EA042392B1 - TYPE III FIBRONECTIN DOMAIN BINDING TO PROSTATE-SPECIFIC MEMBRANE ANTIGEN - Google Patents

Description

Scope of the invention

The present invention relates to molecules that bind to a prostate-specific membrane antigen, and to methods for the preparation and use of these molecules.

Prerequisites for the invention

Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II or N-acetylated alpha-linked acid dipeptidase 1, is a type 2 dimeric transmembrane glycoprotein. PSMA cleaves several substrates, including folate and Nacetyl-L-aspartyl L-glutamate, and is expressed in a number of tissues, with the highest expression observed in the prostate gland and less in the small intestine, central and peripheral nervous system, kidneys and lungs. PSMA is internalized constitutively through clathrin-coated pits.

PSMA is a well-studied cell membrane antigen associated with prostate cancer that is often overexpressed in prostate intraepithelial neoplasia (PIN), a condition in which some prostate cells begin to look and behave abnormally, primary and metastatic cancers, and in newly formed vessels of other solid tumors (eg, breast, lung, bladder, kidney) (Chang et al., Clin Cancer Res 5:2674-2681, 1999, Liu et al., Cancer Res 57:3629-3634, 1997 , Silver et al., Clin Cancer Res 3:81-85, 1997; Bostwick et al., Cancer 82:2256-2261, 1998). PSMA expression levels correlate with disease progression and Gleason scores. PSMA expression is increased in metastatic disease, hormone-refractory cases, and higher-grade lesions, and is further increased in androgen-insensitive tumors (Su et al., Cancer Res 55:1441-1443, 1995, Kawakami et al., 57:2321-2324 , 1997, Wright et al., Urology 48:326-334, 1996).

Prostate cancer is the main cancer in men and is the 2nd leading cause of death from cancer. Worldwide, there are approximately 1,100,000 new cases and 300,000 deaths each year, accounting for about 4% of all deaths due to cancer. It is estimated that out of every 6 men, 1 will be diagnosed with this disease. In the United States, more than 90% of prostate cancer cases are detected at a local or regional stage. In these early stages, the 5-year survival rate is almost 100%. However, with cancer metastasis, the 5-year survival rate drops to about 28%. Localized prostate cancer can often be contained by shutting down endocrine function.

Current treatments for prostate cancer include surgery, radiation, and hormone therapy. However, tumor cells often become androgen insensitive and a limited number of treatment options remain. Generally, the cancer vaccine sipuleucel-T, a radiopharmaceutical agent (such as radium-223 chloride), secondary hormone therapies (such as abiraterone or enzalutamide) and/or chemotherapeutic agents (docetaxel and cabazitaxel) are sequentially added to hormone therapy.

Monoclonal antibodies targeting PSMA have been evaluated in clinical use both as diagnostic imaging agents and as antibody drug conjugates. The most widely used PSMA-targeting antibody drug conjugates (ADCs) use the same humanized/deimmunized monoclonal antibody (mAb) to PSMA J591. At least three different ADCs that use J591 have been evaluated in clinical studies using different linkers and cytotoxic molecules. Millenium Pharmaceuticals has completed a phase 1 clinical study evaluating MLN2704, an anti-PSMA monoclonal antibody disulfide-conjugated to maytansine. Progenics has used Seattle Genetics technology to link J591 to monomethyl auristatin E (MMAE) using a valine-citrulline linker, and ADCT is starting clinical trials of a pyrrolobenzodiazepine (PBD) conjugate with J591. So far, limited clinical efficacy has been associated with severe toxicity and short serum half-lives, likely due to high hepatic uptake (Morris et al., Clin Cancer Res 13:27072713, 2007). However, two separate clinical studies have provided evidence of a decrease in PSA/CTC levels after re-treatment with ADC to PSMA, especially at high doses (D Petrylak, Genitourinary Cancers Symposium, 2014, Galsky et al., J Clin Oncol 26:2147- 2154, 2008, D Petrylak, ASCO 2014). In the case of Progenics, two dose-limiting toxic effects resulted in death due to sepsis due to neutropenia (D Petrylak, ASCO 2014).

Although each of these treatments is able to slow the growth of cancer for several months and reduce the symptoms caused by the disease, eventually the disease becomes resistant to them.

Thus, there is a need for additional and improved therapeutic agents for the treatment of prostate cancer and other PSMA overexpressing cancers.

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The essence of the invention

One embodiment of the invention is an isolated FN3 domain that specifically binds to the human prostate specific membrane antigen (PSMA) SEQ

ID NO: 144.

Another embodiment of the invention is an isolated FN3 domain that specifically binds to human PSMA SEQ ID NO: 144, wherein the FN3 domain cross-reacts with PSMA Macaca Fascicularis SEQ ID NO: 32 or PSMA Pan troglodytes SEQ ID NO: 33.

Another embodiment of the invention is an isolated FN3 domain that specifically binds to human PSMA SEQ ID NO: 144, where the FN3 domain contains an amino acid sequence that is 89% identical to the amino acid sequence of SEQ ID NO: 41 or that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 substitutions compared to the amino acid sequence of SEQ ID NO: 41.

Another embodiment of the invention is an isolated FN3 domain that specifically binds to human PSMA SEQ ID NO: 144; where the FN3 domain contains the amino acid sequence of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 or 140.

One embodiment of the invention is an isolated FN3 domain that specifically binds to human prostate-specific membrane antigen (PSMA) SEQ ID NO: 144 conjugated to a cytotoxic agent or a detectable label.

Another embodiment of the invention is an isolated polynucleotide encoding an FN3 domain of the invention.

Another embodiment of the invention is a vector containing a polynucleotide in accordance with the invention.

Another embodiment of the invention is a host cell containing a vector in accordance with the invention.

Another embodiment of the invention is a method for obtaining an FN3 domain of the invention, comprising culturing an isolated host cell of the invention under conditions that express the FN3 domain of the invention and purifying the FN3 domain.

Another embodiment of the invention is a pharmaceutical composition comprising an FN3 domain of the invention and a pharmaceutically acceptable carrier.

Another embodiment of the invention is a method of treating a patient having a cancer characterized by PSMA overexpression, which comprises administering a therapeutically effective amount of an FN3 domain of the invention conjugated to a cytotoxic agent to the patient for a period of time sufficient to treat the cancer.

Another embodiment of the invention is a diagnostic kit containing the FN3 domain of the invention. Another embodiment of the invention is a diagnostic or capture agent containing an FN3 domain of the invention.

Another embodiment of the invention is a method for detecting PSMA-expressing cells in a biological sample, comprising treating the biological sample with a diagnostic agent containing an FN3 domain of the invention and evaluating the binding of the biological sample to such a diagnostic agent containing an FN3 domain of the invention.

Another embodiment of the invention is a method for isolating PSMA-expressing cells in a biological sample, comprising treating the biological sample with an FN3 domain-containing capture agent of the invention and isolating a portion of the biological sample that binds to such an FN3-domain-containing capture agent.

Another embodiment of the invention is a method for detecting PSMA-expressing tumor cells in a patient, comprising administering to the patient an FN3 domain of the invention and detecting binding of the FN3 domain to PSMA-expressing tumor cells in the patient.

Another embodiment of the invention is a method for delivering a therapeutic molecule to PSMA-expressing tumor cells, comprising administering an FN3 domain of the invention to a patient having a PSMA-expressing tumor.

Brief description of the drawings

In FIG. 1 shows the biodistribution of untargeted ^89Zr -labeled centirin after intravenous administration to male NSG mice.

In FIG. 2A shows the overall crystal structure of the P233FR9_H10 (H10) FN3 PSMA binding domain in complex with the cynomolgus monkey PSMA dimer, demonstrating that H10 binds to the region near the PSMA active site. Zinc atoms (Zn) indicate the position of the PSMA active site, N- and C-termini of the PSMA and H10 molecules are indicated for one of the complexes. The approximate position of the cell membrane is indicated.

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In FIG. 2B shows the crystal structure of the H10 FN3 domain in complex with cynomolgus monkey PSMA. The beta chains A, B, C, D, E, F and G in the FN3 domain of H10 are shown. Shown are the negatively charged residues in the CD H10 loop (residues W38, D39, D40, D41 and E43) that are inserted into the positively charged entry of the PSMA active site. H10 residues are numbered according to SEQ ID NO:

41.

In FIG. 2C shows the crystal structure of the FN3 domain of H10 in complex with cynomolgus monkey PSMA. The figure shows contacting residues W38, D39, D40, D41 and E43 of the H10 domain. Shown are some of the cynomolgus monkey PSMA residues that contact H10 (R511, K514 and K545), coordinate zinc atoms (H377, D387, E424, E425, D453 and H553) or form an active site cavity (R536 and R534). The H10 beta chains C, D, F, and G are marked in the figure. The cynomolgus monkey H10 and PSMA residue numbers correspond to SEQ ID NOS: 41 and 141, respectively.

In FIG. 3A shows a close-up view of the crystal structure integrating the site between the H10 FN3 domain and the cynomolgus monkey PSMA. The contacting residues A32, W36, W38-D41, E43, A44, V46, G64, P68, Y70, A72, W79, F81, P82, A85 and I86 of the H10 FN3 domain are shown. Contact residues Y460, K499-P5O2, P504, R511, K514, N540, W541, K545, F546, F488, K610, N613 and I614 PSMA of the cynomolgus monkey are shown. The numbering of H10 and PSMA residues of the cynomolgus monkey corresponds to SEQ ID NOs: 41 and 141, respectively.

In FIG. 3B shows a map of the interactions between the FN3 domain of H10 and contacting cynomolgus monkey PSMA residues. A 4 A cutoff was used to determine the contact residues. Centirin and cynomolgus monkey PSMA residues are shown in gray and white boxes, respectively, van der Waals interactions are shown as dotted lines, and hydrogen bonds are shown as solid lines with arrows indicating backbone hydrogen bonds and pointing to backbone atoms. Residue numbering according to SEQ ID NO: 41 (H10) and SEQ ID NO: 141 (PSMA cynomolgus monkey).

In FIG. 4A shows the amino acid sequence alignment of the human (h) and cynomolgus monkey (c) PSMA extracellular domains. Contacting H10 residues are underlined and in bold. Residues that differ in human and cynomolgus monkey PSMA are shaded. All cynomolgus PSMA residues interacting with H10 are conserved in human PSMA with the exception of N613. Extracellular domain (ECD) of human PSMA; SEQ ID NO: 143. Cynomolgus monkey ECD PSMA: SEQ ID NO: 32

In FIG. 4B shows H10 FN3 domain residues coming into contact with cynomolgus monkey PSMA. Contact residues are underlined and in bold. The amino acid sequence of H10 is shown in SEQ ID NO: 41.

In FIG. 5 shows the position of residues N6, R11, T22, D25, A26, S52, E53, K62 of centirin H10 and the N and C termini, which are possible sites for chemical conjugation, in the crystal structure of H10 associated with cynomolgus monkey PSMA. Centrin/PSMA contact regions are shown in black. The beta chains of H10 C, D, F and G are marked in the figure. Residues are numbered according to SEQ ID NO: 41 (H10).

In FIG. 6 shows a comparison of the mean fluorescence intensity (MFI) of various tumor cell lines stained with a centirin-phycoerythrin (PE) conjugate to PSMA (black) and an anti-PSMA antibody conjugated to PE (white).

In FIG. 7A shows a series of images in the CellTracks Analyzer II renderer and viewer showing LNCaP cells stained with DAPI, FITC-conjugated anti-cytokeratin antibody, APC-conjugated anti-CD45 antibody, and anti-PSMA centirin-PE conjugate. The thumbnails show from right to left: PSMA-PE staining, CD45-APC signal, DAPI-stained nuclei, cytokeratin-FITC reactivity, and finally, overlay of cytokeratin-FITC and DAPI staining. A cell must have a nucleus, express cytokeratin, and be CD45 negative to be registered as CTC. The CTC must have a positive PSMA signal to be counted as a PSMA-positive CTC.

In FIG. 7B shows a series of images in the CellTracks Analyzer II renderer and viewer showing 22Rv1 cells stained with DAPI, FITC-conjugated anti-cytokeratin antibody, APC-conjugated anti-CD45 antibody, and anti-PSMA centirin-RE conjugate. The thumbnails show from right to left PSMA-PE staining, CD45-APC signal, DAPI-stained nuclei, cytokeratin-FITC reactivity, and finally, cytokeratin-FITC and DAPI staining overlay. A cell must have a nucleus, express cytokeratin, and be CD45 negative to be registered as CTC. The CTC must have a positive PSMA signal to be counted as a PSMA-positive CTC.

In FIG. 7C shows a series of images in the CellTracks Analyzer II renderer and viewer showing PC3 cells stained with DAPI, FITC-conjugated anti-cytokeratin antibody, APC-conjugated anti-CD45 antibody, and anti-PSMA centirin-PE conjugate. The thumbnails show from right to left PSMA-PE staining, CD45-APC signal, DAPI-stained nuclei, cytokeratin-FITC reactivity, and finally, cytokeratin-FITC and DAPI staining overlay.

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A cell must have a nucleus, express cytokeratin, and be CD45 negative to be registered as CTC. The CTC must have a positive PSMA signal to be counted as a PSMA-positive CTC.

In FIG. 7D shows a series of images in the CellTracks Analyzer II renderer and viewer showing SKBR3 cells stained with DAPI, FITC-conjugated anti-cytokeratin antibody, APC-conjugated anti-CD45 antibody, and anti-PSMA centirin-PE conjugate. The thumbnails show from right to left PSMA-PE staining, CD45-APC signal, DAPI-stained nuclei, cytokeratin-FITC reactivity, and finally, cytokeratin-FITC and DAPI staining overlay. A cell must have a nucleus, express cytokeratin, and be CD45 negative to be registered as CTC. The CTC must have a positive PSMA signal to be counted as a PSMA-positive CTC.

Detailed description of the invention

In the context of the present invention, the term fibronectin type III (FN3) domain (FN3 domain) refers to a domain frequently found in proteins, including fibronectins, tenascin, intracellular cytoskeletal proteins, cytokine receptors, and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad Sci USA 89 :8990-8994, 1992; Meinke et al., J Bacteriol 175:1910-1918, 1993; Watanabe et al., J Biol Chem 265:15659-15665, 1990). Examples of FN3 domains are 15 different FN3 domains present in human tenascin C, 15 different FN3 domains present in human fibronectin (FN), and non-naturally occurring synthetic FN3 domains as described in, for example, US Pat. No. 8,278,419. Individual FN3 domains are designated by domain number and protein name, for example tenascin FN3 domain 3 (TN3) or fibronectin FN3 domain 10 (FN10).

In the context of the present invention, the term centirin refers to an FN3 domain based on a consensus sequence of 15 different FN3 domains present in human tenascin C.

The term capture agent refers to substances that bind to a specific cell type and allow that cell to be separated from other cells. Examples of capture agents include, but are not limited to, magnetic beads, ferrofluids, encapsulating agents, and the like.

The term biological sample refers to blood, tissue, bone marrow, sputum, and the like.

The term diagnostic reagent refers to any substance that can be used to analyze a biological sample, whether such substance is supplied in a diagnostic kit as a single substance or in combination with other substances.

In the context of the present invention, the term substitution or substituted, mutation or mutated refers to a change, deletion or insertion of one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to create a variant of that sequence.

The term randomization, randomized, diversified or diversified in the context of the present invention refers to making at least one substitution, insertion or deletion in the sequence of a polynucleotide or polypeptide.

In the context of the present invention, the term variant refers to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications, such as substitutions, insertions or deletions.

In the context of the present invention, the term specifically binds or specific binding refers to the ability of the FN3 domain of the invention to bind to a given antigen with a dissociation constant (KD) of about 1x10'6 M or less, for example about ^1x10'7 M or less, about 1x10'8 M or less than, about 1x10' ⁹ M or less, about 1x10' ¹⁰ M or less, about 1x10'1 M or less, about 1x10' ¹² M or less, or about 1x10' ¹³ M or less. Typically, the FN3 domain of the invention binds to a given antigen (i.e., human PSMA) with a K _D that is at least ten times smaller than its K _D for a non-specific antigen (i.e., BSA or casein) as measured by surface plasmon resonance using, for example, Proteon equipment (BioRad). However, an isolated FN3 domain of the invention that specifically binds to human PSMA may be cross-reactive with other related antigens, e.g. with the same given antigen of another species (homologs), e.g. Macaca Fascicularis (cynomolgus monkey, supo) or Pan troglodytes (chimpanzee) .

As used herein, the term epitope refers to the portion of an antigen to which the FN3 domain of the invention specifically binds. Typically, epitopes are composed of reactive (such as polar, nonpolar, or hydrophobic) surface groupings of moieties, such as amino acid or polysaccharide side chains, and they may have specific three-dimensional structure characteristics as well as specific charge characteristics. An epitope may be formed from contiguous and/or non-contiguous amino acids forming a conformational space block. In the case of a non-contiguous epitope, amino acids from different regions of the linear sequence of the antigen come close to each other in three-dimensional space due to the folding of the protein molecule.

The term library in the present description refers to a group of options. The library may consist of polypeptide or polynucleotide variants.

In the context of the present invention, the term stability refers to the ability of a molecule to maintain its folded state under physiological conditions in such a way as to retain at least one of its normal functionality, eg the ability to bind to a given antigen, such as human PSMA.

Human PSMA in the context of the present invention refers to a well-known type II glycoprotein of about 100 kDa, having a short intracellular domain (residues 1-18), a transmembrane domain (residues 19-43) and an extracellular domain (residues 44-750). The amino acid sequence of mature human PSMA is shown in SEQ ID NO: 144.

In the context of the present invention, the terms overexpressed, overexpressed, and overexpressed, used interchangeably, refer to a cancerous or malignant cell that has elevated levels of PSMA on its surface compared to a normal cell of the same type of tissue. Such overexpression may be due to gene amplification or increased transcription or translation. PSMA overexpression can be measured using well known assays using, for example, ELISA, immunofluorescence, flow cytometry, or radioimmunoassay on live or lysed cells. Alternatively, or additionally, the levels of nucleic acid molecules encoding PSMA can be measured in the cell, for example, using fluorescence in situ hybridization, Southern blot, or PCR. PSMA overexpression means that the level of PSMA on the cell surface is at least 1.5 times higher than that of a normal cell.

