μl / min PBST 0.005% Tween 20. In the second experiment, the same flow channel was first preloaded with 500 nM SEQ ID NO: 25 until the chip surface was saturated. In the next step, 500 nM SEQ ID NO: 15 was applied in the same way as in the first experiment. Alternatively, in a second experiment, the same flow channel was first preloaded with 500 nM SEQ ID NO: 15 until the chip surface was saturated, and then loaded with 500 nM SEQ ID NO:25.

[0153] This experiment showed that the binding of the PSMA binding protein with the motif KHNTW was not affected by the presence of the PSMA binding proteins with the motifs GFAHR and WTTTF and vice versa. Thus, no competition was observed, leading to the conclusion that these PSMA binding proteins bind to different or non-overlapping PSMA epitopes, i.e. to different surface exposed amino acids.

[0154] Example 9: Labeling of fusion protein with DOTA

[0155] The dimeric protein was incubated with a 20-fold excess of maleimide-DOTA (2,2',2"-(10-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, CheMatech) in 50 mM HEPES, 150 mM NaCl, 5 mM EDTA pH 7.0 for 3 hours at room temperature. To reduce metal ions that could interact with DOTA molecules, all columns and AKTA apparatus (GE Healthcare) were incubated with 0.1 M EDTA solution for 30 minutes. For solution preparation, only metal-free or metal-reduced fractions were used. After incubation, the elution was performed by gel filtration (Superdex S200, GE Healthcare) in 100 mM sodium acetate pH 5.0-5.8, the samples were separated from the unbound DOTA molecules. The samples of the labeled proteins were also incubated with 5 mM iron (II) chloride at room temperature for 1 hour to demonstrate that the DOTA molecules can be used for coupling with radioisotopes. After incubation, unbound iron was removed using a HiTrap desalting column (GE Healthcare). MALDI-TOF analysis was used to determine the degree of labeling.

[0156] Example 10: Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOF) mass spectrometry

[0157] Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOF MS) was performed as follows: the fusion protein was purified and concentrated using C18-P10-ZipTips (Millipore; Catalog No. ZTC18S096). The tip was washed with 0.1% (v / v) trifluoroacetic acid (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 (DHAP) matrix (Bruker, catalog No. 8231829). The mass of the fusion protein was measured on an autoflexTM speed mass spectrometer (Bruker). The autoflex speed mass spectrometer was tuned using protein calibration standards (Bruker, catalog No. 8206355 and catalog No. 8207234).

[0158] The fusion proteins with and without DOTA tagging were analyzed by MALDI-TOF mass spectrometry and the peaks were compared. MALDI-TOF analysis showed that DOTA molecules tagged to dimeric PSMA binding protein can be used to couple with iron(II) chloride molecules.

[0159] Although the fusion protein was labeled DThe labeling did not significantly affect the affinity of the fusion protein for the target. The results are summarized in Table 3.

[0160] Table 3. Affinity analysis of labeled PSMA binding proteins using SPR

[0161] SEQ ID NO: mark <![CDATA[Mr 计算 ]]> <![CDATA[Mr 实验 ]]> <![CDATA[Target affinity K D > 5 none 1.8nM 5 DOTA 4.2nM 52 none 43027Da 43019Da 2.94e-13M 52 DOTA 44080Da 44072Da 2.99e-14M 52 DOTA+Iron 44192Da 44178Da Not Detected (nd) 54 none 35209Da 35216Da 6.32e-12M 54 DOTA 36263Da 36266Da 6.39e-10M 54 DOTA+Iron 36374Da 36363Da Not Detected 55 none 27391Da 27384Da 8.27e-14M 55 DOTA 28444Da 28433Da 2.04e-13M 55 DOTA+Iron 28556Da 28535Da Not Detected

[0162] Example 11. Serum stability of PSMA binding protein (flow cytometry)

[0163] The stability of the PSMA binding protein even in the presence of serum was analyzed. The PSMA binding protein included a C-terminal FLAG-tag (DYKDDDDK; see, e.g., SEQ ID NO: 78) for purification and detection purposes. PSMA binding proteins based on ubiquitin mutant proteins with GFAHR or a motif with 80% identity thereto (e.g., SEQ ID NO: 4 or SEQ ID NO: 11) were incubated at 37°C for 0 or 24 hours in 100% mouse serum in a dilution series from 1 μM to 5.6 pM. 100 μl of Affilin-serum solution was used to analyze serum stability on HEK293-PSMA-cells. After removing the supernatant, binding was demonstrated with 1 μg / ml anti-Flag tag-ab (Sigma-Aldrich; F1804) and anti-mouse-IgG-Alexa488 (Invitrogen; A10680). FACS analysis confirmed PSMA binding even after 24 hours of incubation in mouse serum (see Table 4). Further PSMA binding proteins were tested and binding to PSMA was also confirmed in the presence of serum.

[0164] Table 4. Binding of PSMA-binding proteins in the presence of serum (flow cytometry)

[0165]

[0166] Example 12. Serum stability of PSMA binding protein (ELISA)

[0167] High binding 96-well plates (Greiner, 781061) were fixed with 2.5 μg / ml PSMA-Fc overnight at 4°C. A dilution series of Affilin-191871 (with C-terminal SAC; SEQ ID NO: 5) and SEQ ID NO: 5 Dota labeled with lutetium Lu3+ were incubated overnight at 37°C in 100% mouse serum. The ELISA plate was washed with 1xPBS and blocked with 3% BSA / 0.5% Tween / PBS for 2 hours at room temperature. The dilution series after incubation for 0 hours or 24 hours in the presence of serum were 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 using streptavidin-HRP (1: 10.000). PSMA-binding protein did not show significant K after 24 hours of serum incubation. D For example, ELISA analysis confirmed that the K of SEQ ID NO: 5 and SEQ ID NO: 5 Dota labeled with Lu3+ to PSMA was 1.0 fold higher than that of SEQ ID NO: 5 even after 24 hours of incubation in mouse serum. D was also 0.75 + / - 0.02 (compared to K for 0 hours in mouse serum). D is 0.53+ / -0.02). Sequence Listing <110> Navigo Proteins GmbH <120> New PSMA-specific binding proteins for cancer diagnosis and therapy <130> NP35 EP1 <160> 78 <170> PatentIn version 3.5 <210> 1 <211> 76 <212> PRT <213> Artificial sequence <220> <223> Ubiquitin wild type <400> 1 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> 2 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Diubiquitin <400> 2 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 Met Gln Ile Phe 65 70 75 80 Val Lys Thr Leu Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 3 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 162462 (CID167521) <400> 3 Met Gln Ile Phe Val Arg Thr Pro 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 Gly Phe Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Thr Thr Phe Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 4 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 191871 <400> 4 Met Gln Ile Phe Val Arg Thr Gln 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 Gly Trp Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val His Thr Asn Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Glu Thr Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 5 <211> 155 <212> PRT <213> Artificial Sequence <220> <223> Affilin 191871 with c-terminal conjugation site <400> 5 Met Gln Ile Phe Val Arg Thr Gln 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 Gly Trp Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val His Thr Asn Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Glu Thr Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala Ser Ala Cys 145 150 155 <210> 6 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 161421 <400> 6 Met Gln Ile Phe Val Arg Thr Asp 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 Gly Trp Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Thr Thr Phe Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 7 <211> 152 <212> PRT <213> Artificial sequence <220> <223> Affilin 164052 <400> 7 Met Gln Ile Phe Val Arg Thr Gly 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 Gly Phe Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Thr Thr Phe Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 8 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 161438 <400> 8 Met Gln Ile Phe Val Arg Thr His 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 Ser Phe Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Arg Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Thr Thr Phe Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 9 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 164058 <400> 9 Met Gln Ile Phe Val Arg Thr Ile 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 Gly Phe Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Thr Thr Phe Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 10 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 185036 <400> 10 Met Gln Ile Phe Val Arg 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 Gly Phe Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Leu Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Pro Ser Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 11 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 192444 <400> 11 Met Gln Ile Phe Val Arg 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 Thr 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 Gly Phe Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Leu Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Thr Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Pro