In the context of the present invention, the term Tencon refers to a synthetic type III fibronectin (FN3) domain with the sequence shown in SEQ ID NO: 1 and described in US Patent Publication No. US 2010/0216708.

In the context of the present invention, the term cancer cell or tumor cell refers to a cancerous, precancerous or transformed cell, either in vivo or ex vivo, and in cell culture, which has spontaneous or induced phenotypic changes that do not necessarily involve the production of new genetic material. Although transformation may be caused by infection with a transforming virus and the insertion of a new genomic nucleic acid, or by ingestion of an exogenous nucleic acid, it may also occur spontaneously or after exposure to a carcinogen, resulting in mutation of an endogenous gene. Transformation/cancer manifests itself, for example, in morphological changes, cell immortalization, impaired growth control, foci formation, proliferation, malignancy, levels of tumor-specific markers, invasiveness, tumor growth or suppression in acceptable host animals such as nude mice with nude mutation and the like, in vitro, in vivo and ex vivo (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994)).

The expression "growth inhibiting" (for example, in relation to cells such as tumor cells) refers to a measurable reduction in the growth of cells in vitro or in vivo when they are brought into contact with a therapeutic agent or a combination of therapeutics or drugs, compared with the growth of the same cells growing in appropriate control conditions well known to those skilled in the art. The inhibition of cell growth in vitro or in vivo may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Inhibition of cell growth can occur through various mechanisms, such as apoptosis, necrosis, or through inhibition of cell proliferation or through cell lysis.

The term vector refers to a polynucleotide capable of being reproduced within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, a polyadenylation signal, or markers of choice, to facilitate the reproduction or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include cell, virus, animal, plant, and reengineered biological systems using biological components capable of vector doubling. The vector-containing polynucleotide may be a DNA or RNA molecule or a hybrid thereof.

The term expression vector means a vector that can be used in a biological system or a reshaped biological system to directly translate the polypeptide encoded by the polynucleotide sequence present in the expression vector.

The term polynucleotide means a molecule containing a chain of nucleotides covalently linked through a sugar phosphate backbone or other equivalent covalent chemical structure. Double-stranded and single-stranded DNA and RNA are typical examples of polynucleotides.

The term polypeptide or protein refers to a molecule that contains at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides containing less than about 50 amino acids may be referred to as peptides.

In the context of the present invention, the term valency refers to the presence in a molecule of a given number of antigen-specific binding sites. Thus, the terms monovalent, divalent, tetravalent, and hexavalent refer to the presence of one, two, four, and six antigen-specific binding sites, respectively, in a molecule.

In the context of the present invention, the term in combination with means that two or more therapeutic agents can be administered to a patient together in admixture, simultaneously as separate agents, or sequentially as separate agents in any order.

The term synergy, synergy, or synergistic means the effect of a combination that is greater than the expected additive effect.

Compositions according to the present invention

The present invention relates to human PSMA binding FN3 domains and PSMA binding FN3 domains conjugated to toxins or detectable labels. The present invention relates to polynucleotides encoding the FN3 domains of the invention, or to their complementary nucleic acids, vectors, host cells, and methods for their preparation and use.

PSMA binding molecules

The present invention relates to type III fibronectin (FN3) domains that specifically bind to human prostate-specific membrane antigen (PSMA), optionally conjugated to a toxin or a detectable label. These molecules can be widely used for therapeutic and diagnostic purposes. The present invention relates to polynucleotides encoding the FN3 domains of the invention, or to their complementary nucleic acids, vectors, host cells, and methods for their preparation and use.

The FN3 domains of the invention bind to PSMA with high affinity and are internalized into PSMA expressing cells, thus providing an efficient way to deliver therapeutic agents to tumor cells.

One embodiment of the invention is an isolated FN3 domain that specifically binds to human prostate-specific membrane antigen (PSMA) SEQ ID NO: 144.

In some embodiments of the invention described herein, the FN3 domain of the invention cross-reacts with PSMA Macaca Fascicularis SEQ ID NO: 32 or PSMA Pan troglodytes SEQ ID NO: 33.

The FN3 domain of the invention can bind to human PSMA, Macaca Fascicularis and/or Pan troglodytes with a dissociation constant (KD) of less than about 1 x 10' ⁷ M, e.g., less than about 1 x 10' ⁸ M, less than about 1 x 10' ⁹ M, less than about 1x10'1° M, less than about 1x10'1 M, less than about 1x10' ¹² M, or less than about 1x10' ¹³ M as determined by surface plasmon resonance or kinetic exclusion (Kinexa) methods known to those skilled in the art. The measured affinity of interaction in a particular FN3-aHTnreH domain pair may vary when measured under different conditions (eg, osmolarity, pH). Thus, measurements of affinity and other antigen-binding parameters (eg, K _D , K _on , Koff) are performed using standardized solutions of the protein backbone and antigen and a standardized buffer solution, such as the buffer solution described herein.

In some embodiments, the FN3 PSMA binding domains contain an initiating methionine (Met) linked to the N-terminus of the molecule.

In some embodiments, the FN3 PSMA binding domains comprise a cysteine (Cys) bound to the C-terminus of the FN3 domain.

The addition of an N-terminal Met and/or a C-terminal Cys may promote expression and/or conjugation to molecules that increase half-life.

Another embodiment of the invention is an isolated FN3 domain that specifically binds to human PSMA, the FN3 domain inhibiting the enzymatic activity of human PSMA. Enzymatic activity of PSMA can be measured using standard methods. For example, hydrolysis of detectable PSMA or labeled PSMA substrate can be used. Examples of PSMA substrates that can be used are N-acetylaspartylglutamate (NAAG), folate polyglutamate, methotrexate tri-gamma-glutamate, methotrexate di-gamma-glutamate, pteroyl pentaglutamate and derivatives thereof. The substrate may be labeled with, for example, a radioactive marker, a chemiluminescent marker, an enzyme marker, a chromogenic marker, or other detectable markers. Suitable methods for detecting PSMA activity are described in, for example, US Patent No. 5,981,209 or US Patent Publication No. 2006/0009525. The isolated PSMA-binding FN3 domain of the invention inhibits the enzymatic activity of human PSMA if the molecule inhibits the activity of human PSMA by more than about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a sample not containing FN3 domain.

In some embodiments of the invention described herein, the isolated FN3 domain comprises the amino acid sequences of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42,

43, 44, 45, 46, 47, 48, 49, 50, 51, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,

96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,

119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 or 140.

In some embodiments of the invention described herein, the isolated FN3 domain contains an amino acid sequence that is 89% identical to the amino acid sequence of SEQ ID NO: 41.

In some embodiments of the invention described herein, the FN3 domain contains an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions compared to the amino acid sequence of SEQ ID NO: 41.

In some embodiments of the invention described herein, an isolated FN3 domain that specifically binds to human PSMA contains a cysteine residue at at least one residue position corresponding to residue positions 6, 11, 22, 25, 26, 52, 53, 61 in SEQ ID NO 1 or at the C-terminus.

Substitutions that introduce cysteine into a protein sequence can be used to chemically conjugate small molecules such as cytotoxic agents, detectable labels, polyethylene glycol, and/or nucleic acids to the FN3 domain using standard chemical methods.

In some embodiments, the FN3 domain specifically binding to human PSMA competes for binding of human PSMA to the FN3 domain of SEQ ID NO: 41.

In some embodiments, the FN3 domain specifically binding to human PSMA binds to the KKSPSPEFSGMPRISK (SEQ ID NO: 159) and NWETNKF (SEQ ID NO: 160) region of human PSMA.

A human PSMA epitope bound to an FN3 domain of the invention includes some or all of the residues within the amino acid sequences shown in SEQ ID NO: 159 or SEQ ID NO: 160. In some embodiments described herein, the epitope bound with an FN3 domain of the invention, contains at least one amino acid in the KKSPSPEFSGMPRISK (SEQ ID NO: 159) and NWETNKF (SEQ ID NO: 160) regions of human PSMA (SEQ ID NO: 144). In some embodiments described herein, an epitope associated with an FN3 domain of the invention contains at least two, three, four, five, six, or seven amino acids in the KKSPSPEFSGMPRISK region (SEQ ID NO: 159) and at least two , three, four, five or six amino acids in the NWETNKF region (SEQ ID NO: 160) of human PSMA (SEQ ID NO: 144).

In some embodiments described herein, the FN3 domain of the invention binds to human PSMA at residues K499, K500, S501, P502, P504, R511, K514, N540, W541, E542, N544, K545, and F546 (residue numbering according to with SEQ ID NO: 144).

In some embodiments described herein, the FN3 domain of the invention further binds to human PSMA at residues R181, Y460, F488, K610, and/or 1614.

The crystal structure of the FN3 P2 domain of 33FR9 H10 was dissolved in complex with cynomolgus monkey PSMA. Since the contact residues in human and cynomolgus PSMA are identical except for one residue, it is expected that P233FR9 H10 will bind to human PSMA at the same epitope residues where it binds to cynomolgus PSMA.

FN3 domains can be assessed for competition with a reference molecule for binding to human PSMA using well known in vitro methods. In an exemplary method, CHO cells recombinantly expressing human PSMA can be incubated with an unlabeled reference molecule for 15 minutes at 4°C followed by incubation with an excess of fluorescently labeled FN3 test domain for 45 minutes at 4°C. After washing in phosphate buffered saline with bovine serum albumin (PBS/BSA), fluorescence can be measured by flow cytometry using standard methods. In another exemplary method, an extracellular region of human PSMA can be applied to the surface of an ELISA plate. Over about 15 minutes, an excess of unlabeled reference molecule may be added, and subsequently biotinylated FN3 test domains may be added. After washes in PBS/Tween, binding of the test biotinylated FN3 domain can be detected with horseradish peroxidase (HRP) conjugated streptavidin and signal detected using standard methods. Obviously, in competitive assays, the reference molecule can be labeled and the tested FN3 domain unlabeled. The FN3 test domain competes with the reference molecule if the reference molecule inhibits binding of the FN3 test domain or the FN3 test domain inhibits binding of the reference molecule by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%. An epitope of the FN3 test domain can be further determined, for example by peptide mapping or by hydrogen/deuterium protection assays using known methods, or by crystal structure determination. An example of a FN3 reference domain is a domain containing the amino acid sequence of SEQ ID NO: 41.

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FN3 domains that bind to the same region on human PSMA as the FN3 domain of SEQ ID NO: 41 can be obtained, for example, by immunizing mice with peptides having the amino acid sequences shown in SEQ ID NO: 159 and 160 using standard methods, as well as as described in the present description. FN3 domains can be further assessed, for example, by analyzing competition between the FN3 domain of SEQ ID NO: 41 and the test FN3 domain for binding to human PSMA using well known in vitro methods and as described above.

In some embodiments, an isolated FN3 domain that specifically binds to the human PSMA of the invention is conjugated to a detectable label.

A detectable label includes compositions which, when conjugated to an FN3 domain of the invention, render the latter detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Examples of labels include radioactive isotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (eg, often used in ELISA), biotin, digoxigenin, or haptens. Specific radioactive labels include the most common isotopes on the market ^, including, for example, ³ H, ^P C, ¹³ C, ¹⁵ N, ¹⁸ F, ¹⁹ F, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ⁸⁹ Zr, ¹ⁿ In, ^94m Tc, ^99m Tc, ⁶⁴ Cu and ⁶⁸ Ga. Suitable dyes include any commercially available dyes such as, for example, 5(6)-carboxyfluorescein, IRDye 680RD or IRDye 800CW maleimide, ruthenium/polypyridyl complex dyes, and the like.

FN3 domains that specifically bind to human PSMA conjugated to a detectable label can be used as imaging agents to assess tumor distribution, diagnose the presence of tumor cells and/or tumor recurrence.

In some embodiments, the FN3 domains that specifically bind to the human PSMA of the invention are conjugated to a cytotoxic agent.

In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a bacterial, fungal, plant, or animal toxin with enzymatic activity, or fragments thereof), or a radioactive isotope (i.e., a radioactive conjugate ).

FN3 domains that specifically bind to human PSMA conjugated to a cytotoxic agent can be used for targeted delivery of the cytotoxic agent to and intracellular accumulation in a PSMA-expressing tumor cell, and systemic administration of these unconjugated cytotoxic agents can lead to unacceptable levels of toxicity to normal cells.

In some embodiments, the cytotoxic agent is daunomycin, doxorubicin, methotrexate, vindesine, bacterial toxins such as diphtheria toxin, ricin, geldanamycin, maytansinoids, or calicheamicin.

A cytotoxic agent can induce cytotoxic and cytostatic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

In some embodiments, the cytotoxic agent is toxins with enzymatic activity, such as diphtheria toxin A chain, non-binding diphtheria toxin active moieties, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, abrin A chain, modecin, alpha-sarcin, Aleurites fordii proteins, diantine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), omordica charantia inhibitor, curcin, crotin, sapaonaria officinali inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and tricothecenes.

In some embodiments, the implementation of the cytotoxic agent is a radionuclide, such as ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re.

Conjugates of the FN3 domains of the invention and a cytotoxic agent are prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis-(pazidobenzoyl)hexanediamide), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and compounds with two active fluorine groups (such as 1,5-difluoro-2,4-dinitrobenzene).

In some embodiments, the cytotoxic agent is dostatins or peptide analogs and derivatives of dolastatin, auristatin, or monomethylauristatin phenylalanine. Example molecules are described in US Pat. ) and have anticancer and antifungal activity. The functional group of the drug dostatin or auristatin can be attached to the FN3 domain, which includes the molecule of the invention, through the N (amino) terminus or C (carboxyl) terminus of the functional group of the peptide drug

- 8 042392 (WO 02/088172) or through any cysteine artificially introduced into the FN3 domain.

In some embodiment, an FN3 domain that specifically binds to a human

PSMA is removed from the blood by renal clearance.

Isolation of PSMA-binding FN3 domains from a library based on the Tencon sequence

Tencon (SEQ ID NO: 1) is a non-naturally occurring fibronectin type III (FN3) domain developed from a consensus sequence of fifteen human tenascin-C FN3 domains (Jacobs et al., Protein Engineering, Design, and Selection, 25:107- 117, 2012; US Patent Publication No. 2010/0216708). The Tencon crystal structure shows six surface-exposed loops connecting seven beta-strands characteristic of FN3 domains, with beta-strands designated as A, B, C, D, E, F, and G, and loops designated as AB, BC, CD , DE, EF, and FG (Bork and Doolittle, Proc Natl Acad Sci USA 89:8990-8992, 1992; US Pat. No. 6,673,901). These loops, or selected residues within each loop, can be randomized to construct libraries of type III fibronectin (FN3) domains that can be used to select new PSMA-binding molecules. In table. 1 shows the positions and sequences of each loop and beta strand in Tencon (SEQ ID NO: 1).

Thus, a library designed from the Tencon sequence may have a randomized FG loop or randomized BC and FG loops, such as the TCL1 or TCL2 libraries described below. The Tencon BC loop is 7 amino acids long. Thus, a BC loop diversified Tencon sequence library can randomize 1, 2, 3, 4, 5, 6, or 7 amino acids. The Tencon FG loop is 7 amino acids long, so a Tencon FG loop diversified sequence library can randomize 1, 2, 3, 4, 5, 6, or 7 amino acids.

Additional diversity in the loops of the Tencon libraries can be achieved by insertion and/or deletion of residues in the loops. For example, FG and/or BC loops can be extended by 1-22 amino acids or shortened by 1-3 amino acids. The Tencon FG loop is 7 amino acids long, while the corresponding loop in antibody heavy chains ranges from 4 to 28 residues. To maximize diversity, the FG loop can be diversified in sequence as well as in length to fit the antibody CDR3 length range of 4-28 residues. For example, the FG loop can be further diversified in length by extending the loop by an additional 1, 2, 3, 4, or 5 amino acids.

A library designed based on the Tencon sequence can also have alternative randomized surfaces formed lateral to the FN3 domain and containing two or more beta strands and at least one loop. One such alternative surface is formed by the amino acids in the C and F beta strands and in the CD and FG loops (C-CD-F-FG surface). The library configuration based on the Tencon C-CD-F-FG alternative surface is described in US Patent Publication No. US 2013/0226834. Libraries developed from the Tencon sequence also include libraries based on Tencon variants, such as Tencon variants having substitutions at residue positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering according to SEQ ID NO: 1), and such variants exhibit improved thermal stability. Examples of Tencon variants are described in US Patent Publication No. 2011/0274623 and include Tencon27 (SEQ ID NO: 4) having substitutions E11R, L17A, N46V and E86I compared to Tencon with SEQ ID NO: 1.

Libraries based on Tencon and another FN3 sequence can be randomized to selected residue positions using a random or specific set of amino acids. For example, library variants having random substitutions can be generated using codons

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NNK, which code for all 20 naturally occurring amino acids. In other diversification schemes, DVK codons can be used to code for the amino acids Ala, Tip, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys. Alternatively, NNS codons can be used to obtain all 20 amino acid residues and simultaneously reduce the frequency of stop codons. Libraries of FN3 domains with a shifted distribution of amino acids at diversifiable positions can be synthesized, for example, using Slonomics® technology (http:_//www_sloning_com). This technology uses a library of pre-produced double-stranded triplets that serve as versatile building blocks sufficient for the synthesis of thousands of genes. The library of triplets contains all possible combinations of sequences needed to build any desired DNA molecule. The well-known IUB code is used to designate codons.

FN3 domains of the invention that specifically bind to human PSMA can be isolated by preparing a FN3 library, such as the Tencon library, using a cis display to ligate DNA fragments encoding scaffold proteins with a DNA fragment encoding RepA to generate a pool of protein- DNA formed after in vitro translation, with each protein stably associated with its encoding DNA (US patent No. 7842476; Odegrip et al., Proc Natl Acad Sci USA 101, 2806-2810, 2004), and analysis of the library for specific binding to PSMA by any method known in the art or described in the example. Examples of well-known methods that can be used are ELISA, sandwich ELISA, and competitive and non-competitive assays (see, for example, Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons , Inc., New York). The identified FN3 domains specifically binding to PSMA are further evaluated for PSMA activity inhibition, internalization, stability, and other desired characteristics.