Ser Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 12 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 191822 <400> 12 Met Gln Ile Phe Val Arg Thr Leu Thr Gly Gln 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 Ala Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gly Phe Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Leu Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Pro Ser Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 13 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 185678 <400> 13 Met Gln Ile Phe Val Arg 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 Gly Phe Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gly His Glu Tyr Leu Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 14 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 185152 <400> 14 Met Gln Ile Phe Val Arg Thr Met 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 Gly Phe Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gly Asp Gly Asp Val Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 15 <211> 152 <212> PRT <213> Artificial sequence <220> <223> Affilin 164668 (164000) <400> 15 Met Gln Ile Phe Val Arg Thr Met 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 Gly Phe Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Thr Thr Phe Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 16 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 185071 <400> 16 Met Gln Ile Phe Val Arg Thr Met 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 Trp Gly Gly 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Leu Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Pro Ser Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 17 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 157424 <400> 17 Met Gln Ile Phe Val Arg Thr Pro 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 Pro Ala 50 55 60 His Gln Leu His Leu Val Leu Arg Leu Cys Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Thr Thr Phe Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 18 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 157423 <400> 18 Met Gln Ile Phe Val Arg Thr Pro 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 Pro Ala 50 55 60 His Gln Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Arg Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Thr Thr Phe Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 19 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 161644 <400> 19 Met Gln Ile Phe Val Arg Thr Pro 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 Ser Tyr Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Thr Thr Phe Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 20 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 191829 <400> 20 Met Gln Ile Phe Val Arg Thr Gln 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 Gly Trp Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Ala Thr Phe Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Pro Thr Ile Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 21 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 185113 <400> 21 Met Gln Ile Phe Val Arg Thr Trp 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 Gly Phe Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr His Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gly Asp Gly Asp Val Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 22 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> Affilin 185104 <400> 22 Met Gln Ile Phe Val Arg Thr Trp 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 Gly Phe Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gly Asp Gly Asp Val Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 23 <211> 152 <212> PRT <213> Artificial sequence <220> <223> Affilin 162633 <400> 23 Met Gln Ile Phe Val Arg Thr Trp 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 Trp Trp Ala 50 55 60 Asp Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Thr Thr Phe Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 24 <211> 152 <212> PRT <213> Artificial sequence <220> <223> Affilin 163970 <400> 24 Met Gln Ile Phe Val Trp Thr Pro 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 Gly Phe Ala 50 55 60 His Leu Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Thr Thr Phe Leu His 130 135 140 Leu Val Leu Arg Leu Arg Ala Ala 145 150 <210> 25 <211> 82 <212> PRT <213> Artificial sequence <220> <223> Affilin 164667 (161984) <400> 25 Met Gln Ile Phe Val Lys Thr Leu Thr Phe Glu His Pro Ser Phe Gly 1 5 10 15 Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val 20 25 30 Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln His 35 40 45 Leu