FN3 domains of the invention that specifically bind human PSMA can be generated using any FN3 domain as a template for library construction and screening of a library of molecules that specifically bind human PSMA using the proposed methods. Examples of suitable FN3 domains for use include tenascin C (TN3) FN3 3 (SEQ ID NO: 145), Fibcon (SEQ ID NO: 146) and fibronectin FN3 10 (FN10) (SEQ ID NO: 147). Standard cloning and expression techniques are used to clone libraries into a vector or synthesize double-stranded cDNA library cassettes to express libraries or translate them in vitro. For example, one can use a ribosome display (Hanes and Pluckthun, Proc Natl Acad Sci USA, 94, 4937-4942, 1997), an mRNA display (Roberts and Szostak, Proc Natl Acad Sci USA, 94, 12297-12302, 1997), or others. cell-free systems (US patent No. 5643768). FN3 domain variant libraries can be expressed as fusion proteins that display on the surface of, for example, any suitable bacteriophage. Well-known methods for displaying fused polypeptides on the surface of a bacteriophage (US Patent Publication No. 2011/0118144; International Patent Publication No. WO 2009/085462; US Patent No. 6969108; US Patent No. 6172197; US Patent No. 5223409; US Patent No. 6582915; US Patent No. 6472147).

In some embodiments of the invention described herein, the FN3 domain specifically binding to human PSMA is based on the Tencon sequence of SEQ ID NO: 1 or the Tencon27 sequence of SEQ ID NO: 4, wherein SEQ ID NO: 1 or SEQ ID NO: 4 optionally has substitutions at residue positions 11, 14, 17, 37, 46, 73 and/or 86;

The FN3 domains of the invention that specifically bind to human PSMA can be modified to improve their properties, such as improving thermal stability and reversibility of thermal folding and unfolding. A number of methods have been used to increase the effective thermostability of proteins and enzymes, including rational configuration based on comparison with highly similar thermostable sequences, configuration of stabilizing disulfide bridges, mutations to increase the propensity to form alpha helices, salt bridge engineering, alteration of protein surface charge, directed evolution and composition containing consensus sequences (Lehmann and Wyss, Curr Opin Biotechnol, 12, 371-375, 2001). High heat stability can increase expressed protein yield, solubility or activity, reduce immunogenicity, and minimize the need for a cold line during manufacture. Residues at which substitutions can be made to improve thermal stability Tencon (SEQ ID NO: 1) are residue positions 11, 14, 17, 37, 46, 73, or 86 as described in US Patent Publication No. 2011/0274623. Substitutions corresponding to these residues can be introduced into molecules of the invention containing FN3 domains.

The measurement of protein stability and protein lability can be considered as one or different aspects of protein integrity. Proteins are sensitive or labile to denaturation caused by heat, ultraviolet or ionizing radiation, changes in environmental osmolarity and pH when in liquid solution, mechanical shear caused by filtration through small pores, ultraviolet radiation, ionizing radiation such as gamma irradiation, chemical or thermal dehydration or any other exposure or force

- 10 042392 eat, which can lead to the destruction of the protein structure. The stability of the molecule can be determined using standard methods. For example, the stability of a molecule can be determined by measuring the thermal melting point (T _m )—the temperature in degrees Celsius (°C) at which half of the molecules unfold—using standard methods. Generally, the higher the T _m , the more stable the molecule is. In addition to heating, the ability of a protein to maintain a particular three-dimensional structure can also be changed by means of a chemical environment.

In one embodiment, an FN3 domain of the invention specifically binding to human PSMA may exhibit at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% increased stability. , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more compared to the same pre-design domain as measured by Tm rise.

Chemical denaturation can similarly be measured in a variety of ways. Chemical denaturants include guanidine hydrochloride, guanidine thiocyanate, urea, acetone, organic solvents (dimethylformamide, benzene, acetonitrile), salts (ammonium sulfate, lithium bromide, lithium chloride, sodium bromide, calcium chloride, sodium chloride); reducing agents (e.g. dithiothreitol, beta-mercaptoethanol, dinitrotiobenzene and hydrides such as sodium borohydride), non-ionic and ionic detergents, acids (e.g. hydrochloric acid (HCl), acetic acid (CH3COOH), haloacetic acids), hydrophobic molecules (e.g. phospholipids) and specialized denaturing compounds. Quantification of the degree of denaturation can be based on the loss of a functional property, such as the ability to bind to a target molecule, or on the analysis of physicochemical properties, such as the tendency to aggregate, access to residues previously inaccessible to the solvent, or on the destruction or formation of disulfide bonds.

The FN3 domain of the invention can be designed as monomers, dimers, or multimers, for example, as a means of increasing valence and thus avidity to bind a target molecule, or to create bi- or multispecific scaffolds that simultaneously bind two or more different target molecules. Dimers and multimers can be created by linking monospecific, bi- or multispecific protein scaffolds, for example by including an amino acid linker, such as a linker containing polyglycine, glycine and serine or alanine and proline. Examples of linkers include (GS)2, (SEQ ID NO: 148), (GGGS^ (SEQ ID NO: 149), (GGGGS)5 (SEQ ID NO: 150), (AP) ₂ (SEQ ID NO: 151) , (AP) ₅ (SEQ ID NO: 152), (AP) ₁₀ (SEQ ID NO: 153), (AP) ₂₀ (SEQ ID NO: 154) and A(EAAAK) ₅ AAA (SEQ ID NO: 142) The use of natural and artificial peptide linkers to link polypeptides into novel linked fused polypeptides is well known in the literature (Hallewell et al., J Biol Chem 264, 5260). -5268, 1989; Alfthan et al., Protein Eng. 8, 725-731, 1995; Robinson & Sauer, Biochemistry 35, 109-116, 1996; U.S. Patent No. 5,856,456).

Fragments that prolong half-life

The FN3 domain of the invention specifically binding to human PSMA may include other subunits, eg, through covalent interaction. In one aspect of the invention, the FN3 domain of the invention further comprises a half-life increasing functional group. Examples of fragments that increase half-life are albumin, albumin variants, albumin-binding proteins and/or domains, transferrin and fragments and analogs thereof, and Fc regions. An example of an albumin variant is shown in SEQ ID NO: 155. The amino acid sequences of human Fc regions are well known and include the Fc regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE.

All or part of an antibody constant region can be attached to the FN3 domain of the invention to confer antibody-like properties, especially those associated with the Fc region, such as Fc effector functions such as C1q binding, complement dependent cytotoxicity (CDC), Fc- receptor, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, suppression of inhibition of cell surface receptors (e.g., B-cell receptor; BCR), and can be further modified by modifying the residues that in Fc are responsible for these functions (see review in Strohl , Curr Opin Biotechnol 20: 685-691, 2009).

Additional moieties can be inserted into the FN3 domain of the invention, such as polyethylene glycol (PEG) molecules such as PEG-5000 or PEG-20000, fatty acids and fatty acid esters of various chain lengths, e.g. laurate, myristate, stearate, arachidate, behenate , oleate, arachidonate, octandioic acid, tetradecanedioic acid, octadecanedioic acid, docosandioic acid and the like, polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) to obtain the desired properties. These fragments may be direct fusions to scaffold-encoding sequences and may be generated using standard cloning and expression techniques. Alternatively, well-known chemical linkage techniques can be used to attach fragments to recombinantly produced molecules of the present invention.

- 11 042392

A pegyl functional group can, for example, be added to the FN3 domain of the invention by inserting a cysteine residue at the C-terminus of the molecule, or artificially incorporating cysteines into positions of residues facing away from the surface of the human-binding molecule.

PSMA, and the addition of a pegyl group to cysteine using well known methods.

The FN3 domain of the invention with built-in additional fragments can be compared in functionality using several well-known assays. For example, property changes due to insertion of Fc domains and/or Fc domain variants can be analyzed in Fc receptor binding assays using soluble forms of the receptor, such as FcyRI, FcyRII, FcyRIII, or FcRn receptors, or using known cellular assays to measure e.g. ADCC or CDC, or evaluation of the pharmacokinetic properties of the molecules of the invention in in vivo models.

Polynucleotides, vectors, host cells

The invention provides nucleic acids encoding FN3 domains of the invention specifically binding to human PSMA, as isolated polynucleotides, or as regions of expression vectors, or as regions of linear DNA sequences, including linear DNA sequences used for in vitro transcription/translation , vectors compatible with the expression, secretion and/or presentation of the compositions or the results of their site-directed mutagenesis in prokaryotes, eukaryotes or filamentous phages. Some polynucleotides are described herein, however, other polynucleotides which, given the degeneracy of the genetic code or the preference for codon usage in a given expression system, encode FN3 domains in accordance with the invention are also contemplated as part of the invention.

One embodiment of the invention is an isolated polynucleotide encoding a FN3 domain specifically binding to human PSMA, which contains the amino acid sequence of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 , 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 .

One embodiment of the invention is an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 156, 157, 158, or 159.

The polynucleotides of the invention can be prepared by chemical synthesis, such as solid phase polynucleotide synthesis, on an automatic polynucleotide synthesizer and assembly into complete single or double stranded molecules. Alternatively, the polynucleotides of the invention can be obtained by other techniques such as PCR followed by standard cloning. Techniques for producing or obtaining polynucleotides of a given known sequence are well known in the art.

The polynucleotides of the invention may contain at least one non-coding sequence such as a promoter or enhancer sequence, an intron, a polyadenylation signal, a cis sequence to facilitate binding to RepA, and the like. The polynucleotide sequences may also contain additional sequences encoding additional amino acids that encode, for example, a marker or tag sequence such as a histidine tag or a HA tag to facilitate purification or protein detection, a signal sequence, a fusion protein partner, such as RepA, Fc or bacteriophage coat protein such as pIX or pIII.

Another embodiment of the invention is a vector containing at least one polynucleotide of the invention. Such vectors may be plasmid vectors, viral vectors, baculovirus expression vectors, transposon vectors, or any other vectors suitable for introducing the polynucleotides of the invention into a given organism or genetic environment in any way. Such vectors may be expression vectors containing elements of the nucleotide sequence that can control, regulate, cause or permit the expression of the polypeptides encoded by such a vector. Such elements may contain transcriptional enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that facilitate the expression of encoded polypeptides in a given expression system. Such expression systems may be cellular or cell-free systems well known in the art.

Another embodiment of the invention is a host cell containing a vector in accordance with the invention. The FN3 domain of the invention specifically binding to human PSMA can optionally be generated by a cell line, a mixed cell line, an immortalized cell, or a clonal population of immortalized cells, as is well known in the art. See, for example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, ^2nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc.,

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NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (19972001).

The host cell chosen for expression may be derived from a mammalian, or it may be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, He G2, SP2/0, HeLa, myeloma, lymphoma, yeast cells. , insects or plants or any derivative, immortalized or transformed cells mentioned. Alternatively, the host cell may be selected from a species or organism incapable of polypeptide glycosylation, e.g. a prokaryotic cell or an organism such as BL21, BL21(DE3), BL21-GOLD (DE3), XL1-Blue, JM109, HMS174, HMS174(DE3) , and any of the natural or engineered strains of E. coli, Klebsiella, or Pseudomonas.

Another embodiment of the invention is a method for obtaining an isolated FN3 domain of the invention that specifically binds to human PSMA, comprising culturing the isolated host cell of the invention under conditions that express the isolated FN3 domain that specifically binds to human PSMA, and purifying the FN3 domain.

The FN3 domain specifically binding to human PSMA can be purified from recombinant cell cultures by well-known methods, e.g., protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, lectin chromatography or high performance liquid chromatography (HPLC).

Uses of FN3 Domains of the Invention Binding to Human PSMA

The FN3 domains of the invention, which specifically bind to human PSMA, can be used to diagnose, monitor, modulate, treat, attenuate, prevent the development or reduce the symptoms of a human disease or specific cell, tissue, organ, fluid, or host pathologies in general. The methods of the invention may be used to treat an animal patient within any classification. Examples of such animals include mammals such as humans, rodents, dogs, cats, and farm animals.

One embodiment of the invention is a method of treating a patient having a PSMA overexpressing cancer which comprises administering to the patient an FN3 domain of the invention specifically binding to human PSMA conjugated to a cytotoxic agent for a period of time sufficient to treat the patient.

In some embodiments, the cancer is prostate cancer, colorectal cancer, gastric cancer, clear cell kidney cancer, bladder cancer, lung cancer, or kidney cancer.

In some embodiments, the cancer is a solid tumor.

In some embodiments, the cancer is a disorder of the prostate, such as prostate cancer or benign prostatic hyperplasia (BPH).

In some embodiments, the cancer is prostate cancer.

In some embodiments, the cancer is colorectal cancer.

In some embodiments, the cancer is stomach cancer.

In some embodiments, the cancer is clear cell kidney cancer.

In some embodiments, the cancer is bladder cancer.

In some embodiments, the cancer is kidney cancer.

In some embodiments, the cancer is a neovascular disorder, such as cancer characterized by the growth of a solid tumor. Examples of cancers with tumor vasculature characterized by PSMA overexpression and treatable according to the present invention include, for example, clear cell kidney cancer (CCRCC), colorectal cancer, breast cancer, bladder cancer, lung cancer, and pancreatic cancer (see ., e.g., Baccala et al., Urology 70:385.390, 2007 (PSMA expression in CCRCC); Liu et al., Cancer Res. 57:3629-3634, 1997 (PSMA expression in various non-prostatic cancers including kidney cancer, urothelial, lung, colon, breast and liver adenocarcinoma), and Milowsky et al., J. Clin. Oncol. 25:540-547, 2007.

One embodiment of the invention is a method of treating a patient having PSMA overexpressing prostate cancer, which comprises administering to the patient an FN3 domain of the invention specifically binding to human PSMA conjugated to a cytotoxic agent for a period of time sufficient to treat the patient.

Patients administered with an FN3 domain of the invention that specifically binds to human PSMA as described herein include those at high risk of developing a particular disorder characterized by PSMA overexpression, as well as patients with pre-existing such disorder. Typically, a patient is diagnosed with a disorder that requires treatment. Additionally, patients may be monitored for any changes in the disorder (eg, an increase or decrease in the clinical symptoms of the disorder) over the course of treatment.

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In prophylactic use, the pharmaceutical compositions or medicaments are administered to a patient predisposed or otherwise at risk of developing a particular disorder in an amount sufficient to eliminate or reduce the risk or delay the onset of the disorder. In therapeutic use, the compositions or medicaments are administered to a patient with a suspected or already suffering disease in an amount sufficient to cure or at least partially alleviate the symptoms of the disorder and its complications. An amount sufficient to accomplish the foregoing is referred to as a therapeutically effective dose or amount. In both prophylactic and therapeutic regimens, agents are typically administered in multiple doses until a sufficient response (eg, inhibition of abnormal angiogenic activity) is achieved. As a rule, the response to treatment is monitored and, if the desired response begins to wane, repeated doses are administered.

To identify patients to be treated in accordance with the methods of the invention, suitable screening methods can be used to determine risk factors associated with specific disorders, or to determine the status of an existing disorder identified in a patient. Such methods may include, for example, determining whether a patient has relatives who have been diagnosed with a particular disorder. Screening methods may also include, for example, standard tests to determine a family history for a particular disorder known to have an inherited component. For example, it is known that various cancers also have certain inherited components. Inherited components of cancer include, for example, mutations in a variety of genes that are transformative (e.g., Ras, Raf, EGFR, cMet, and others), the presence or absence of specific HLA and cytotoxicity suppression receptor (KIR) molecules, or the mechanisms by which cancer cells cells are able to modulate the suppression of immune cells such as NK cells and T cells directly or indirectly (see, for example, Ljunggren and Malmberg, Nature Rev. Immunol. 7:329-339, 2007; Boyton and Altmann, Clin. Exp Immunol 149:1-8, 2007). Therefore, nucleotide probes can usually be used to identify patients carrying genetic markers associated with a particular disorder of interest. In addition, many different immunological methods are known in the art that are used to detect markers of a particular disorder. For example, various immunoassay (ELISA) methods are available and well known in the art that use monoclonal antibody probes to detect antigens associated with specific tumors. If necessary, screening can be performed for the patient's known symptomology, age factors, associated risk factors, and the like. These methods allow the clinician to routinely select patients requiring treatment by the methods described herein. In accordance with these methods, as an independent treatment program or as a follow-up, ancillary or coordinating treatment regimen in relation to other treatments, targeting of pathological cells expressing PSMA can be performed.

In some of the methods described herein, FN3 domains of the invention specifically binding to human PSMA conjugated to a cytotoxic agent can be used to treat a patient with prostate cancer in combination with a second therapeutic agent.

In some of the methods described herein, FN3 domains of the invention specifically binding to human PSMA conjugated to a cytotoxic agent can be used to treat a patient who has or has developed resistance to treatment with a second therapeutic agent.

The second therapeutic agent may be a drug approved for the treatment of prostate cancer such as abiraterone acetate (Zytiga), bicalutamide, cabazitaxel, casodex (bicalutamide), degarelix, docetaxel, enzalutamide, goserelin acetate, jevtana (cabasitaxel), leuprolide acetate, lupron (leuprolide acetate), lupron-depot (leuprolide acetate), luprondepo for 3 months (leuprolide acetate), lupron-depot for 4 months (leuprolide acetate), lupron-depot pediatric (leuprolide acetate), mitoxantrone hydrochloride, prednisone, provenge ( sipuleucel-T), radium-223 dichloride, sipuleucel-T, taxotere (docetaxel), viadur (leuprolide acetate), xofigo (radium-223 dichloride), xtandi (enzalutamide), or zoladex (goserelin acetate) (source: National Cancer Institute ).

Various qualitative and/or quantitative methods can be used to determine whether a patient has resistance, acquired resistance, or a predisposition to acquire resistance to treatment. Symptoms that may be associated with resistance include, for example, worsening or no improvement in the health of the patient, an increase in tumor size, cessation or slowing of tumor growth inhibition, and/or spread of cancer cells in the body from one site to other organs, tissues, or cells. An indicator that a patient has developed resistance or has a predisposition to develop resistance to treatment can also be the reappearance or worsening of various cancer-related symptoms,

- 14 042392 such as anorexia, cognitive disorders, depression, shortness of breath, fatigue, hormonal disorders, neutropenia, pain, peripheral neuropathy and sexual dysfunction. Symptoms associated with cancer may vary depending on the type of cancer. For example, symptoms associated with prostate cancer may include difficulty urinating or frequent urination, painful urination, blood in the urine or semen, aching pain in the pelvis, back and/or hips. Symptoms associated with lung cancer may include a persistent cough, coughing up blood, shortness of breath, wheezing and chest pain, loss of appetite, weight loss without weight loss, and fatigue. An oncologist can easily identify the symptoms associated with a particular type of cancer.

The terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventive measures, the aim being to prevent or slow down (reduce) an undesired physiological change or disorder, such as the development or spread of cancer. For the purposes of the present invention, advantageous or desirable clinical results include, but are not limited to, relief of symptoms, reduction in the extent of the disease, stabilization of the condition (i.e., no worsening), delay or slowing of the progression of the disease, improvement or temporary improvement in the condition, and remission (complete or partial) both detectable and non-discoverable. The term treatment can also refer to the prolongation of survival compared to life expectancy in the absence of treatment. Patients in need of treatment include those who already have the condition or disorder, as well as those who are predisposed to developing the condition or disorder, or those in whom the condition or disorder is to be prevented.

The term "therapeutically effective amount" refers to an amount effective at the dosages and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of the PSMA binding FN3 domain of the invention may vary depending on such factors as the disease state, age, sex and body weight of the patient, and the ability of the PSMA binding FN3 domain of the invention to elicit the desired response in the patient. Examples of PSMA-binding FN3 efficacy measures that may be reduced or attenuated by resistance include, for example, improved patient health, reduced or reduced tumor size, halted or slowed tumor growth, and/or no cancer cell metastasis elsewhere in the body. .

Introduction/pharmaceutical compositions

The invention provides pharmaceutical compositions that contain FN3 domains that specifically bind to human PSMA, optionally conjugated to a second molecule of the invention, and a pharmaceutically acceptable carrier. For therapeutic use, it is possible to obtain the FN3 domains of the invention as pharmaceutical compositions containing an effective amount of the domain or molecule as the active ingredient in a pharmaceutically acceptable carrier. The term carrier refers to the diluent, adjuvant, excipient or carrier vehicle with which the active compound is administered. Such a carrier may be liquid, such as, for example, water or oils, including oils derived from petroleum, animal, vegetable or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, a 0.4% saline solution and a 0.3% glycine solution can be used. These solutions are sterile and essentially free of particulate matter. They can be sterilized using well known standard sterilization techniques (eg filtration). The compositions may contain pharmaceutically acceptable adjuvants necessary for the respective physiological conditions, such as pH adjusting agents, buffering agents, stabilizers, thickeners, lubricants and colorants, and the like. The concentration of the molecules of the invention in such a pharmaceutical composition can vary within wide limits, i. e. from less than about 0.5%, typically at least about 1% and up to 15 or 20 wt.%, and is selected primarily based on the required dose, fluid volumes, viscosity, and the like. according to the specific route of administration chosen. Suitable carrier media and formulations, including other human proteins, such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, ^21st Edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006 , Part 5, Pharmaceutical Manufacturing pp 691-1092, see especially pp. 958-989.

The route of administration for therapeutic use of the FN3 domains of the invention may be any suitable route that delivers the agent to the host, such as parenteral administration, eg intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary; transmucosal (oral, intranasal, intravaginal, rectal) using a composition in the form of a tablet, capsule, solution, powder, gel, corpuscle; and contained in a syringe, implanted device, osmotic pump, cartridge, micropump; or by other means, obvious to the skilled person, which are well known in this field. Localized administration can be achieved, for example, by delivery to the joint, bronchi, abdominal cavity, inside the capsule, cartilage, cavity, cell, cerebellum, cerebral ventricle, colon, cervix, stomach, liver, myocardium, bone, pelvis, pericardium, abdominal cavity, pleura, prostate, lungs, rectum, kidney, retina, spine, bursa, thorax, uterus, vessel, inside the bladder, damaged tissue, vaginally, rectally, buccally, sublingually, intranasally, or transdermally.

Thus, a pharmaceutical composition of the invention for intramuscular injection can be prepared containing 1 ml of sterile buffered water and about 1 ng to about 100 mg, such as about 50 ng to about 30 mg, or more preferably about 5 to about 25 mg of domain FN3 according to the invention.

The FN3 domains of the invention may be administered to a patient by any suitable route, such as parenterally by intravenous (IV) infusion or bolus injection, intramuscularly, subcutaneously, or intraperitoneally. Intravenous infusion can be carried out for as little as 15 minutes, but more often for 30, 60, 90 minutes, or even 2 or 3 hours. The PSMA-binding FN3 domains of the invention can also be injected directly into the site of the disease (for example, into the tumor itself). The dose administered to a patient having cancer is sufficient to attenuate or at least partially inhibit the disease being treated (therapeutically effective amount), and may sometimes be from 0.1 to 10 mg/kg of body weight, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may be even higher, such as 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg. It is also possible to provide a fixed unit dose, eg 50, 100, 200, 500 or 1000 mg, or the dose may be dependent on the patient's body surface area, eg 400, 300, 250, 200 or 100 mg/m ² . For the treatment of cancer, 1 to 8 doses (eg, 1, 2, 3, 4, 5, 6, 7, or 8) are usually administered, but 10, 12, 20 or more doses may be administered. Administration of the FN3 domains of the invention can be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or more. Repeated courses of treatment in the form of long-term administration are also possible. Repeated administration can be carried out at the same dose or at a different dose.

For example, a pharmaceutical composition containing FN3 domains of the invention for administration to an 80 kg patient by intravenous infusion may be formulated to contain about 200 ml of sterile Ringer's solution and about 8 to about 2400 mg, from about 400 to about 1600 mg or about 400 to about 800 mg PSMA-binding FN3 domains. Methods for preparing compositions for parenteral administration are well known and described in more detail, for example, in Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, PA.

The FN3 domains of the invention may be lyophilized for storage and reconstituted in an appropriate vehicle prior to use. This technique has been shown to be effective for standard protein preparations, and lyophilization and reconstitution methods known in the art can be used.

The FN3 domains of the invention may be administered to a patient in a single dose, or administration may be repeated, e.g., one day, two days, three days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks , seven weeks, two months or three months. Repeated administration can be carried out at the same dose or at a different dose. Administration can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more.

The FN3 domains of the invention may be administered in combination with a second therapeutic agent simultaneously, sequentially or separately.

The FN3 domain of the invention, optionally in combination with a second therapeutic agent, can be administered with any form of radiation therapy, including external beam radiation, intensity modulated radiation therapy (IMRT), as well as any form of radiosurgery, including gamma knife, cyberknife , Linac, and interstitial irradiation (eg, implanted radioactive seeds, GliaSite balloon), and/or surgical treatment.

When considering the treatment of solid tumors specifically, protocols for evaluating measurable endpoints and antitumor activity are well known in the art. Although each tumor response assessment protocol can be defined differently, the RECIST criteria (criteria for evaluating response to treatment of solid tumors) are currently the guidelines for assessing tumor response to treatment recommended by the National Cancer Institute (see Therasse et al. ., J Natl Cancer Inst 92: 205-216, 2000). According to the RECIST criteria, tumor response to treatment means the reduction or elimination of all measurable lesions or metastases. A disease is considered to be substantially measurable if it contains at least one of the dimensions of which, when accurately measured, is = 20 mm using standard methods or = 10 mm using a helical CT scan with well-defined borders on a medical image or radiograph, computer axial tomography (CT), magnetic resonance imaging (MRI) or clinical examination (in case of superficial lesions). Non-measurable disease means that the disease contains lesions < 20 mm using standard measurement methods

- 16 042392 or < 10 mm when using helical CT scanning and true lesions that cannot be measured (too small for accurate measurement). Diseases that cannot be measured include pleural exudates, ascites, and circumstantial documented disease.

Objective status criteria are required for protocols to assess the response of a solid tumor to treatment. Typical criteria include the following: (1) complete response (CR), defined as the complete disappearance of all measurable diseases; no new lesions; absence of disease-related symptoms; no evidence of an unmeasurable disease; (2) partial response (40), defined as a total reduction of 30% in the longest diameter of the target lesions; (3) progressive disease (PD), defined as a total increase of 20% in the longest diameter of the target lesions or the appearance of any new lesion; (4) stable or non-response, defined as failure to categorize as PO, PR, or progressive disease. (See Therasse et al., supra.).

Additional measurable endpoints that are accepted in oncology include overall survival (OS), disease-free survival (RFS), objective response rate (400), time to progression (TRT), and progression-free survival (PFS) (see Guidance for Industry: Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologics, April 2005, Center for Drug Evaluation and Research, FDA, Rockville, Md.)

Pharmaceutical compositions can be supplied in the form of a kit containing a container that contains the pharmaceutical composition described in the present description. The pharmaceutical composition may be provided, for example, in the form of a solution for injection in single or multiple doses, or as a sterile powder which is reconstituted prior to injection. In an alternative embodiment, such a kit may include a dry powder dispenser, a liquid aerosol generator, or a nebulizer for administering a pharmaceutical composition. Such a kit may further contain written information about the indications and use of the pharmaceutical composition.

Although the invention has been described in general terms, embodiments of the invention will be further described in the following examples, which should not be construed as limiting the scope of the claims.

Reagents and constructs

The extracellular PSMA domains of cynomolgus monkey (cynomolgus monkey protein database accession number EH56646.1, SEQ ID NO: 32) and chimpanzee (Uniprot accession number H2Q3K5, SEQ ID NO: 33) were cloned into the pUnder expression vector along with 6His tags. and Avi. The proteins were transiently expressed in 293HEK-expi cells. The supernatants were collected and purified by centrifugation. Proteins were purified using a two-step purification method: 1) IMAC purification using a HisTrap HP column and 2) size exclusion purification (Superdex 200) in which the elution buffer was DPBS containing Mg ²⁺ , Ca ²⁺ and 0, 5 mM ZnCl2 to stabilize PSMA dimerization. Fractions containing the protein of interest were pooled and the protein concentration determined by A280.

The gene encoding S. aureus sortase A was created using the DNA2.0 vector and subcloned into the pJexpress401 (DNA2.0) vector for expression under the control of the T5 promoter. The sortase construct for soluble protein expression is not present in the 25 amino acid N-terminal domain of the natural protein because this domain is membrane associated (Ton-That et al., Proc Natl Acad Sci USA 96:12424-12429, 1999). Sortase was expressed as an N-terminal His6 tag (HHHHH, SEQ ID NO: 34) followed by a TEV protease site to remove the tag (ENLYFQS, SEQ ID NO: 54), resulting in sortase having the amino acid sequence of SEQ ID NO: 52 The sortase protein used also includes 5 mutation sequences that are reported in the literature to increase the catalytic efficiency of the enzyme compared to wild-type proteins (SEQ ID NO: 53) (Chen et al., Proc Natl Acad Sci USA 108:11399-11404, 2011). The plasmid was transformed into E. coli BL21 Gold cells (Agilent Technologies) for expression. One colony was selected and grown in Luria Broth (Teknova) enriched with kanamycin and incubated for 18 h at 37°C and 250 rpm. 250 ml of Terrific Broth (Teknova) broth enriched with kanamycin were seeded with these subcultures and grown at 37° C. for 4 hours with stirring. Protein expression was induced with 1 mM IPTG and the protein was expressed for 18 hours at 30°C. Cells were harvested by centrifugation at 6000 g and stored at -20°C until purification. The frozen cell pellet was thawed for 30 min at room temperature and suspended in BugBusterHT Protein Extraction Reagent (EMD Millipore) enriched with 1 μl of recombinant lysozyme (EMD Millipore) per 30 ml at a ratio of 5 ml per gram of cell paste and incubated for 30 min on a shaker at room temperature. The lysate was purified by centrifugation at 74600 g for 30 minutes.

The supernatant was applied to a gravity column packed with 3 ml Qiagen Superflow NiNTA resin preconditioned with buffer A (50 mM sodium phosphate buffer, pH 7.0, containing 0.5 M NaCl and 10 mM imidazole). After loading, the column was washed

- 17 042392

100 ml buffer A. The protein was eluted with buffer A enriched with 250 mM imidazole and loaded onto a preparative gel filtration column, TSK Gel G3000SW 21.5x600 mm (Tosoh), equilibrated in phosphate buffered saline (PBS) (Gibco) . Size exclusion chromatography was performed at room temperature in PBS with a flow rate of 10 ml/min using an AKTA-AVANT chromatography system. The purified sortase was then digested with TEV protease to remove the His6 tag. 28 mg of sortase were incubated in 10 ml with 3000 units of AcTEV protease (Invitrogen) in the supplied buffer solution enriched with 1 mM DTT for 2 hours at 30°C. The unlabeled sortase was purified with Ni-NTA resin. The reaction was buffer exchanged with TBS (50 mM Tris pH 7.5, 150 mM NaCl) using PD-10 columns (GE Healthcare) and the reaction was applied to a gravity column packed with 0.5 ml Qiagen Superflow Ni- NTA pre-equilibrated with buffer A. The eluent was collected and the resin was washed with 3 ml of buffer A, which was added to the eluent. The eluent was concentrated to ~0.5 ml in a 10 kDa Amicon 15 concentrator (EMD Millipore). Additional TBS buffer was added and the sample was concentrated again (repeated twice) to replace the buffer with TBS. 1/3 volume of 40% glycerol was added (final glycerol concentration: 10%) and the sortase was stored at -20°C for short term storage or at -80°C for long term storage.

Example 1: Creating Tencon Libraries with Randomized Loops

Tencon (SEQ ID NO: 1) is a type III fibronectin (FN3) framework immunoglobulin-like domain developed from a consensus sequence of fifteen human tenascin-C FN3 domains (Jacobs et al., Protein Engineering, Design, and Selection, 25: 107-117, 2012; US patent No. 8278419). Tencon's crystal structure shows six surface loops connecting seven beta strands. These loops or selected residues within each loop can be randomized to create a library of type III fibronectin (FN3) domains that can be used to select new molecules that bind to specific targets.

Tencon: LPAPKNLWSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO 1):

Various libraries were generated using the tencon framework and various construction strategies. In general, the TCL1 and TCL2 libraries produced good binders. The creation of the TCL1 and TCL2 libraries is described in detail in International Patent Publication No. WO 2014081944A2.

Creation of the TCL1 library

A library designed with only FG loop randomization in Tencon (SEQ ID NO: 1), TCL1, was created for use with a cis display system (Jacobs et al., Protein Engineering, Design and Selection, 25:107-117, 2012) . This system generates a single stranded DNA that includes the sequences for the Tac promoter, the Tencon library code sequence, the RepA code sequence, the cis element, and the ori element. When expressed in an in vitro transcription/translation system, a Tencon-RepA fusion protein complex is formed that is cis-linked to the DNA that encodes it. The complexes binding to the target molecule are then isolated and amplified by polymerase chain reaction (PCR) as described below.

The construction of the TCL1 library for use with cis display was performed by sequential PCR cycles to obtain the final linear double-stranded DNA molecules from the two halves; the 2 m (5') fragment contains the promoter and Tencon sequences, and the 1 m (3') fragment contains the repA gene and the cis and ori elements. These two halves are combined with a restriction fragment to obtain a complete construct. The TCL1 library was designed to include random amino acids only in the FG loop in Tencon, KGGHRSN (SEQ ID NO: 55). When creating this library, NNS codons were used, which made it possible to insert all 20 amino acids and one stop codon into the FG loop. The TCL1 library contains six separate sub-libraries, each with a different randomized FG loop length, from 7 to 12 residues, to further increase diversity.

TCL1 Library (SEQ ID NO: 2)

LPAPKNLWSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLT GLKPGTEYTVSIYGVX ₇ _ ₁₂ PLSAEFTT;

where X1, X ₂ , X ₃ , X ₄ , X ₅ , X6, X ₇ represent any amino acid and

X ₈ , X ₉ , X ₁₀ , X _p and X ₁₂ are any amino acid or have been deleted.

Creation of the TCL2 library

A TCL2 library was created in which both the BC loop and the FG loop were randomized in Tencon, and the distribution of amino acids at each position was tightly controlled. In table. 3 shows the distribution of amino acids at desired loop positions in the TCL2 library. The developed distribution of amino acids had two purposes. First, the library was targeted to residues predicted to be structurally significant for Tencon folding and stability based on Tencon crystal structure analysis and/or homology modeling. For example, position 29 was only intended for a subset of hydrophobic amino acids, as this

- 18 042392 the remainder is immersed in the hydrophobic core of the folded Tencon. The second level of development involved shifting the distribution of amino acids to a distribution of residues that are preferentially found in the heavy chain of HCDR3 antibodies to efficiently produce compounds with high binding affinity (Birtalan et al., J Mol Biol 377:1518-28, 2008; Olson et al ., Protein Sci 16:476-84, 2007). Thus, the developed distribution from Table. 2 refers to the following distribution: 6% alanine, 6% arginine, 3.9% asparagine, 7.5% aspartic acid, 2.5% glutamic acid, 1.5% glutamine, 15% glycine, 2.3% histidine , 2.5% isoleucine, 5% leucine, 1.5% lysine, 2.5% phenylalanine, 4% proline, 10% serine, 4.5% threonine, 4% tryptophan, 17.3% tyrosine and 4% valine . This distribution lacks methionine, cysteine, and STOP codons.

TCL2 Library (SEQ ID NO: 3)

LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8SFLIQYQESEKVGEAINLTVPGSER SYDLTGLKPGTEYTVSIYGVX ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ SX ₁₄ X ₁₅ LSAEFTT; Where

X1 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

X ₂ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

X3 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

X ₄ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

X ₅ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

X ₆ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

X7 is Phe, Ile, Leu, Val or Tyr;

X ₈ is Asp, Glu or Thr;

X9 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

X ₁₀ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

X11 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

X ₁₂ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

X ₁₃ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

X ₁₄ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val and

X ₁₅ is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

table 2

Position Remains of the wild type Distribution in TCL2 library remainder * 22 T Designed Distribution 23 A Designed Distribution 24 R 50% P+designed distribution 25 D Designed Distribution 26 A 20% A + 20% G + developed distribution 27 A Designed Distribution 28 F 20% F, 20% I, 20% L, 20% V, 20% Y 29 D 33% D, 33% E, 33% T 75 TO Designed Distribution 76 G Designed Distribution 77 G Designed Distribution 78 H Designed Distribution 79 R Designed Distribution 80 S 100% S 81 N Designed Distribution 82 R 50% P+designed distribution

*Residue numbering based on Tencon SEQ ID NO: 1

Subsequently, these libraries have been improved in various ways, including building libraries on a stabilized Tencon scaffold (US Pat. No. 8,569,227) that introduces E11R/L17A/N46V/E86I (Tencon27; SEQ ID NO: 4) substitutions compared to tencon wild-type, as well as changing positions randomized in loops BC and FG. A description of Tencon27 is provided in International Patent Application No. WO 2013049275. New libraries were then developed capable of randomizing Tencon's FG loop alone (TCL9 library) or a combination of BC and FG loops (TCL7 library). Such libraries have been designed for use with a cis display system (Odegrip et al., Proc Natl Acad Sci USA 101:2806-2810, 2004). Detailed information about this configuration is given below.