Tyr Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp 50 55 60 Tyr Asn Ile Lys His Asn Thr Trp Leu Glu Leu Met Leu Phe Leu Arg 65 70 75 80 Ala Ala <210> 26 <211> 82 <212> PRT <213> Artificial sequence <220> <223> Affilin 157434 <400> 26 Met Gln Ile Phe Val Lys Thr Leu Thr Asp Met Tyr Arg Phe Met Gly 1 5 10 15 Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val 20 25 30 Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Ile 35 40 45 Leu Trp Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp 50 55 60 Tyr Asn Ile Val Ala Tyr Arg Pro Leu Tyr Leu Thr Leu Ala Leu Arg 65 70 75 80 Ala Ala <210> 27 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> Afffilin 161717 <400> 27 Met Gln Ile Phe Val Lys Thr Leu Thr Phe Glu His His Ser Phe Gly 1 5 10 15 Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val 20 25 30 Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln His 35 40 45 Leu Tyr Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp 50 55 60 Tyr Asn Ile Lys His Asn Thr Trp Leu Glu Leu Met Leu Phe Leu Arg 65 70 75 80 Ala Ala <210> 28 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> Affilin 161776 <400> 28 Met Gln Ile Phe Val Lys Thr Leu Thr Phe Glu His Lys Ser Phe Gly 1 5 10 15 Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val 20 25 30 Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln His 35 40 45 Leu Tyr Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp 50 55 60 Tyr Asn Ile Lys His Asn Thr Trp Leu Glu Leu Met Leu Phe Leu Arg 65 70 75 80 Ala Ala <210> 29 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> Affilin 162055 <400> 29 Methionine Glutamine Isoleucine Phenylalanine Valine Lysine Threonine Leucine Threonine Phenylalanine Glutamic Acid Histidine Asparagine Serine Phenylalanine Glycine 1 5 10 15 Lysine Threonine Isoleucine Threonine Leucine Glutamic Acid Valine Glutamic Acid Proline Serine Aspartic Acid Threonine Isoleucine Glutamic Acid Asparagine Valine 20 25 30 Lysine Alanine Lysine Isoleucine Glutamine Aspartic Acid Lysine Glutamic Acid Glycine Isoleucine Proline Proline Aspartic Acid Glutamine Glutamine Histidine 35 40 45 Leucine Tyrosine Tryptophan Valine Glycine Lysine Glutamine Leucine Glutamic Acid Aspartic Acid Glycine Arginine Threonine Leucine Serine Aspartic Acid 50 55 60 Tyrosine Asparagine Isoleucine Lysine Histidine Asparagine Threonine Tryptophan Leucine Glutamic Acid Leucine Methionine Leucine Phenylalanine Leucine Arginine 65 70 75 80 Alanine Alanine <210> 30 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> Affilin 162102 <400> 30 Methionine Glutamine Isoleucine Phenylalanine Valine Lysine Threonine Leucine Threonine Proline Isoleucine Proline Proline Aspartic Acid Tryptophan Glycine 1 5 10 15 Lysine Threonine Isoleucine Threonine Leucine Glutamic Acid Valine Glutamic Acid Proline Serine Aspartic Acid Threonine Isoleucine Glutamic Acid Asparagine Valine 20 25 30 Lysine Alanine Lysine Isoleucine Glutamine Aspartic Acid Lysine Glutamic Acid Glycine Isoleucine Proline Proline Aspartic Acid Glutamine Glutamine Histidine 35 40 45 亮氨酸 酪氨酸 色氨酸 丙氨酸 甘氨酸 赖氨酸 谷氨酰胺 亮氨酸 谷氨酸 天冬氨酸 甘氨酸 精氨酸 苏氨酸 亮氨酸 丝氨酸 天冬氨酸 50 55 60 酪氨酸 天冬酰胺 异亮氨酸 赖氨酸 组氨酸 天冬酰胺 苏氨酸 色氨酸 亮氨酸 谷氨酸 亮氨酸 甲硫氨酸 亮氨酸 苯丙氨酸 亮氨酸 精氨酸 65 70 75 80 丙氨酸 丙氨酸 <210> 31 <211> 82 <212> PRT <213> Artificial sequence <220> <223> Affilin 161912 <400> 31 甲硫氨酸 谷氨酰胺 异亮氨酸 苯丙氨酸 缬氨酸 赖氨酸 苏氨酸 亮氨酸 苏氨酸 脯氨酸 脯氨酸 苯丙氨酸 丙氨酸 苯丙氨酸 色氨酸 甘氨酸 1 5 10 15 赖氨酸 苏氨酸 异亮氨酸 苏氨酸 亮氨酸 谷氨酸 缬氨酸 谷氨酸 脯氨酸 丝氨酸 天冬氨酸 苏氨酸 异亮氨酸 谷氨酸 天冬酰胺 缬氨酸 20 25 30 赖氨酸 丙氨酸 赖氨酸 异亮氨酸 谷氨酰胺 天冬氨酸 赖氨酸 谷氨酸 甘氨酸 异亮氨酸 脯氨酸 脯氨酸 天冬氨酸 谷氨酰胺 谷氨酰胺 组氨酸 35 40 45 亮氨酸 酪氨酸 色氨酸 丙氨酸 甘氨酸 赖氨酸 谷氨酰胺 亮氨酸 谷氨酸 天冬氨酸 甘氨酸 精氨酸 苏氨酸 亮氨酸 丝氨酸 天冬氨酸 50 55 60 酪氨酸 天冬酰胺 异亮氨酸 赖氨酸 组氨酸 天冬酰胺 苏氨酸 色氨酸 亮氨酸 谷氨酸 亮氨酸 甲硫氨酸 亮氨酸 苯丙氨酸 亮氨酸 精氨酸 65 70 75 80 丙氨酸 丙氨酸 <210> 32 <211> 82 <212> PRT <213> Artificial sequence <220> <223> Affilin 162034 <400> 32 Methionine Glutamine Isoleucine Phenylalanine Valine Lysine Threonine Leucine Threonine Proline Glutamine Proline Proline Glutamic Acid Phenylalanine Glycine 1 5 10 15 Lysine Threonine Isoleucine Threonine Leucine Glutamic Acid Valine Glutamic Acid Proline Serine Aspartic Acid Threonine Isoleucine Glutamic Acid Asparagine Valine 20 25 30 Lysine Alanine Lysine Isoleucine Glutamine Aspartic Acid Lysine Glutamic Acid Glycine Isoleucine Proline Proline Aspartic Acid Glutamine Glutamine Histidine 35 40 45 Leucine Tyrosine Tryptophan Alanine Glycine Lysine Glutamine Leucine Glutamic Acid Aspartic Acid Glycine Arginine Threonine Leucine Serine Aspartic Acid 50 55 60 Tyrosine Asparagine Isoleucine Lysine Histidine Asparagine Threonine Tryptophan Leucine Glutamic Acid Leucine Methionine Leucine Phenylalanine Leucine Arginine 65 70 75 80 Alanine Alanine <210> 33 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> Affilin 161843 <400> 33 Methionine Glutamine Isoleucine Phenylalanine Valine Lysine Threonine Leucine Threonine Proline Glutamine Proline Proline Glutamic Acid Tryptophan Glycine 1 5 10 15 Lysine Threonine Isoleucine Threonine Leucine Glutamic Acid Valine Glutamic Acid Proline Serine Aspartic Acid Threonine Isoleucine Glutamic Acid Asparagine Valine 20 25 30 Lysine Alanine Lysine Isoleucine Glutamine Aspartic Acid Lysine Glutamic Acid Glycine Isoleucine Proline Proline Aspartic Acid Glutamine Glutamine Histidine 35 40 45 亮氨酸 酪氨酸 色氨酸 丙氨酸 甘氨酸 赖氨酸 谷氨酰胺 亮氨酸 谷氨酸 天冬氨酸 甘氨酸 精氨酸 苏氨酸 亮氨酸 丝氨酸 天冬氨酸 50 55 60 酪氨酸 天冬酰胺 异亮氨酸 赖氨酸 组氨酸 天冬酰胺 苏氨酸 色氨酸 亮氨酸 谷氨酸 亮氨酸 甲硫氨酸 亮氨酸 苯丙氨酸 亮氨酸 精氨酸 65 70 75 80 丙氨酸 丙氨酸 <210> 34 <211> 82 <212> PRT <213> Synthetic sequence <220> <223> Affilin 161374 <400> 34 甲硫氨酸 谷氨酰胺 异亮氨酸 苯丙氨酸 缬氨酸 赖氨酸 苏氨酸 亮氨酸 苏氨酸 脯氨酸 谷氨酰胺 脯氨酸 脯氨酸 谷氨酸 酪氨酸 甘氨酸 1 5 10 15 赖氨酸 苏氨酸 异亮氨酸 苏氨酸 亮氨酸 谷氨酸 缬氨酸 谷氨酸 脯氨酸 丝氨酸 天冬氨酸 苏氨酸 异亮氨酸 谷氨酸 天冬酰胺 缬氨酸 20 25 30 赖氨酸 丙氨酸 赖氨酸 异亮氨酸 谷氨酰胺 天冬氨酸 赖氨酸 谷氨酸 甘氨酸 异亮氨酸 脯氨酸 脯氨酸 天冬氨酸 谷氨酰胺 谷氨酰胺 组氨酸 35 40 45 亮氨酸 酪氨酸 色氨酸 丙氨酸 甘氨酸 赖氨酸 谷氨酰胺 亮氨酸 谷氨酸 天冬氨酸 甘氨酸 精氨酸 苏氨酸 亮氨酸 丝氨酸 天冬氨酸 50 55 60 酪氨酸 天冬酰胺 异亮氨酸 赖氨酸 组氨酸 天冬酰胺 苏氨酸 色氨酸 亮氨酸 谷氨酸 亮氨酸 甲硫氨酸 亮氨酸 苯丙氨酸 亮氨酸 精氨酸 65 70 75 80 丙氨酸 丙氨酸 <210> 35 <211> 76 <212> PRT <213> Synthetic sequence <220> <223> Affilin 187093 <400> 35 Methionine Glutamine Isoleucine Phenylalanine Valine Tryptophan Threonine Arginine Glutamic Acid Phenylalanine Lysine Glutamine Isoleucine Threonine Leucine Glutamic Acid 1 5 10 15 Valine Glutamic Acid Proline Serine Aspartic Acid Threonine Isoleucine Glutamic Acid Asparagine Valine Lysine Alanine Lysine Isoleucine Glutamine Aspartic Acid 20 25 30 Lysine Glutamic Acid Glycine Isoleucine Proline Proline Aspartic Acid Glutamine Glutamine Methionine Leucine Phenylalanine Tryptophan Lysine Glycine Lysine 35 40 45 Glutamine Leucine Glutamic Acid Aspartic Acid Glycine Arginine Threonine Leucine Serine Aspartic Acid Tyrosine Asparagine Isoleucine Tryptophan Tryptophan Asparagine 50 55 60 Proline Asparagine Leucine Serine Leucine Asparagine Leucine Aspartic Acid Leucine Arginine Alanine Alanine 65 70 75 <210> 36 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> Affilin 187092 <400> 36 Methionine Glutamine Isoleucine Phenylalanine Valine Tyrosine Threonine Alanine Glutamic Acid Leucine Lysine Glutamine Isoleucine Threonine Leucine Glutamic Acid 1 5 10 15 Valine Glutamic Acid Proline Serine Aspartic Acid Threonine Isoleucine Glutamic Acid Asparagine Valine Lysine Alanine Lysine Isoleucine Glutamine Aspartic Acid 20 25 30 Lysine Glutamic Acid Glycine Isoleucine Proline Proline Aspartic Acid Glutamine Glutamine Methionine Leucine Phenylalanine Tryptophan Lysine Glycine Lysine 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asn Leu Asp Leu Arg Ala Ala 65 70 75 <210> 37 <211> 76 <212> PRT <213> Synthetic sequence <220> <223> Affilin 187064 <400> 37 Met Gln Ile Phe Val Tyr Thr Lys Gln Leu Lys Gln 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 Lys Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asn Leu Asp Leu Arg Ala Ala 65 70 75 <210> 38 <211> 76 <212> PRT <213> Synthetic sequence <220> <223> Affilin 187154 <400> 38 Met Gln Ile Phe Val Tyr Thr Arg Ala Leu Lys Gln Ile Thr Leu Glu 1 5 10 15 Val Gly Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Arg Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Met Leu Phe Trp Lys Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asp Leu Asp Leu Arg Ala Ala 65 70 75 <210> 39 <211> 76 <212> PRT <213> Synthetic Sequence <220> <223> Affilin 187165 <400> 39 Met Gln Ile Phe Val Tyr Thr Arg Ala Leu Lys Gln Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Val 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 Lys Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asp Leu Asp Leu Arg Ala Ala 65 70 75 <210> 40 <211> 76 <212> PRT <213> artificial sequence <220> <223> Affilin 187191 <400> 40 Met Gln Ile Phe Val Tyr Thr Arg Ala Leu Lys Gln 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 Lys Gly Arg 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asn Leu Asp Leu Arg Ala Ala 65 70 75 <210> 41 <211> 76 <212> PRT <213> artificial sequence <220> <223> Affilin 187210 <400> 41 Met Gln Ile Phe Val Tyr Thr Arg Ala Leu Lys Gln 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 Arg Glu Gly Ile Pro Pro Asp Gln Gln Met Leu Phe Trp Lys Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asn Leu Asp Leu Arg Ala Ala 65 70 75 <210> 42 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> Affilin 187131 <400> 42 Met Gln Ile Phe Val Tyr Thr Arg Ala Leu Lys Gln 