Стабилизированный Tencon (Tencon27) (SEQ ID NO: 4) LPAPKNLWSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTTTCL 7 (рандомизированные петли FG и ВС) (SEQ ID NO: 5) LPAPKNLWSRVTEDsarlswx ₁ x ₂ x ₃ x ₄ x ₅ x ₆ x ₇ x ₈ x ₉ fdsfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvx ₁₀

X11X12X13X14X15X16X17X18X19SNPLSAIFTT;

Where

X1, X2, X3, X4, X5, X6, X10, X11, X12, X13, X14, X15 and X16 are A, D, E, F, G, H, I, K, L, N, P, Q , R, S, T, V, W or Y and

X ₇ , X ₈ , X ₉ , X ₁₇ , X ₁₈ and X ₁₉ are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V , W, Y or removed.

TCL9 (randomized FG loop) (SEQ ID NO: 6) LPAPKNLWSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLKPGTEYTVSIYGV X ₁ X2X ₃ X 4 X ₅ X ₆ X ₇ X ₈ X _{9 X10X11X12SNPLSAIFTT} ;

where X ₁ , X2, X ₃ , X4, X ₅ , X ₆ and X ₇ represent A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T , V, W or Y; And

X ₈ , X ₉ , X ₁₀ , X ₁₁ and X12 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or removed.

For library construction, DNA fragments encoding randomized BC loops (6-9 positions long) or FG loops (7-12 positions long) were synthesized using Slonomics technology (Sloning Biotechnology GmbH) to control library amino acid distribution and remove stop codons. Two different sets of DNA molecules randomizing either the BC loop or the FG loops were independently synthesized and subsequently combined by PCR to create a complete library product.

Building FG Loop Libraries (TCL9)

Created a set of synthetic DNA molecules, consisting of the promoter 2 m (5') Tac and the subsequent complete sequence of the Tencon gene except for randomized codons in the FG loop (SEQ ID NO: 26-31). For FG loop randomization, all amino acids except cysteine and methionine were encoded in equal percentages. The lengths of the diversified region are such that they encode 7, 8, 9, 10, 11 or 12 amino acids in the FG loop. Sub-libraries of each length variant were synthesized individually at 2 μg and then amplified by PCR using Sloning-FOR (SEQ ID NO: 9) and Sloning-Rev (SEQ ID NO: 10) oligonucleotides.

The 1 m (3') fragment of the library is a constant DNA sequence containing display elements, including the PspOMI restriction site, the coding region of the repA gene, and cis and ori elements. PCR was performed to amplify this fragment using plasmid (pCR4Blunt) (Invitrogen) as a template with primers M13 Forward (forward) and M13 Reverse (reverse). The resulting PCR products were digested with PspOMI overnight and purified from the gel. To ligate the 2 m (5') region of library DNA with the 1 m (3') DNA fragment containing the repA gene, 2 pmol (~540 to 560 ng) of the 2 m (5') DNA fragment was ligated with an equimolar amount (~1 .25 μg) of a 1 m repA DNA fragment (3') in the presence of NotI and PspOMI enzymes and T4 ligase at 37°C overnight. The ligated library product was amplified by 12 cycles of PCR using POP2250 (SEQ ID NO: 11) and DigLigRev (SEQ ID NO: 12) oligonucleotides. For each sublibrary, DNAs obtained from 12 PCRs were pooled and purified using a Qiagen spin column. The yield for each TCL9 sublibrary was in the range of 32-34 µg.

Building FG/BC Loop Libraries (TCL7)

The TCL7 library is a library with randomized BC and FG Tencon loops. In this library, BC loops of 6-9 amino acids were combinatorially mixed with randomized FG loops of 7-12 amino acids. Synthetic fragments of Tencon BC6, BC7, BC8 and BC9 (SEQ ID NO: 13-16) were created, including the Tencon gene encoding the N-terminal region of the protein up to and including the VX residue in such a way that the BC loop is substituted with 6, 7, 8 or 9 randomized amino acids. These fragments were synthesized prior to the discovery of the L17A, N46V and E83I (CEN5243) mutations, but these mutations were introduced at the molecular biological steps described below. The following steps were performed to combine this fragment with fragments encoding randomized FG loops.

- 20 042392

First, a DNA fragment encoding the Tac promoter and the 2 m (5') Tencon sequence up to the nucleotide encoding the amino acid A17 (130mer-L17A, SEQ ID NO: 17) was created by PCR using the oligonucleotides POP2222ext (SEQ ID NO: 18) and LS1114 (SEQ ID NO: 19). This was done to include the L17A mutation in the library (CEN5243). Then, DNA fragments encoding Tencon residues R18-V75, including randomized BC loops, were amplified by PCR using BC6, BC7, BC8 or BC9 as a template and oligonucleotides LS1115 (SEQ ID NO: 20) and LS1117 (SEQ ID NO: 21) . At this PCR step, the BsaI site was added to the 1 m (3') end. These DNA fragments were subsequently coupled by overlapping PCR using POP2222ext and LS1117 oligonucleotides as primers. The resulting PCR product is 240 bp in size. were pooled and purified using the Qiagen PCR Purification Kit. Purified DNA was digested with BsaI-HF and gel purified.

The FG loop encoding fragments were amplified by PCR using FG7, FG8, FG9, FG10, FG11 and FG12 as templates with SDG10 (SEQ ID NO: 22) and SDG24 (SEQ ID NO: 23) oligonucleotides to include a BsaI restriction site and options N46V and E86I (CEN5243).

The cleaved BC fragments and the FG fragments were ligated together in one step using triple ligation. Four ligation reactions were performed in 16 possible combinations, with each ligation reaction combining two BC loops of different lengths and 2 FG loops of different lengths. Each ligation pool contained ~300 ng of the total BC fragment and 300 ng of the FG fragment. Subsequently, 4 ligation pools were amplified by PCR using oligonucleotides POP2222 (SEQ ID NO: 24) and SDG28 SEQ ID NO: 25). Subsequently, 7.5 μg of each reaction product was digested with NotI and purified using a Qiagen PCR purification column. 5.2 μg of this DNA was ligated with an equimolar amount of the RepA DNA fragment (~14 mg) digested with PspOMI and the product amplified by PCR using POP2222 oligonucleotides.

Example 2: Creating Tencon Libraries with Alternate Link Surfaces

The choice of residues for randomization in the configuration of a particular library determines the overall shape of the generated interaction surface. X-ray crystallographic analysis of the FN3-domain-containing scaffold protein selected to bind the maltose-binding protein (MBP) from a library with randomized BC, DE, and FG loops showed a significantly curved interaction surface corresponding to the MBP active site (Koide et al., Proc Natl Acad Sci USA, 104:6632-6637, 2007). In contrast, the ankyrin repeat scaffold protein chosen to bind to MBP was found to have a significantly flatter interaction surface, binding to the outer surface of MBP, which is distant from the active one (Binz et al., Nat Biotechnol, 22 : 575-582, 2004). These results suggest that the shape of the binding surface of the scaffold molecule (curvilinear or planar) may determine which target proteins or specific epitopes on those target proteins can be effectively bound by the scaffold. Published attempts to construct FN3-domain-containing protein scaffolds for protein binding have relied on the construction of adjacent loops for target binding, thus allowing curved binding surfaces to be obtained. This approach may limit the number of targets and epitopes available for such scaffolds.

Tencon and other FN3 domains contain two sets of CDR-like loops lying on opposite sides of the molecule, with the first set formed by BC, DE, and FG loops, and the second set formed by AB, CD, and EF loops. The two sets of loops are separated by beta strands that form the center of the FN3 structure. By rotating the Tencon image by 90 degrees, an alternate surface can be rendered. This somewhat concave surface is formed by loops CD and FG and two antiparallel beta strands C and F and is referred to herein as the C-CD-F-FG surface. The CCD-F-FG surface can be used as a template for designing libraries of protein backbone interaction surfaces by randomizing a subset of the residues that form the surface. Beta strands have a repeating structure in which the side chain of every second residue is open on the surface of the protein. Thus, a library can be generated by randomizing some or all of the exposed beta-strand residues on the surface. By selecting appropriate residues in the beta strands, a unique scaffold surface for interaction with other proteins can be obtained with minimal loss of the inherent stability of the Tencon scaffold.

The TCL14 library (SEQ ID NO: 7) was developed in the Tencon27 framework (SEQ ID NO: 4).

For a full description of the methods used to develop this library, see US Patent Publication No. US 2013/0226834.

Library TCL14 (SEQ ID NO: 7): LPAPKNLWSRVTEDSARLSWTAPdaafdsfx ₁ ix ₂ yx ₃ ex4X ₅ x ₆ x ₇ geaivltvpgsersydltglkpgteyx ₈ vx ₉ ix ₁₀ gvkggx ₁₁ x ₁₂ sx ₁₃ pls AIFTT; Where

Xi, X ₂ , X ₃ , X4 X ₅ , X6, X ₇ , X ₈ , X ₉ , Xu, Xu, X ₁₂ and X ₁₃ are A, D, E, F, G, H, I, K, L, N, P,

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Q, R, S, T, V, W, Y, C or M.

The two beta strands that form the C-CD-F-FG surface in Tencon27 have a total of 9 open surface residues that can be randomized; strand C: S30, L32, Q34, Q36; strand F: E66, T68, S70, Y72 and V74, while the CD loop has 6 potential residues: S38, E39, K40, V41, G42 and E43, and the FG loop has 7 potential residues: K75, G76, G77, H78, R79, S80 and N81. Only selected residues were selected for inclusion in the TCL14 configuration due to the larger theoretical library size when all 22 residues were randomized.

Thirteen positions in Tencon were selected for randomization: L32, Q34 and Q36 in the C loop, S38, E39, K40 and V41 in the CD loop, T68, S70 and Y72 in the F band, H78, R79 and N81 in the FG loop. In strands C and F, S30 and E66 were not randomized because they are directly behind the CD and FG loops and are not clearly part of the C-CD-F-FG surface. In the CD loop, G42 and E43 were not randomized because glycine, which provides flexibility, may be valuable in loop regions, and E43 is located at the junction with the surface. K75, G76, G77 and S80 were eliminated in the FG loop. Glycines were excluded for the above reasons, while careful examination of the crystal structures showed that S80 forms key contacts with the nucleus and helps to form a stable FG loop. K75 faces away from the C-CD-F-FG surface and was a less attractive candidate for randomization. Although the above residues were not randomized in the original TCL14 configuration, they could be included in subsequent library configurations to provide additional diversity for de novo selection or, for example, an affinity enhancing library based on the selected TCL14 specific target variant.

After the creation of TCL14, 3 additional Tencon libraries of a similar configuration were created. These two libraries, TCL19, TCL21 and TCL23, are randomized at the same positions as TCL14 (see above), but the distribution of amino acids appearing at these positions is changed (Table 3). TCL19 and TCL21 were designed to include an equal distribution of the 18 naturally occurring amino acids at each position (5.55% each) except for cysteine and methionine. TCL23 was designed such that each randomized position roughly corresponds to the distribution of amino acids found in the HCDR3 loops of functional antibodies (Birtalan et al., J Mol Biol 377:1518-1528, 2008) as shown in Table 1. 3. As in the TCL21 library, cysteine and methionine were excluded.

A third additional library was built to expand the potential target binding surface of other library libraries. In this library (TCL24), 4 additional Tencon positions were randomized compared to the TCL14, TCL19, TCL21 and TCL23 libraries. These positions include N46 and T48 from the D-strand and S84 and 186 from the G-strand. Positions 46, 48, 84, and 86 were chosen, in part, because the side chain surfaces of these residues do not touch the D and G beta strands and are located structurally adjacent to the randomized regions of the C and F strands, resulting in an increase in the area surface available for binding to target proteins. The distribution of amino acids used at each position for TCL24 is identical to that described for TCL19 and TCL21 in Table. 3.

Library TCL24 (SEQ ID NO: 8): LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX ₁ IX ₂ YX ₃ EX ₄ X ₅ X ₆ X ₇ GEAIX ₈ LX ₉ VPGSERSYDLTGLKPGTEYX ₁₀ VX ₁₁ IX ₁ 2GVKGGX ₁₃ X ₁ 4SX ₁ 5P LX ₁₆ AX ₁₇ FTT;

where Xi, X2 X ₃ , X4, X5, X6, X10, X _n , X12, X13, X14, X15, X16 and X ₁₇ are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, Y, or W.

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Table 3. Amino acid frequency (%) at each randomized position for TCL21, TCL23 and TCL24

Amino acid TCL19 TCL21 TCL23 TCL24 Ala 5.6 5.6 6.0 5.6 Arg 5.6 5.6 6.0 5.6 As η 5.6 5.6 3.9 5.6 asp 5.6 5.6 7.5 5.6 Cys 0.0 0.0 0.0 0.0 gin 5.6 5.6 1.5 5.6 Glu 5.6 5.6 2.5 5.6 gly 5.6 5.6 15.0 5.6 His 5.6 5.6 2.3 5.6 lie 5.6 5.6 2.5 5.6 Leu 5.6 5.6 5.0 5.6 Lys 5.6 5.6 1.5 5.6 Met 0.0 0.0 0.0 0.0 Phe 5.6 5.6 2.5 5.6 Pro 5.6 5.6 4.0 5.6 Ser 5.6 5.6 10.0 5.6 Thr 5.6 5.6 4.5 5.6 trp 5.6 5.6 4.0 5.6 Tyr 5.6 5.6 17.3 5.6 Vai 5.6 5.6 4.0 5.6

Creation of TCL21, TCL23 and TCL24 libraries

The TCL21 library was created using Colibra library engineering technology (Isogenica) to control amino acid distribution. TCL19, TCL23, and TCL24 gene fragments were generated using Slonomics technology (Morphosys) to control amino acid distribution. PCR was used to amplify each library after initial synthesis followed by ligation to the RepA gene to be used in selections using the CIS display system (Odegrip et al., Proc Natl Acad Sci USA 101: 2806-2810, 2004) as described above for loop libraries.

Example 3 Selection of type III fibronectin (FN3) domains that bind to PSMA.

Selection on tablets.

A CIS display was used to select PSMA-binding centirins from the TCL7, TCL9, TCL19, and TCL21 libraries. To perform in vitro transcription and translation (ITT), 3 µg of the DNA library was incubated at 30°C with 0.1 mM total amino acids, premixed S30 1X components, and 15 µl of S30 extract (Promega) in a total volume of 50 µl. After 1 h, 375 µl blocking solution (1 x TBS pH 7.4, 0.01% I-block (Life Technologies, no. T2015), 100 µg/ml herring milt DNA) was added and the reactions were incubated in an ice bath for 15 min. ITT reaction mixtures were incubated with recombinant chimpanzee PSMA (well 229) or cynomolgus macaque (well 230) or cynomolgus monkey PSMA-Fc fusion protein (well 231) immobilized on 96-well Maxisorb plates coated with antibodies to human PSMA (Lifespan Bioscience, catalog number LC-C150527). Unbound library elements were removed by successive washes with TBST and TBS. After washing, the DNA was eluted from the target protein by heating to 85°C for 10 min and amplified by PCR for additional rounds of panning. Compounds with high binding affinity were isolated by successively lowering the PSMA target concentration in each cycle from 400 to 100 nM and increasing the stringency of the washing conditions.

After panning, the selected FN3 domains were amplified by PCR, subcloned into a pET vector modified to include a ligase-free cloning site, and transformed into BL21-GOLD (DE3) cells (Stratagene) to express the soluble product in E.cob using standard molecular biology techniques. A gene sequence encoding a C-terminal polyhistidine tag was added to each FN3 domain to allow purification and detection. The cultures were grown to achieve an optical density of 0.6-0.8 in TB medium with the addition of 100

- 23 042392 μg/ml carbenicillin in 96-well blocks with a volume of 1 ml at 37°C, then IPTG was added to 1 mm and at this point the temperature was reduced to 30°C. Cells were harvested approximately 16 hours later by centrifugation and frozen at -20°C. Cell lysis was performed by incubating each pellet in 0.6 ml BugBuster® HT lysis buffer (Novagen EMD Biosciences) with shaking at room temperature for 45 min.

Selection using granules

Centirins were also sampled using a bead-based capture system. ITT reaction mixtures were prepared as described above and subsequently incubated with biotinylated recombinant proteins: chimpanzee or cynomolgus monkey PSMA. Biotinylated recombinant proteins and associated library elements were captured on neutravidin or streptavidin coated magnetic beads. Unbound library elements were removed by successive washes with TBST and TBS. After washing, the DNA was eluted from the target protein by heating to 85°C for 10 min and amplified by PCR for additional rounds of panning. Compounds with high binding affinity were isolated by successively lowering the PSMA target concentration in each cycle from 400 to 100 nM and increasing the stringency of the washing conditions.

Selection by dissociation rate

The products of the fifth selection cycle using pellets were subjected to four cycles of dissociation rate selection. After incubation of ITT reaction mixtures with biotinylated recombinant chimpanzee or cynomolgus monkey proteins, proteins and associated library elements were captured on neutravidin or streptavidin-coated magnetic beads and washed copiously in TBST, bound complexes were washed in 5 μM cold recombinant PSMA proteins for 1 h. Subsequently, ITT associated with the beads were washed copiously in TBST and TBS, followed by elution. The concentration of the biotinylated target antigen was gradually lowered from 25 nM in cycles 6 and 7 to 2.5 nM in cycles 8 and 9. Selection products from cycles 7 and 9 were subcloned into the modified pET15 vector for expression and screening.

Selection of affinity maturation libraries

An affinity maturation library (TCL25) was created based on the sequence of clone P229CR9P819-H11 (SEQ ID NO: 40) using Slonomics technology at Morphosys (Munich, Germany), in which positions 23-30 from the BC loop and positions 78- 83 from the FG loop. Target binding in the library was maintained by introducing nucleotides encoding the original amino acid (from P229CR9P819-H11) at a target frequency of 65% for each randomized position. The remaining 35% of the nucleotides were designed to contain a mixture of codons encoding all the other 20 naturally occurring amino acids with equal probability, except for cysteine and methionine, which were not included. In table. 4 shows the configuration of the TCL25 maturation library. In the table, numbers in brackets indicate the percentage of molecules in the library designed to contain the corresponding amino acid at each position. The scheme of introduction (65% of the original in 14 positions) creates a theoretical distribution of molecules containing predominantly 3, 4, 5, 6 or 7 changes compared to the original molecule.

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Table 4

Position Source amino acid Amino acid distribution (%) 23 asp (2.05), arg(2.05), asn(2.05), asp(65), gin (2.05), glu (2.05), gly (2.05), his (2.05), He (2.05), leu (2.05), lys (2.05), phe (2.05), pro (2.05), ser (2.05), thr (2.05), tyr (2.05), trp (2.05), val(2.05) 24 Not ala(2.05), arg(2.05), asn(2.05), asp(2.05), gin(2.05), glu(2.05), gly(2.05), his (2.05), ile (65), leu (2.05), lys (2.05), phe (2.05), pro (2.05), ser (2.05), thr (2, 05), tyr(2.05), trp(2.05), val(2.05) 25 asp ala(2.05), arg(2.05), asn(2.05), asp(65), gin (2.05), glu (2.05), gly (2.05), his (2.05), ile (2.05), leu (2.05), lys (2.05), phe (2.05), pro (2.05), ser (2.05), thr (2.05), tyr (2.05), trp (2.05), val(2.05) 26 Glu ala (2.05), arg (2.05), asn (2.05), asp (2.05), gin (2.05), glu (65), gly (2.05), his (2 .05), ile (2.05), leu (2.05), lys (2.05), phe (2.05), pro (2.05), ser (2.05), thr (2, 05), tyr(2.05), trp(2.05), val(2.05) 27 gin ala (2.05), arg (2.05), asn (2.05), asp (2.05), gin (65), glu (2.05), gly (2.05), his (2 .05), ile (2.05), leu (2.05), lys (2.05), phe (2.05), pro (2.05), ser (2.05), thr (2, 05), tyr(2.05), trp(2.05), val(2.05) 28 Arg ala (2.05), arg (65), asn (2.05), asp (2.05), gin (2.05), glu (2.05), gly (2.05), his (2 .05), ile (2.05), leu (2.05), lys (2.05), phe (2.05), pro (2.05), ser (2.05), thr (2.05), tyr (2.05), trp (2.05), val(2.05) 29 asp ala(2.05), arg(2.05), asn(2.05), asp(65), gin (2.05), glu (2.05), gly (2.05), his (2.05), ile (2.05), leu (2.05), lys (2.05), phe (2.05), pro (2.05), ser (2.05), thr (2.05), tyr (2.05), trp (2.05), val(2.05) thirty trp ala(2.05), arg(2.05), asn(2.05), asp(2.05), gin (2.05), glu (2.05), gly (2.05), his (2.05), ile (2.05), leu (2.05), lys (2.05), phe (2.05), pro (2.05), ser (2.05), thr (2.05), tyr (2.05), trp (65), val(2.05)

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78 Tugh ala(2.05), arg(2.05), asn(2.05), asp(2.05), gin (2.05), glu (2.05), gly (2.05), his (2.05), ile (2.05), leu (2.05), lys (2.05), phe (2.05), pro (2.05), ser (2.05), thr (2.05), tyr (65), trp (2.05), val(2.05) 79 His ala(2.05), arg(2.05), asn(2.05), asp(2.05), gin (2.05), glu (2.05), gly (2.05), his (65), ile (2.05), leu (2.05), lys (2.05), phe (2.05), pro (2.05), ser (2.05), thr (2.05), tyr (2.05), trp (2.05), val(2.05) 80 Vai ala(2.05), arg(2.05), asn(2.05), asp(2.05), gin (2.05), glu (2.05), gly (2.05), his (2.05), ile (2.05), leu (2.05), lys (2.05), phe (2.05), pro (2.05), ser (2.05), thr (2.05), tyr (2.05), trp (2.05), val(65) 81 Tugh ala(2.05), arg(2.05), asn(2.05), asp(2.05), gin (2.05), glu (2.05), gly (2.05), his (2.05), ile (2.05), leu (2.05), lys (2.05), phe (2.05), pro (2.05), ser (2.05), thr (2.05), tyr (65), trp (2.05), val(2.05) 82 Arg ala (2.05), arg (65), asn (2.05), asp (2.05), gin (2.05), glu (2.05), gly (2.05), his (2 .05), ile (2.05), leu (2.05), lys (2.05), phe (2.05), pro (2.05), ser (2.05), thr (2.05), tyr (2.05), trp (2.05), val(2.05) 83 Ser ala(2.05), arg(2.05), asn(2.05), asp(2.05), gin(2.05), glu(2.05), gly(2.05), his (2.05), ile (2.05), leu (2.05), lys (2.05), phe (2.05), pro (2.05), ser (65), thr (2, 05), tyr(2.05), trp(2.05), val(2.05)

CIS displays were used to select PSMA-binding centirins from the TCL25 library. ITT reaction mixtures were incubated with biotinylated recombinant proteins: chimpanzee and cynomolgus monkey PSMA. Biotinylated recombinant proteins and associated library elements were captured on neutravidin or streptavidin coated magnetic beads. Unbound library elements were removed by successive washes with TBST and TBS. After washing, the DNA was eluted from the target protein by heating to 85°C for 10 min and amplified by PCR for additional rounds of panning. Centirin binding compounds were isolated by successively lowering the PSMA target concentration in each cycle from 400 nM to 100 nM and increasing the stringency of the washing conditions.

The products of the second selection cycle were subjected to four cycles of dissociation rate selection. After incubation of ITT reaction mixtures with biotinylated PSMA recombinant proteins, proteins and bound library elements were captured on neutravidin or streptavidin coated magnetic beads and washed copiously in TBST, bound complexes were washed in 5 μM cold PSMA recombinant proteins for 1 h. Subsequently, ITT associated with beads, washed copiously in TBST and TBS, followed by elution. The concentration of the biotinylated target antigen was gradually lowered from 25 nM in cycles 3 and 4 to 2.5 nM in cycles 5 and 6. Selection products from cycles 7 and 9 were subcloned into the modified pET15 vector for expression and screening.

Biochemical screening for centirins that bind to PSMA

Neutravidin-coated plates were blocked for 1 h in Starting Block T20 (Pierce) and subsequently coated with biotinylated PSMA (using the same antigen as in panning) or a negative control for 1 h. Plates were washed with TBST and diluted lysate was applied on the plates for 1 hour. After additional washing steps, the wells were treated with horseradish peroxidase-conjugated anti-centirin antibody (PAB25) for 1 hour and subsequently analyzed using POD (Roche). Centirins with signals at least 10 times background were selected for additional analysis.

SEC Analysis

Size exclusion chromatography (SEC) was used to evaluate the aggregation status of PSMA-binding centirin. Aliquots (10 μl) of each purified centirin were injected into a Superdex 75 5/150 column (GE Healthcare) at a flow rate of 0.3 ml/min in PBS pH 7.4 mobile phase. The elution from the column was monitored by absorbance at 280 nm. Wild type Tencon was included in each assay as a control. Elution profiles were analyzed using Agilent ChemStation (Rev. B.

- 26 042392

04.02). For further characterization, only proteins with elution profiles similar to those of the wild-type protein in the same assay were selected.

High throughput expression, conjugation and purification of centirins

Isolated clones from unique hits identified by biochemical binding ELISA were pooled in a single hit plate for growth in 96-well block plates; clones were grown in 1 ml cultures (LB medium enriched with kanamycin for selection) at 37° C. overnight with shaking. For protein expression in 96-well block plates in 1 ml of TB medium enriched with kanamycin, 50 μl of the culture incubated overnight were inoculated and grown at 37°C with continuous stirring at 300 rpm until an optical density of OD600=0.6 was reached. -1. After reaching the target OD, protein expression was induced by adding up to 1 mM IPTG; the plates were transferred to 30° C. (300 rpm) conditions for overnight growth. The overnight incubated cultures were centrifuged to collect cells; bacterial pellets were stored at -80°C until use. Both positive and negative controls were applied to each plate in duplicate.

For sortase-labeled conjugation, bacterial pellets were thawed, resuspended, and lysed in BugBusterHT (EMD, cat. no. 70922) enriched with recombinant human lysozyme (EMD, cat. no. 71110). Lysis was performed at room temperature with gentle agitation, after which the plate was transferred to a temperature of 42°C to precipitate host cell proteins. The degradation products were precipitated by centrifugation and the supernatants were transferred to a new block plate for sortase-catalyzed labeling. A stock mixture containing Gly3-vcMMAF (Concortis) de-labeled sortase A and sortase buffer (Tris, sodium chloride and calcium chloride) was prepared at 2-fold concentration and added in equal volumes to the lysate supernatants. The labeling reaction was performed for two hours at room temperature, after which the proteins were purified using a Ni-NTA multi-trap HP plate (GE, cat. no. 28-4009-89). Protein conjugates were recovered by stepwise elution using an elution buffer containing imidazole (50 mM Tris, pH 7.5, 500 mM NaCl, 250 mM imidazole), sterilized by filtration, and used directly for cellular cytotoxicity assays.

High Throughput Cytotoxicity Assay of Centirin Drug Conjugates 96-well black tissue culture coated plates (BD/Corning, cat. no. 353219) were seeded with LNCaP FGC cells (ATCC, cat. no. CRL-1740) at a density of 10,000 cells/well in the assay. media (RPMI free of phenol red (Life Technologies, cat. no. 11835-030) supplemented with 5% fetal bovine serum). The seeded plates were incubated overnight at 37° C. with 5% CO ₂ to allow cell attachment. Twenty-four hours later, CDC was diluted in assay medium (1:100, 1:300, 1:1000, or 1:3000) and applied directly to LNCaP cells. Subsequently, LNCaP cells were incubated at 37° C., 5% CO 2 for 66-72 hours. Cellular toxicity was assessed using the CellTiter-Glo reagent (Promega, cat. no. G7571); 100 µl of the prepared reagent was added to the treated wells and incubated for ten minutes with gentle agitation, protected from light. Luminescence was measured using a SpectraMax M5 plate reader. Values were normalized to untreated controls and selected for further analysis if the achieved toxicity exceeded 50%.

Example 4 Characterization of anti-PSMA centirins Expression and purification on a large scale

The gene sequences encoding the centirin mutants were identified by panning and cloned into the pET15b vector for expression under the control of the T7 promoter or generated by DNA2.0 and subcloned into the pJexpress401 vector (DNA2.0) for expression under the control of the T5 promoter. The resulting plasmids were transformed into E. coli BL21 Gold (Agilent) or BL21DE3 Gold (Agilent) for expression. One colony was selected and grown in Luria Broth (Teknova) enriched with kanamycin and incubated for 18 h at 37°C and 250 rpm. One liter of Terrific Broth (Teknova) enriched with kanamycin was inoculated with these subcultures and grown at 37° C. for 4 hours with stirring. Protein expression was induced with 1 mM IPTG when the absorbance at 600 nm reached 1.0. The protein was expressed for 4 hours at 37°C or for 18 hours at 30°C. Cells were collected by centrifugation at 6000 g and stored at -20°C until purification. Frozen cell pellet (~15-25 g) was thawed for 30 min at room temperature and suspended in BugBusterHT Protein Extraction Reagent (EMD Millipore) enriched with 0.2 mg/ml recombinant lysozyme (Sigma) at a ratio of 5 ml per gram cell paste and incubated for 1 h on a shaker at room temperature. The lysate was purified by centrifugation at 74,600 g for 25 min. The supernatant was applied to a 5 ml Qiagen Ni-NTA cartridge submerged in ice at a flow rate of 4 ml/min using an AKTA AVANT chromatography system. All other Ni-NTA chromatography steps were performed at a flow rate of 5 ml/min. The Ni-NTA column was equilibrated in 25.0 ml of 50 mM Tris-HCl buffer, pH 7.0, containing 0.5 M NCl and 10 mM imidazole (buffer A). After loading, the column was washed with 100 ml of buffer solution A, with

- 27 042392 followed by washing with 100 ml of 50 mM Tris-HCl buffer, pH 7.0, containing 10 mM imidazole, 1% CHAPS detergents, and 1% n-octyl-vO-glucopyranoside, and 100 ml of buffer A. The protein was eluted buffer A enriched with 250 mM imidazole and loaded onto a preparative gel filtration column, TSK Gel G3000SW 21.5x600 mm (Tosoh), equilibrated in PBS (Gibco). Size exclusion chromatography was performed at room temperature in PBS with a flow rate of 10 ml/min using an AKTA-AVANT chromatography system.

Definition of thermal stability

Thermal stability was measured by capillary differential scanning calorimetry (DSC). Each sample was diluted in PBS, pH 7.4 to reach a concentration of 1 mg/mL. Melting points were measured for these samples using a VP-DSC equipped with an autosampler (MicroCal, LLC). The samples were heated from 10 to 95°C or 100°C at a rate of 1°C per minute. Between scans of each sample, only the buffer solution was scanned to calculate a baseline for integration. After subtracting the signal from the buffer solution only, the data were fitted according to the two-state folding model. The reversibility of thermal denaturation was determined by rescanning each sample without removing it from the cell.

Selective Cytotoxicity of Centirin Anti-PSMA Drug Conjugates on PSMA Positive Cells

Centirins were conjugated to vc-MMAF using a cysteine-maleimide chemistry (Brinkley, Bioconjugate Chemistry 3:2-13, 1992) or using the sortase reaction described above. The cytotoxicity of centirin conjugates with vcMMAF was evaluated in LNCaP, VCAP, MDA-PC2B, and PC3 cells in vitro. Cells were plated in 96-well black plates for 24 hours and subsequently treated with various doses of centirin-vcMMAF conjugates. Cells were incubated with centirin drug conjugates (CDC) for 66-72 hours. The CellTiterGlo reagent was used to assess toxicity as described above. Luminescence values were imported into Excel from where they were copied and pasted into the Prism software for graphical analysis. The data was transformed according to the equation X=Log(x), then analyzed using non-linear regression using a 3-parameter model to determine the IC ₅₀ .

In table. 6 combines the unique hits identified during panning, covering many families of sequences. Centirins showed thermal stability in the range of 55-85°C and were cytotoxic against LNCaP cells when conjugated to vcMMAF, with IC ₅₀ values of 22.6-0.38 nM.

Example 4 Characterization of Centirins to PSMA

Expression and purification on a large scale

The gene sequences encoding the centirin mutants were identified by panning and cloned into the pET15b vector for expression under the control of the T7 promoter or generated by DNA2.0 and subcloned into the pJexpress401 vector (DNA2.0) for expression under the control of the T5 promoter. The resulting plasmids were transformed into E. coli BL21 Gold (Agilent) or BL21DE3 Gold (Agilent) for expression. One colony was selected and grown in Luria Broth (Teknova) broth enriched with kanamycin and incubated for 18 h at 37°C and 250 rpm. One liter of Terrific Broth (Teknova) enriched with kanamycin was inoculated with these subcultures and grown at 37° C. for 4 hours with stirring. Protein expression was induced with 1 mM IPTG when the absorbance at 600 nm reached 1.0. The protein was expressed for 4 hours at 37°C or for 18 hours at 30°C. Cells were collected by centrifugation at 6000 g and stored at -20°C until purification. Frozen cell pellet (~15-25 g) was thawed for 30 min at room temperature and suspended in BugBusterHT Protein Extraction Reagent (EMD Millipore) enriched with 0.2 mg/ml recombinant lysozyme (Sigma) at a ratio of 5 ml per gram cell paste and incubated for 1 h on a shaker at room temperature. The lysate was purified by centrifugation at 74,600 g for 25 min. The supernatant was applied to a 5 ml Qiagen Ni-NTA cartridge submerged in ice at a flow rate of 4 ml/min using an AKTA AVANT chromatographic system. All other Ni-NTA chromatography steps were performed at a flow rate of 5 ml/min. The Ni-NTA column was equilibrated in 25.0 ml of 50 mM Tris-HCl buffer, pH 7.0, containing 0.5 M NCl and 10 mM imidazole (buffer A). After loading, the column was washed with 100 ml of buffer solution A, followed by washing with 100 ml of 50 mM Tris-HCl buffer, pH 7.0, containing 10 mM imidazole, 1% CHAPS detergents and 1% n-octyl-β-D-glucopyranoside, and 100 ml of buffer A. The protein was eluted with buffer A enriched with 250 mM imidazole and loaded onto a preparative gel filtration column, TSK Gel G3000SW 21.5x600 mm (Tosoh), equilibrated in PBS (Gibco). Size exclusion chromatography was performed at room temperature in PBS with a flow rate of 10 ml/min using an AKTA-AVANT chromatography system.

Definition of thermal stability

Thermal stability was measured using a capillary differential scanning calorie.

- 28 042392 metrics (DSC). Each sample was diluted in PBS, pH 7.4 to reach a concentration of 1 mg/mL. Melting points were measured for these samples using a VP-DSC equipped with an autosampler (MicroCal, LLC). The samples were heated from 10 to 95°C or 100°C at a rate of 1°C per minute. Between scans of each sample, only the buffer solution was scanned to calculate a baseline for integration. After subtracting the signal from the buffer solution only, the data were fitted according to the two-state folding model. The reversibility of thermal denaturation was determined by rescanning each sample without removing it from the cell.

Selective Cytotoxicity of Centirin Anti-PSMA Drug Conjugates on PSMA Positive Cells

Centirins were conjugated to vc-MMAF using a cysteine-maleimide chemistry (Brinkley, Bioconjugate Chemistry 3:2-13, 1992) or using the sortase reaction described above. The cytotoxicity of centirin conjugates with vcMMAF was evaluated in LNCaP, VCAP, MDA-PC2B, and PC3 cells in vitro. Cells were plated in 96-well black plates for 24 hours and subsequently treated with various doses of centirin-vcMMAF conjugates. Cells were incubated with centirin drug conjugates (CDC) for 66-72 hours. The CellTiterGlo reagent was used to assess toxicity as described above. Luminescence values were imported into Excel from where they were copied and pasted into the Prism software for graphical analysis. The data were transformed according to the equation X=Log(x), then analyzed using non-linear regression using a 3-parameter model to determine the IC50.