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 Lys Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asn Leu Gly Leu Arg Ala Ala 65 70 75 <210> 43 <211> 76 <212> PRT <213> Artificial sequence <220> <223> Affilin 187170 <400> 43 Met Gln Ile Phe Val Tyr Thr Arg Ala Leu Lys Gln 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 Lys Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asn Leu Asn Leu Arg Ala Ala 65 70 75 <210> 44 <211> 76 <212> PRT <213> Artificial sequence <220> <223> Affilin 187096 <400> 44 Met Gln Ile Phe Val Tyr Thr Arg Glu Leu Lys Gln 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 Lys Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asn Leu Asp Leu Arg Ala Ala 65 70 75 <210> 45 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> Affilin 181514 <400> 45 Met Gln Ile Phe Val Tyr Thr Arg Val Leu Lys Gln 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 Lys Trp Lys Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asn Leu Asp Leu Arg Ala Ala 65 70 75 <210> 46 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> Affilin 181515 <400> 46 Met Gln Ile Phe Val Tyr Thr Arg Val Leu Lys Gln 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 Lys Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asn Leu Asp Leu Arg Ala Ala 65 70 75 <210> 47 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> Affilin 187134 <400> 47 Met Gln Ile Phe Val Tyr Thr Arg Val Leu Lys Gln Ile Thr Leu Ala 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 Lys Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asn Leu Asp Leu Arg Ala Ala 65 70 75 <210> 48 <211> 76 <212> PRT <213> artificial sequence <220> <223> Affilin 187186 <400> 48 Met Gln Ile Phe Val Tyr Thr Arg Val Leu Lys Gln Ile 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 Met Leu Phe Trp Lys Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asn Leu Asp Leu Arg Ala Ala 65 70 75 <210> 49 <211> 76 <212> PRT <213> artificial sequence <220> <223> Affilin 187283 <400> 49 Met Gln Ile Phe Val Tyr Thr Arg Val Leu Lys Gln 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 Lys Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asn Arg Asp Leu Arg Ala Ala 65 70 75 <210> 50 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> Affilin 187295 <400> 50 Met Gln Ile Phe Val Tyr Thr Arg Val Leu Lys Gln 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 Lys Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asn Leu Asp Arg Arg Ala Ala 65 70 75 <210> 51 <211> 76 <212> PRT <213> Artificial sequence <220> <223> Affilin 187178 <400> 51 Met Gln Ile Phe Val Tyr Thr Arg Val Leu Lys Gln 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 Thr Pro Pro Asp Gln Gln Thr Leu Phe Trp Arg Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Trp Asn 50 55 60 Pro Asn Leu Ser Leu Asn Leu Asp Leu Arg Ala Ala 65 70 75 <210> 52 <211> 404 <212> PRT <213> Artificial sequence <220> <223> Homodimer of 167528 (162462) <400> 52 Met Gln Ile Phe Val Arg Thr Pro 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 Gly Phe Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Thr Thr Phe Leu His 130 135 140 Leo Val Leo Arg Leo Arg Ala Ala Ser Ala Pro Ala Ala Ser Pro Ser 145 150 155 160 Pro Ala Ala Pro Ala Pro Ser Pro Ala Ser Pro Ala Pro Ala Ser Pro 165 170 175 Ala Ser Ala Pro Ser Ala Pro Ala Ser Ala Pro Pro Ala Ala Ser Ala 180 185 190 Met Gln Ile Phe Val Arg Thr Pro Thr Gly Lys Thr Ile Thr Leu Glu 195 200 205 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 210 215 220 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys 225 230 235 240 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gly Phe Ala 245 250 255 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 260 265 270 Val Met Thr Gln Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser 275 280 285 Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile 290 295 300 Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 305 310 315 320 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Thr Thr Phe Leu His 325 330 335 Leu Val Leu Arg Leu Arg Ala Ala Ser Ala Pro Ala Pro Ser Ala Pro 340 345 350 Ala Ala Ser Ala Pro Pro Ala Pro Ala Ala Pro Cys Ala Pro Ala Ala 355 360 365 Pro Ala Ser Ala Pro Ala Pro Ala Ser Ala Pro Ala Ala Ser Pro Cys 370 375 380 Pro Ala Ala Pro Ala Pro Ser Pro Ala Ser Pro Ala Pro Ala Ser Pro 385 390 395 400 Ala Ser Ala Pro <210> 53 <211> 264 <212> PRT <213> Artificial sequence <220> <223> Homopolymer 161984 (164661) <400> 53 Met Gln Ile Phe Val Lys Thr Leu Thr Phe Glu His Pro Ser Phe Gly 1 5 10 15 Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val 20 25 30 Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln His 35 40 45 Leu Tyr Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp 50 55 60 Tyr Asn Ile Lys His Asn Thr Trp Leu Glu Leu Met Leu Phe Leu Arg 65 70 75 80 Ala Ala Ser Ala Pro Ala Ala Ser Pro Ser Pro Ala Ala Pro Ala Pro 85 90 95 Ser Pro Ala Ser Pro Ala Pro Ala Ser Pro Ala Ser Ala Pro Ser Ala 100 105 110 Pro Ala Ser Ala Pro Pro Ala Ala Ser Ala Met Gln Ile Phe Val Lys 115 120 125 Thr Leu Thr Phe Glu His Pro Ser Phe Gly Lys Thr Ile Thr Leu Glu 130 135 140 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 145 150 155 160 Lys Glu Gly Ile Pro Pro Asp Gln Gln His Leu Tyr Trp Ala Gly Lys 165 170 175 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Lys His Asn 180 185 190 Thr Trp Leu Glu Leu Met Leu Phe Leu Arg Ala Ala Ser Ala Pro Ala 195 200 205 Pro Ser Ala Pro Ala Ala Ser Ala Pro Pro Ala Pro Ala Ala Pro Cys 210 215 220 Ala Pro Ala Ala Pro Ala Ser Ala Pro Ala Pro Ala Ser Ala Pro Ala 225 230 235 240 Ala Ser Pro Cys Pro Ala Ala Pro Ala Pro Ser Pro Ala Ser Pro Ala 245 250 255 Pro Ala Ser Pro Ala Ser Ala Pro 260 <210> 54 <211> 334 <212> PRT <213> Artificial Sequence <220> <223> Heterodimer 42 of 161984 and 162462 (CID167526) <400> 54 Met Gln Ile Phe Val Lys Thr Leu Thr Phe Glu His Pro Ser Phe Gly 1 5 10 15 Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val 20 25 30 Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln His 35 40 45 Leu Tyr Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp 50 55 60 Tyr Asn Ile Lys His Asn Thr Trp Leu Glu Leu Met Leu Phe Leu Arg 65 70 75 80 Ala Ala Ser Ala Pro Ala Ala Ser Pro Ser Pro Ala Ala Pro Ala Pro 85 90 95 Ser Pro Ala Ser Pro Ala Pro Ala Ser Pro Ala Ser Ala Pro Ser Ala 100 105 110 Pro Ala Ser Ala Pro Pro Ala Ala Ser Ala Met Gln Ile Phe Val Arg 115 120 125 Thr Pro Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr 130 135 140 Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro 145 150 155 160 Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg 165 170 175 Thr Leu Ser Asp Tyr Asn Ile Gly Phe Ala His Arg Leu His Leu Val 180 185 190 Leu Arg Leu Arg Ala Ala Met Gln Ile Phe Val Met Thr Gln Thr Gly 195 200 205 Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val 210 215 220 Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg 225 230 235 240 Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp 245 250 255 Tyr Asn Ile Trp Thr Thr Thr Phe Leu His Leu Val Leu Arg Leu Arg 260 265 270 Ala Ala Ser Ala Pro Ala Pro Ser Ala Pro Ala Ala Ser Ala Pro Pro 275 280 285 Ala Pro Ala Ala Pro Cys Ala Pro Ala Ala Pro Ala Ser Ala Pro Ala 290 295 300 Pro Ala Ser Ala Pro Ala Ala Ser Pro Cys Pro Ala Ala Pro Ala Pro 305 310 315 320 Ser Pro Ala Ser Pro Ala Pro Ala Ser Pro Ala Ser Ala Pro 325 330 <210> 55 <211> 334 <212> PRT <213> Artificial Sequence <220> <223> Heterodimer 24 162462 and 161984 (CID167525) <400> 55 Met Gln Ile Phe Val Arg Thr Pro 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 Gly Phe Ala 50 55 60 His Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Met Gln Ile Phe 65 70 75 80 Val Met Thr Gln Thr Gly Lys Thr 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 Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp 115 120 125 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Thr Thr Thr Phe Leu His 130 135 140 Leo Val Leo Arg Leo Arg Ala Ala Ser Ala Pro Ala Ala Ser Pro Ser 145 150 155 160 Pro Ala Ala Pro Ala Pro Ser Pro Ala Ser Pro Ala Pro Ala Ser Pro 165 170 175 Ala Ser Ala Pro Ser Ala Pro Ala Ser Ala Pro Pro Ala Ala Ser Ala 180 185 190 Met Gln Ile Phe Val Lys Thr Leu Thr Phe Glu His Pro Ser Phe Gly 195 200 205 Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val 210 215 220 Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln His 225 230 235 240 Leu Tyr Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp 245 250 255 Tyr Asn Ile Lys His Asn Thr Trp Leu Glu Leu Met Leu Phe Leu Arg 260 265 270 Ala Ala Ser Ala Pro Ala Pro Ser Ala Pro Ala Ala Ser Ala Pro Pro 275 280 285 Ala Pro Ala Ala Pro Cys Ala Pro Ala Ala Pro Ala Ser Ala Pro Ala 290 295 300 Pro Ala Ser Ala Pro Ala Ala Ser Pro Cys Pro Ala Ala Pro Ala Pro 305 310 315 320 Ser Pro Ala Ser Pro Ala Pro Ala Ser Pro Ala Ser Ala Pro 325 330 <210> 56 <211> 5 <212> PRT <213> artificial sequence <220> <223> Motif <400> 56 Gly Phe Ala His Arg 1 5 <210> 57 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Motif <400> 57 Gly Trp Ala His Arg 1 5 <210> 58 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Motif <400> 58 Trp Thr Thr Thr Phe 1 5 <210> 59 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Motif <400> 59 Lys His Asn Thr Trp 1 5 <210> 60 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Motif <400> 60 Val Ala Tyr