In table. 5 combines the unique hits identified during panning, covering many families of sequences. Centirins showed thermal stability in the range of 55-85°C and were cytotoxic against LNCaP cells when conjugated to vcMMAF, with IC ₅₀ values of 22.6-0.38 nM. In table. 6, 7 and 8 show the amino acid sequences of loop BC, C, CD, F and FG of selected clones. In table. 9 shows the amino acid sequences of the clones.

Table 5

clone ID SEQID NO: Types of antigen LNCaP 1С ₅₀ (nM) Tm (°C) P229CR5P819_H11 40 Chimpanzee 20.7 78.1 P258AR6P1071_G03 35 Javanese macaque 5.8 83.1 P258AR6P1070_A05 36 Javanese macaque 4.6 83 P258AR6P1071_F04 37 Javanese macaque 5.4 80.8 P258AR6P1070_F09 38 Javanese macaque 0.9 79.8 P258AR6P1071_D02 39 Javanese macaque 0.8 78.5 P234CR9_H01 46 Javanese macaque 22.6 74.1 P234CR9_A7 45 Javanese macaque 8.8 No data P233FR9_H10 41 Chimpanzee 0.4 65.5 P233FR9P1001_D9 44 Chimpanzee 1.4 58.1 P233FR9P1001_B5-5 42 Chimpanzee 0.5 65 P233FR9P1001_H3-l 43 Chimpanzee 0.4 64.5

Table 6

clone ID Sun loop Loop C clone ID SEQID NO: Subsequence SEQID NO: Subsequence SEQID NO: P229CR5P819 NOR 40 DIDEQRDW 56 FDSFLIQYQE 63 P258AR6P1071 G03 35 DIDEQRDW 56 FDSFLIQYQE 63 P258AR6P1070 A05 36 TIDEQRDW 57 FDSFLIQYQE 63 P258AR6P1071 F04 37 VIDEQRDW 58 FDSFLIQYQE 63 P258AR6P1070 F09 38 TIDEQRDW 57 FESFLIQYQE 64 P258AR6P1071 D02 39 AIDEQRDW 59 FESFLIQYQE 64 P234CR9 H01 46 EWWVIPGD 60 FDSFLIQYQE 63 P234CR9 A7 45 GEQFTI 61 FDSFLIQYQE 63 P233FR9 H10 41 TAPDAA 62 FDSFAIGYWE 65 P233FR9P1001 D9 44 TAPDAA 62 FDSFPIGYWE 66 P233FR9P1001 B5-5 42 TAPDAA 62 FDSFTIGYWE 67 P233FR9P1001 NZ-1 43 TAPDAA 62 FDSFPIGYWE 66

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Table 7

clone ID CD loop Loop F clone ID SEQID NO: Subsequence SEQID NO: Subsequence SEQID NO: P229CR5P819_H11 40 SEKVGE 68 TEYTVSIYGV 70 P258AR6P1071_G03 35 SEKVGE 68 TEYTVSIYGV 70 P258AR6P1070_A05 36 SEKVGE 68 TEYTVSIYGV 70 P258AR6P1071_F04 37 SEKVGE 68 TEYTVSIYGV 70 P258AR6P1070_F09 38 SEKVGE 68 TEYTVSIYGV 70 P258AR6P1071_D02 39 SEKVGE 68 TEYTVSIYGV 70 P234CR9_H01 46 SEKVGE 68 TEYTVSIYGV 70 P234CR9_A7 45 SEKVGE 68 TEYTVSIYG 71 P233FR9_H10 41 WDDDGE 69 TEYPVYIAGV 72 P233FR9P1001_D9 44 WDDDGE 69 TEYWVYIAGV 73 P233FR9P1001_B5-5 42 WDDDGE 69 TEYPVYIAGV 72 P233FR9P1001_H3-l 43 WDDDGE 69 TEYHVYIAGV 74

Table 8

clone ID Hinge FG clone ID SEQID NO: Subsequence SEQID NO: P229CR5P819_H11 40 YHVYRSSN 75 P258AR6P1071_G03 35 YHVYRSN 76 P258AR6P1070_A05 36 YHVYRSN 76 P258AR6P1071_F04 37 YHVYRSN 76 P258AR6P1070_F09 38 YHVYRSN 76 P258AR6P1071_D02 39 YHVYRSN 76 P234CR9_H01 46 VNSGQWNDTSN 77 P234CR9_A7 45 ASGYEWFHAFGSSN 78 P233FR9_H10 41 KGGQWSF 79 P233FR9P1001_D9 44 KGGQWSF 79 P233FR9P1001_B5-5 42 KGGQWSF 79 P233FR9P1001_H3-l 43 KGGQWSF 79

Table 9

Clone clone ID SEQID NO: Subsequence P229CR5P819_H11 40 LPAPKNLWSRVTEDSARLSWDIDEQRDWFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTE YTVSIYGVYHVYRSSNPLSAIFTT P258AR6P1071_G03 35 LPAPKNLWSRVTEDSARLSWDIDEQRDWFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTE YTVSIYGVYHVYRSNPLSAIFTT P258AR6P1070_A05 36 LPAPKNLWSRVTEDSARLSWTIDEQRDWFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTE YTVSIYGVYHVYRSNPLSAIFTT P258AR6P1071_F04 37 LPAPKNLWSRVTEDSARLSWVIDEQRDWFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTE YTVSIYGVYHVYRSNPLSAIFTT P258AR6P1070_F09 38 LPAPKNLWSRVTEDSARLSWTIDEQRDWFESFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTE YTVSIYGVYHVYRSNPLSAIFTT P258AR6P107l_D02 39 LPAPKNLWSRVTEDSARLSWAIDEQRDWFESFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTE YTVSIYGVYHVYRSNPLSAIFTT P234CR9_H01 46 LPAPKNLWSRVTEDSARLSWEWWVIPGDFDSFL IQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTE YTVSIYGWNSGQWNDTSNPLSAIFTT P234CR9_A7 45 LPAPKNLWSRVTEDSARLSWGEQFTIFDSFLIQYQ ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GASGYEWFHAFGS SNPLSAIFTT P233FR9_H10 41 L PAP KN L WS RVT EDSARLSWTAP DAAFD S FAI G YWEWDDDGEAIVLTVPGSERSYDLTGLKPGTEYP VYIAGVKGGQWSFPLSAIFTT P233FR9P1001_D9 44 L P AP KN L WS RVT E D S ARL S WT AP DAAFD S F PIG YWEWDDDGEAIVLTVPGSERSYDLTGLKPGTEYW VYIAGVKGGQWSFPLSAIFTT P233FR9P1001_B5-5 42 L PAP KN L WS RVT EDSARLSWTAP DAAFD S FT IG YWEWDDDGEAIVLTVPGSERSYDLTGLKPGTEYP VYIAGVKGGQWSFPLSAIFTT P233FR9P1001_H3-l 43 LPAPKNLWSRVTEDSARLSWTAPDAAFDSFPIG YWEWDDDGEAIVLTVPGSERSYDLTGLKPGTEYH VYIAGVKGGQWSFPLSAIFTT

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Selected centirin drug conjugates were tested against a panel of cell lines. In table. 10 shows IC ₅₀ values for several vcMMAF-conjugated centirins. Data are averages from one to nine curve fittings. Data are presented as means ± SPE. CDCs were most active in LNCaP cells, which are known to express high concentrations of PSMA. CDCs were also active in MDAPCA-2B and VCAP cells, which are prostate cancer cell lines with lower PSMA concentrations. The activity was absent in PC3 cells (PSMA-negative cell line), indicating selectivity.

Table 10

Clone SEQ ID NO: Cytotoxicity Assays for Centirin Drug Conjugates 1C ₅ 0 (NM) LNCaP cells Cells MDAPCA-2B 1C ₅₀ (nM) Cells VCAP 1C ₅₀ (nM) Cells RSZ 1C ₅₀ (nM) P233FR9P1001-H3-1 43 0.4 4.6 ± 1.2 15.2±1.0 > 500 P234CR9_H01 46 22.6 150.8±4.4 401.0 ± 130.0 > 500 P233FR9_H10 41 0.5±0.1 5.8±2.3 25.9±15.0 > 500 P229CR5P819_H11 40 9.3 ± 1.9 106.8 ± 13.6 231.0 ± 38.0 > 500

Example 5 Construction of Centirins to PSMA

Scanning with cysteine

The genes encoding the anti-PSMA centirin, P233FR9_10, with cysteine residues introduced at various positions in the protein, were generated by DNA2.0 and used to express and purify proteins as described above. The resulting centirins were evaluated for thermal stability (with and without vcMMAF conjugate) and cytotoxicity to LNCaP as described above. The results are presented in table. eleven.

Table 11

clone ID SEQ ID NO: Position of cysteine * Thermal stability (NEM capped) thermal stability (conjugated to vcMMAF) Cytotoxicity to LNCaP (1C ₅₀ ; nM) P233FR9_H10(End) 47 C-terminus TBD TBD No data P233FR9_H10(K62C) 51 K62 56.91 54.03 0.69 P233FR9_H10(R11C) 50 Rll 65.72 63.58 0.40 P233FR9_H10 (E53C) 49 E53 66.75 65, 98 0.66 * Residue numbering according to SEQ ID NO: 41

Example 6 Visualization of Biodistribution of Untargeted Centirins

Centirin, unable to specifically bind to the target antigen, designed to include a cysteine at position 62, was conjugated to DOTA and subsequently to the zirconium-89 radioisotope at IsoTherapeutics Group, LLC (Angleton, Texas). Castrated male NSG mice (Jackson laboratories) were anesthetized with 1.5% isoflurane and examined on a Siemens Inveon micro PET/CT system. Mice were treated with approximately 0.2 mCi of [89Zr] centirin (SEQ ID 51) via tail vein injection (adjusted to 1 mg/kg with cold centirin) and continuous imaging was performed for the first 60 min and subsequently at 3, 6, and 24 hours after centirin injection.

3D PET images were reconstructed using the 2D ordered subset EM algorithm (Siemens Healthcare, Knoxville, TN, USA) in a 768x768x512 tomographic volume with a voxel size of 0.107mmx0.107mmx0.107mm. Images were processed and analyzed using PMOD v3.0 software (PMOD Technologies, Zurich, Switzerland). A cylinder of known activity was scanned in a PET scanner to cross-calibrate between the injected dose as measured by the dose calibrator and the number of pulses per voxel in the PET images. Each PET image was recorded along with a CT image to obtain an anatomical landmark using the PMOD image fusion program. Regions of interest (ROI) were delineated around every 4th section for each analyzed tissue. Received the average number of pulses per voxel and converted to percent injected by dose per gram of body weight, using a correction factor obtained from a calibration cylinder of known activity. All measured radioactivity values were corrected for decay using the known half-life of Zr-89 (78.41 hours).

In FIG. 1 shows the tissue distribution of the FN3 domain labeled with a radioactive isotope during

- 31 042392 time. Found rapid accumulation in the kidneys and bladder, and only a small amount accumulated in the liver, indicating the excretion of centirins through the kidneys.

Example 7 Crystal structure of anti-PSMA antibody P233FR9-H10 in complex with cynomolgus monkey PSMA

Histidine-tagged P233FR9-H10 centirin (referred to herein as centirin H10) was expressed in E. coli and purified using affinity and size exclusion chromatography. Centirin was taken in dPBS, pH 7.2.

The cynomolgus monkey PSMA extracellular domain as a C-terminal fusion protein with the huIgGI Fc domain was expressed in GnTI ^- cells and purified by affinity and size exclusion chromatography. The fusion protein was taken up in dPBS, 0.5 mM ZnCl ₂ , pH 7.2. Subsequently, the Fc domain was removed by treatment with Prescission protease followed by affinity and size exclusion chromatography. The isolated cynomolgus monkey PSMA extracellular domain (cynoPSMA) was stored in dPBS, 0.5 mM ZnCl2, pH 7.2.

The cynomolgus centirin H10/PSMA complex was prepared by mixing cynomolgus monkey PSMA with centirin H10 in a 1:3 molar ratio (excess centirin) while dialyzing for 48 hours at 4°C with 20 mM Hepes, pH 7.0.0 .5 mM ZnCl ₂ . Subsequently, the complex was eluted from a monoS column with a gradient of 48-68 mM NaCl, 20 mM Hepes, pH 7.5, 10% glycerol and concentrated to 3.4 mg/ml. Crystals suitable for X-ray diffraction were prepared from 25% PEG 3 kDa, 0.2 M NH ₄ Cl, 0.1 M Na acetate pH 4.5 using the sessile drop vapor diffusion method at 20°C.

To collect X-ray data, one crystal was soaked for several seconds in a mother liquor containing a cryoprotectant solution with the addition of 20% glycerol and flash-frozen in liquid nitrogen. X-ray diffraction data were obtained using a Dectris Pilatus 6M pixel array detector in the 17-ID emission channel of the Advanced Synchrotron Radiation Source (APS) at Argonne National Laboratory. Diffraction data were processed using HKL2000 software (Otwinowski & Minor, 1997). The statistics of X-ray analysis data are presented in Table. 12.

The structure was restored by molecular displacement (M3) using a Phaser (Read, 2001). The study models for MZ were human PSMA crystal structures (PDB code: 2C6G) and P114AR7P94-A3 W33A centirin structure. Structures were purified using PHENIX (Adams et al, 2004) and model adjustments were performed using COOT (Emsley & Cowtan, 2004). All other crystallographic calculations were performed using the CCP4 software package (CCP4, 1994). All molecular plots were obtained using PyMol (DeLano, 2002). The structure refinement statistics are presented in Table 1. 12.

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Table 12

PS42 Crystal parameters crystallizing solution 0.1 M buffer solution Acetate, pH 4.5 Precipitating agent 25% PEG, 3 kDa Additive 0.2 M NH ₄ C1 space group p2 ₄ 2 ₄ 2 Complex/asymmetric unit 2 elementary cell a (A) 84.0 b (A) 109.9 c (A) 261.6 V _m (A ³ / Yes) 3.32 Solvent content (%) 63 X-ray diffraction data * Resolution (A) 50.00-2.80 High Definition Shell (A) (2.85-2.80) Measured Reflections 335 467 Unique Reflections 57 166 Completeness (%) 93.2 (69.3) Excess 5.9 (4.4) R-sym. (%) 25.1 (64.1) <ΐ/σ> 6.4 (1.6) Optimization Resolution (A) 40.0-2.8 Number of reflections 57 063 Number of all atoms 12 330 Number of water atoms 8 R-factor (%) 25.14 R-free (%) 31.28 RMSD LINK LENGTHS (A) 0.003 connection angles (°) 0.998 Average B-factor (A ² ) 78.9 Ramachandran schedule favorable area (%) 94.9 allowable area (%) 4.8 deviation Values (%) 0.3

*Values in parentheses are for the high resolution shell.

The structure of homodimeric cynomolgus monkey PSMA includes residues 57-750, corresponding to the protease (residues 57-116 and 352-590), apical (residues 117-351), and helical (residues 591-750) domains, and eight of the eleven possible N- linked glycans (in Asn-76, -121, -140, 195, -459, -476, 613 and -638) per dimer subunit. The cynomolgus monkey PSMA active site is located at the interface between three domains and contains two zinc atoms coordinated by histidine (H377 and H553) and glutamate/aspartate residues (D387, catalytic E424, E425 and D453) and a water molecule. The structure of centirin H10 (SEQ ID NO: 41) contains residues 2-92. H10 residues are sequentially numbered according to SEQ ID NO: 41. Cynomolgus monkey PSMA residues are numbered according to the full-length cynomolgus monkey PSMA sequence of SEQ ID NO: 141. Mature cynomolgus monkey PSMA (without signal peptide) starts at residue 44 of SEQ ID NO: 141.

There is one cynomolgus monkey PSMA homodimer in the asymmetric unit and one H10 centirin associated with each PSMA subunit (Fig. 2A). The two centirin/PSMA complexes are structurally very similar as indicated by a standard deviation (CKO) of 0.72 A for the superposition of all equivalent atoms in the PSMA subunits. There is also a high degree of structural similarity between human and cynomolgus PSMA and the absence of large conformational changes caused by binding to centirin, as indicated by an SD of 0.5 A for the superposition of the Ca atom between the cynomolgus PSMA molecule in the centirin complex and unbound human PSMA ( PDB code: 2OOT, 1.6A resolution structure). An interesting feature is that the 541-547 loop region is only visible in the cynomolgus monkey protein due to stabilization of the loop conformation through interactions with centirin.

The site of association of centirin with PSMA is well marked on the electron density map 2Fobs-F _cal c, which allows reliable positioning of binding residues. The following section only describes the interactions between the B and C chains (the PSMA and centirin chains, respectively).

Centirin H10 binds to a region near the PSMA active site (FIG. 2A) and spans the cynomolgus monkey PSMA around 1170 A ² , namely centirin recognizes cynomolgus monkey PSMA residues in the protease (Y460, F488, K499-P502, P504, R511, K514, N540-E542 and N544-F546), apical (R181 residue) and helical (K610, N613 and I614 residues) domains as shown in FIG. 3 and 4.

The front side of the four-stranded centirin β-sheet folds on the PSMA surface in such a way

- 33 042392 such that one CD loop is deeply inserted into the active site entry (FIGS. 2B and 2C). Namely, H10 FN3 domain residues involved in PSMA binding are located in the β-strands C (A32 and G34), D (V46), F (G64, P68, Y70 and A72) and G (S84-I86) and loops CD (W36, W38-D41, E43 and A44) and FG (W79, F81 and P82). Residues D39, D40, D41, and E43 transfer a negative charge to the CD centirin loop, and these residues are inserted into a positively charged funnel ~20 A deep, which leads to zinc ions in the active site, likely blocking substrate entry into the funnel and PSMA enzymatic activity (Fig. 2B and 2C). However, centirin does not interact directly with zinc ions or their coordinating residues.