Arg Pro 1 5 <210> 61 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Motif <400> 61 Trp Trp Asn Pro Asn 1 5 <210> 62 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Connectors <400> 62 Ser Ala Pro Ala Ala Ser Pro Ser Pro Ala Ala Pro Ala Pro Ser Pro 1 5 10 15 Ala Ser Pro Ala Pro Ala Ser Pro Ala Ser Ala Pro Ser Ala Pro Ala 20 25 30 Ser Ala Pro Pro Ala Ala Ser Ala 35 40 <210> 63 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Connectors <400> 63 Pro Ala Ala Pro Ala Pro Ser Pro Ala Ser Pro Ala Pro Ala Ser Pro 1 5 10 15 Ala Ser Ala Pro Ser 20 <210> 64 <211> 61 <212> PRT <213> Artificial sequence <220> <223> Coupling domain <400> 64 Ser Ala Pro Ala Pro Ser Ala Pro Ala Ala Ser Ala Pro Pro Ala Pro 1 5 10 15 Ala Ala Pro Cys Ala Pro Ala Ala Pro Ala Ser Ala Pro Ala Pro Ala 20 25 30 Ser Ala Pro Ala Ala Ser Pro Cys Pro Ala Ala Pro Ala Pro Ser Pro 35 40 45 Ala Ser Pro Ala Pro Ala Ser Pro Ala Ser Ala Pro Ser 50 55 60 <210> 65 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Coupling domain <400> 65 Ser Ala Pro Ala Pro Ser Ala Pro Ala Ala Ser Ala Pro Pro Ala Pro 1 5 10 15 Ala Ala Pro Cys Ala Pro Ala Ala Pro Ala Ser Ala Pro Ala Pro Ala 20 25 30 Ser Ala Pro Ala Ala Ser Pro Cys 35 40 <210> 66 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Coupling domain <400> 66 Ser Ala Pro Ala Pro Ser Ala Pro Ala Ala Ser Ala Pro Pro Ala Pro 1 5 10 15 Ala Ala Pro Cys 20 <210> 67 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Coupling domain <400> 67 Ala Pro Ala Ala Pro Ala Ser Ala Pro Ala Pro Ala Ser Ala Pro Ala 1 5 10 15 Ala Ser Pro Cys 20 <210> 68 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Coupling domain <400> 68 Ser Ala Pro Ala Pro Ser Ala Pro Ala Ala Ser Ala Pro Pro Ala Pro 1 5 10 15 Ala Ala Pro Ala Ala Pro Ala Ala Pro Ala Ser Ala Pro Ala Pro Ala 20 25 30 Cys <210> 69 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Coupling domain <400> 69 Ala Pro Ala Ala Ser Pro Ser Pro Ala Ala Pro Ala Pro Ser Pro Ala 1 5 10 15 Ser Pro Ala Pro Ala Ser Pro Ala Ser Ala Pro Ser Ala Pro Ala Ser 20 25 30 Cys <210> 70 <211> 98 <212> PRT <213> Artificial sequence <220> <223> Coupling domain <400> 70 Ser Ala Pro Ala Pro Ser Ala Pro Ala Ala Ser Ala Pro Pro Ala Pro 1 5 10 15 Ala Ala Pro Ala Ala Pro Ala Ala Pro Ala Ser Ala Pro Ala Pro Ala 20 25 30 Pro Ala Pro Ala Ala Ser Pro Ser Pro Ala Ala Pro Ala Pro Ser Pro 35 40 45 Ala Ala Pro Ala Pro Ala Ser Pro Ala Ser Ala Pro Ser Ala Pro Ala 50 55 60 Ser Ala Pro Pro Ala Ala Pro Ser Ala Ala Ala Ser Pro Ala Pro Ser 65 70 75 80 Ala Pro Ala Pro Ala Ala Ser Pro Ala Ala Ala Pro Ala Ser Ala Ala 85 90 95 Pro Ala <210> 71 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Streptococcus <400> 71 Ser Ala Trp Ser His Pro Gln Phe Glu Lys 1 5 10 <210> 72 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Motif <400> 72 Ser Phe Ala His Arg 1 5 <210> 73 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Motif <400> 73 Ser Tyr Ala His Arg 1 5 <210> 74 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Motif <400> 74 Gly Phe Ala His Leu 1 5 <210> 75 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Motif <400> 75 Trp Thr Pro Ser Ile 1 5 <210> 76 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Motif <400> 76 Trp Thr Glu Thr Ile 1 5 <210> 77 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Motif <400> 77 Gly Asp Gly Asp Val 1 5 <210> 78 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Flag Tags <400> 78 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5

Claims

1. A prostate specific membrane antigen (PSMA) binding protein consisting of any one amino acid sequence selected from the group consisting of SEQ ID NO: 3-12, 15-19, 21, 23-25, 27-44, 47-55, wherein the PSMA binding protein has a specific binding affinity for the extracellular domain of PSMA of 500 nM or less.

2. The PSMA-binding protein according to claim 1, for use in diagnosing or treating PSMA-positive tumors.

3. The PSMA-binding protein according to claim 2, for use in PSMA-positive tumor imaging and radiotherapy treatment of PSMA-positive tumors.

4. A composition comprising the PSMA-binding protein according to any one of claims 1 to 3.

5. The composition according to claim 4, for use in the diagnosis or treatment of PSMA-positive tumors.

6. The composition according to claim 5, for use in PSMA-positive tumor imaging and radiotherapy treatment of PSMA-positive tumors.

7. A method for producing a PSMA-binding protein according to any one of claims 1 to 3, comprising the following steps: a) culturing the host cell under conditions suitable for obtaining the PSMA-binding protein and b) isolating the produced PSMA-binding protein.

Images