The conserved PSMA residues W541, Y460, F488, P502, and P504 form an aromatic cluster across the fusion site with centirin residues W36, P68, Y70, W79, F81, and P82 (FIG. 3A). The conserved R511 is in the central position of the fusion site and is hydrogen bonded to Y70, which is the central residue in the four-stranded β-sheet of centirin. In FIG. 3B shows in drawing the remains of a paratope and an epitope.

The human and cynomolgus PSMAs are 97% identical, and with the exception of the S613N change, all residues interacting with H10 are conserved between the two species (FIG. 4). The change in S613N leads to glycosylation of N613 in PSMA of the cynomolgus monkey and the creation of van der Waals contacts between the carbohydrate and residues E66, 186, T88 (β-strands F and G) of centirin, which will not be in the human enzyme.

Centirin residues for conjugation

Various H10 centirin residues outside the fusion site can be modified for conjugation to small molecules (toxins) without disrupting PSMA binding or centirin folding. Cysteines were already located and conjugated to toxins at the C-terminus (behind the histidine tag) and at positions R11, E53 and K62, and all of these variants showed similar effective cytotoxicity. In addition, residues T22, D25 and A26 in loop BC, terminal residue N6 and S52 in loop DE are potentially good sites for mutagenesis followed by chemical conjugation (FIG. 5). These solvent-exposed residues are located away from the centirin/PSMA interface and are located in structurally flexible regions.

Moreover, the N- and C-terminal regions do not contain fusions with the domains of other proteins. The N-terminus is directed to the protease domain of PSMA and is reachable by the fusion linker, and the other also accessible C-terminus is directed to the PSMA helical domain. The optimal length of the linker to a molecule that fuses with centirin will depend on the structure of the fusion molecule and the location of its binding site on the target molecule.

Mechanism of action

Centirin H10 is a potential molecule for targeted delivery of toxins (toxic small molecules, nucleic acids, etc.) to prostate cancer cells by internalization of the centirin/PSMA complex. Moreover, Centirin H10 is a potential molecule for redirecting immune cells to prostate cancer cells when in a multispecific form.

Centirin H10 probably also inhibits PSMA enzymatic activity, which may lead to reduced cell fitness and survival. In the centirin/PSMA structure of the cynomolgus monkey, centirin is associated with an active site entry, which may prevent substrate interaction with PSMA through steric hindrance and direct competition for the binding site.

Example 8. Creation of additional variants of centirin to PSMA

The selected centirin to PSMA was further engineered to improve the properties of the parent centirin. PSMA binding FN3 domains were then created using the libraries described above and tested for PSMA binding.

In table. 13 shows the amino acid sequences of the generated molecules.

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Table 13

clone ID SEQID NO: Subsequence P258AR6P1071_D02_vl 75 LPAPKNLWSRVTEDSARLSWAADEQRDWFESFLIQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYHVYRSN P L SAIFT T P2 5 8AR6 P1071_D 0 2_v2 76 LPAPKNLWSRVTEDSARLSWAIAEQRDWFESFLIQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYHVYRSNPL SAIFT T P258AR6P1071_D02_v3 77 LPAPKNLWSRVTEDSARLSWAIDAQRDWFESFLIQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYHVYRSN P L SAIFT T P258AR6P1071_D02_v4 78 L PAP KN L WS RVT E D SARL S WAI D EARDW F E S FL IQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYHVYRSN P L SAIFT T P258AR6P1071_D02_v5 79 L PAP KN L WS RVT ED SARL S WAI D EQADWFE S FL IQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYHVYRSN P L SAIFT T P258AR6P1071_D02_v6 80 LPAPKNLWSRVTEDSARLSWAIDEQRAWFESFLIQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYHVYRSN P L SAIFT T P258AR6P1071_D02_v7 81 L PAP KN L WS RVT ED SARL S WAI D EQRDAFE S FL IQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYHVYRSN P L SAIFT T P258AR6P1071_D02_v8 82 LPAPKNLWSRVTEDSARLSWAIDEQRDWFESFLIQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVAHVYRSNPLSAIFTT

- 35 042392

P258AR6P1071_D02_v9 83 LPAPKNLWSRVTEDSARLSWAIDEQRDWFESFLIQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYAVYRSN P L SAIFT T P258AR6P1071_D02_vl0 84 LPAPKNLWSRVTEDSARLSWAIDEQRDWFESFLIQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYHAYRSNPLSAIFTT P258AR6P1071_D02_vll 85 LPAPKNLWSRVTEDSARLSWAIDEQRDWFESFLIQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYHVARSN P L SAIFT T P258AR6P1071_D02_vl2 86 LPAPKNLWSRVTEDSARLSWAIDEQRDWFESFLIQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYH VYASNPLSAIFTT P258AR6P1071_D02_vl3 87 L PAP KN LWS RVT ED S ARL S WAI D EQRDWFAS FL IQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYHVYRSN P L SAIFT T P258AR6P1071_D02_vl4 88 LPAPKNLWSRVTEDSARLSWDIDEQRDWFESFLIQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYHVYRSN P L SAIFT T P258AR6P1071_D02_vl5 89 LPAPKNLWSRVTEDSARLSWAIDEQRDWFDSFLIQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYHVYRSN P L SAIFT T P258AR6P1071_D02_vl6 90 LPAPKNLWSRVTEDSARLSWAIDEQRDWFESFL IQYQESEKVGEAIVLTVPGSCRSYDLTGLKPGTE YTVSIYGVYHVYRSSNPLSAIFTT P258AR6P1071_D02_vl7 91 LPAPKNLWSRVTEDSARLSWDIDEQRDWFDSFLIQ YQESEKVGEAIVLTVPGSCRSYDLTGLKPGTEYTVS IYGVYHVYRSN P L SAIFT T P258AR6P1071_D02_vl8 92 LPAPKNLWSRVTEDSARLSWDIDEQRDWFESFL IQYQESEKVGEAIVLTVPGSCRSYDLTGLKPGTE YTVSIYGVYHVYRSSNPLSAIFTT P258AR6P1071_D02_vl9 93 LPAPKNLWSRVTEDSARLSWAIDEQRDWFDSFL IQYQESEKVGEAIVLTVPGSCRSYDLTGLKPGTE YTVSIYGVYHVYRSSNPLSAIFTT P233FR9_H10_vl 94 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYRVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v2 95 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYKVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v3 96 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYEVYIA GVKGGQWSFPLSAIFTT

- 36 042392

P233FR9_H10_v4 97 L PAP KN LWS RVT ED S ARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v5 98 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYDVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v6 99 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYAVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v7 100 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYGVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v8 101 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYWYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v9 102 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYLVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_vl0 103 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYIVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_vll 104 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYFVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_vl2 105 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYWVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_vl3 106 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYNVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_vl4 107 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYQVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_vl5 108 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYSVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_vl6 109 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYTVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_vl7 110 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYYVYIA GVKGGQWSFPLSAIFTT

- 37 042392

P233FR9_H10_vl8 111 L PAP KN LWS RVT ED S ARL S WTAP DAAFD S FAI AYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYPVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_vl9 112 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI S YW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYPVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v20 113 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDTDGEAIVLTVPGSCRSYDLTGLKPGTEYPVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v21 114 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDSDGEAIVLTVPGSCRSYDLTGLKPGTEYPVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v22 115 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYY EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYPVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v23 116 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYF EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYPVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v24 117 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYL EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYPVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v25 118 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EYDDDGEAIVLTVPGSCRSYDLTGLKPGTEYPVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v26 119 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EFDDDGEAIVLTVPGSCRSYDLTGLKPGTEYPVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v27 120 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW ELDDDGEAIVLTVPGSCRSYDLTGLKPGTEYPVYIA GVKGGQWSFPLSAIFTT P233FR9_H10_v28 121 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYPVYIA GVKGGQYSFPLSAIFTT P233FR9_H10_v29 122 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYPVYIA GVKGGQFSFPLSAIFTT P233FR9_H10_v30 123 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYPVYIA GVKGGQLSFPLSAIFTT P233FR9P1001-H3-l_vl 124 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FRI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT

- 38 042392

P233FR9P1001-H3-l_v2 125 L PAP KN LWS RVT ED S ARL S WTAP DAAFD S FKIGYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_v3 126 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FE I GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_v4 127 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FHI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_v5 128 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FDI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_v6 129 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FAI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_v7 130 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FGI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_v8 131 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FVI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_v9 132 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FL I GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_vl0 133 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FI I GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_vll 134 L PAP KN LWS RVT EDSARLSWTAP DAAFD S F FIG YW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_vl2 135 L PAP KN LWS RVT EDSARLSWTAP DAAFD S FWIG YWEWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYH VYIAGVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_vl3 136 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FNI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_vl4 137 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FQI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_vl5 138 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FSI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_vl6 139 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FT I GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT P233FR9P1001-H3-l_vl7 140 L PAP KN LWS RVT ED SARL S WTAP DAAFD S FYI GYW EWDDDGEAIVLTVPGSCRSYDLTGLKPGTEYHVYIA GVKGGQWSFPLSAIFTT

Example 9 Detection of PSMA expression on tumor cells using anti-PSMA centirin conjugated with a fluorescent dye.

This example shows the detection of PSMA present on cells using centirin to PSMA conjugated to a fluorescent dye. C-terminated his-tag centirin to PSMA P233FR9_H10 (SEQ ID NO: 49) with a free cysteine at amino acid position 53 was conjugated to R-phycoerythrin (PE) (Prozyme, cat. no. PB31). Phycoerythrin (PE) was activated with sulfo-SMCC (Pierce, catalog # 22122) for 60 min and activated PE was separated from free sulfo-SMCC by size exclusion chromatography using a Sephadex G25 column and PBS/EDTA buffer. Centirin was reduced with TCEP (Sigma cat. no. 646547) for 30 minutes. The reduced centirin was separated from the free TCEP by size exclusion chromatography using a Se column.

- 39 042392 phadex G25 and PBS/EDTA buffer. Activated R-PE was covalently coupled to reduced centirin for 90 minutes followed by quenching with N-ethylmaleimide (Sigma, Cat #04260) for 20 minutes. PE-conjugated centirin was purified by size exclusion chromatography (SEC) using a Tosoh TSKgel G3000SW column in 100 mM sodium phosphate, 100 mM sodium sulfate, sodium azide 0.05%, pH 6.5 on a high performance liquid chromatograph (FPLC) AKTA explorer ( General Electric).

PE-conjugated centirin was tested for sensitivity and specificity using PSMA positive and negative cell lines by flow cytometry and circulating tumor cell (CTC) CellSearch analysis. The following prostate cell lines were purchased from the ATCC collection and used to determine the specificity of centirin for PSMA: LNCaP (high PSMA expression), 22Rv1 (low PSMA expression) and PC3 (no PSMA expression).

Detection of PSMA on cell lines by flow cytometry

Prostate cell lines were harvested using standard cell line preparation procedures. Cells (~30,000) were stained in 0.1 ml PBS containing 1% bovine serum albumin (BSA) with PE-conjugated centirin for 20 min. Anti-PSMA PE conjugate from Biolegend (clone LNI-17, cat. no. 342504) was used as a positive control. After incubation, 3 ml of PBS/BSA buffer solution was added, and unbound PE conjugate was removed by centrifugation at 800 g for 5 min. The supernatant was aspirated and the cells were resuspended in 0.3 ml PBS/BSA. Samples were analyzed using FACSCalibur from BD Biosciences. The average fluorescence intensity (MFI) of PSMA staining in each cell line was determined and compared with the MIF obtained using an anti-PSMA antibody. SIF is directly related to the level of PSMA expression, with higher SIF observed in a cell line with high PSMA expression. In FIG. 6 shows SIF values in various cell lines obtained using PE-conjugated centirin compared to SIF values obtained using an anti-PSMA antibody complex with PE.

The results show that PE-conjugated centirin binds to PSMA-positive cell lines and does not non-specifically bind to PSMA-negative cells. As expected, SIF is higher in a cell line (LNCaP) with high PSMA expression compared to low SIF in a cell line (22Rv1) with low PSMA expression. SIF in PSMA-negative cell line (PC3) is almost equal to the background signal. In addition, the binding performance of PE-conjugated Centirin E to various cell lines is similar to that of PE-conjugated anti-PSMA antibody, as similar CIF values were obtained using both conjugates (centirin and antibody). This example shows that PE-conjugated centirin has sensitivity and specificity for the detection of PSMA on tumor cells.

PSMA detection through analysis of circulating tumor cells

The above results were further confirmed by testing the RE-conjugated centirin in the CELLSEARCH assay to detect and enumerate circulating tumor cells (CTCs) in 7.5 ml of blood. Circulating tumor cell counts using CELLSEARCH (Veridex LLC, Raritan, NJ, USA) were performed after training according to the manufacturer's instructions. The CELLSEARCH assay uses anti-EpCAM antibodies conjugated to magnetic particles in ferrofluid to capture CTCs and visualizes anti-cytokeratin antibodies specific to cytokeratins 8, 18 and 19 conjugated to fluorescein. The CELLSEARCH assay uses a CELLSEARCH AutoPrep for sample preparation and a CELLTRACKS Analyzer II® (CTA II) for analysis. STA II is a four-color semi-automatic fluorescence microscope that is used to detect and count CTCs using 3 colors. The fourth color in CTA II can be used to phenotype CTCs with additional markers of interest. In this example, tissue culture tumor cells were placed in normal blood to mimic CTC in blood. Approximately 500 tumor cells (LNCaP, 22Rv1, PC3-9 or SKBR3 cells) were added to 7.5 ml of normal donated blood collected in a CELLSAVE tube (Janssen Diagnostics). Also, a breast cancer cell line (SKBR3) was used as a PSMA-negative cell line. Samples were processed in AutoPrep using the CELLSEARCH CXC kit and PE-conjugated centirin as a marker.

Sample preparation system

AutoPrep increases the concentration of tumor cells by capturing tumor cells with ferrofluid to EpCAM. CTC-enriched samples were stained with nucleic acid stain (DAPI) to detect nucleated cells, anti-cytokeratin antibodies conjugated to fluorescein isothiocyanate (FITC) to detect tumor cells, and anti-leukocyte antibodies conjugated to allophycocyanin (APC) to detect leukocytes. sample image

- 40 042392 was diluted to a final volume of 0.32 ml and transferred to the sample chamber inside the MagNest® Cell Viewer. The MagNest® device presents magnetically labeled cells for analysis in the CELLTRACKS Analyzer II®. Samples were analyzed using CTAII for CTC count and detection on CTC PSMA. The analyzer automatically analyzes the samples and presents potential DAPI and cytokeratin positive tumor cells as thumbnail images for viewing. The results of evaluating tumor cells stained with PE-conjugated centirin in the CellSearch assay are shown in FIG. 7. Images of CTCs stained with PE-conjugated centirin are shown in the columns labeled PSMA-PE.

In FIG. 7A shows PSMA expression on LNCaP tumor cells with 100% of these cells being PSMA positive. 26% of the cell line with low PSMA expression (22Rv1) are PSMA positive (Fig. 7B). On the other hand, PSMA-negative cell lines (PC3-9 and SKBR3) are PSMA-negative (FIGS. 7C and 7D). These results are consistent with the results of flow cytometry. This example shows that Centirin can be used to detect PSMA expression on CTC and further confirms the sensitivity and specificity of Centirin for PSMA.

Sequences

SEQ ID NO: 1=Tencon source sequence

LPAPKNLWSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLT

GLKPGTEYTVSIYGVKGGHRSNPLSAEFTT

SEQ ID NO: 2=TCL1 Library

LPAPKNLWSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLT

GLKPGTEYTVSIYGV(X) ₇ -i ₂ PLSAEFTT ;

Where

Xi, X ₂ , Xs, X ₄ , X5, X ₆ , X? are any amino acid; And

X ₈ , X ₉ , Xu, Chi and X ₂₂ are any amino acid or have been deleted.

SEQ ID NO: 3=TCL2 Library

LPAPKNLWSEVTEDSLRLSWXiX ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ SFLIQYQESEKVGEAINLTVPGSER

SYDLTGLKPGTEYTVSIYGVX ₉ Xi ₀ XiiX12X13SXi ₄ X15LSAEFTTT;

Where

Xi is Ala, Arg, Asn, Asp, Glu, Gin, Gly,

His, lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Vai;

X ₂ is Ala, Arg, Asn, Asp, Glu, Gin, Gly,

His, lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Vai;

X ₃ is Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, lie, Leu,

Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Vai;

X ₄ is Ala, Arg, Asn, Asp, Glu, Gin, Gly,

His, lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Vai;

X ₅ is Ala, Arg, Asn, Asp, Glu, Gin, Gly,

His, lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Vai;

X ₆ is Ala, Arg, Asn, Asp, Glu, Gin, Gly,

His, lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Vai;

X ₇ is Phe, lie, Leu, Vai or Tyr;

X ₈ is Asp, Glu or Thr;

X ₉ is Ala, Arg, Asn, Asp, Glu, Gin, Gly,

His, lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Vai;

Hu is Ala, Arg, Asn, Asp, Glu, Gin, Gly,

His, lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Vai;

Xn is Ala, Arg, Asn, Asp, Glu, Gin, Gly,

His, lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Vai;

X ₂₂ is Ala, Arg, Asn, Asp, Glu, Gin, Gly,

His, lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Vai;

Xis is Ala, Arg, Asn, Asp, Glu, Gin, Gly,

His, lie, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Vai;

- 41 042392

X14 represents ala, arg, asn, asp, Glu, Gin, gly his, lie, , Leu, Lys, Phe, Pro , Ser, , Thr, trp, Tyr or Vai; And X15 represents ala, arg, asn, asp, Glu, Gin, gly his, lie, , Leu, Lys, Phe, Pro , Ser, , Thr, trp, Tyr or wai.

SEQ ID NO: 4=Tencon Stabilized

LPAPKNLWSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLKPGTEYTVSIYGVKGGHRSNPLSAIFTT

SEQ ID NO: 5=TCL7 (EG and BC loops)

LPAPKNLWSRVTEDSARLSWXiX ₂ X ₃ X4X ₅ X ₆ X ₇ X ₈ X ₉ FDSFLIQYQESEKVGEAIVLTVP

GSERSYDLTGLKPGTEYTVSIYGVX10X11X12X13X14X15X16X17X18X19SNPLSAI FTT ; Where

Xl, X ₂ , Xs, X ₄ , X ₅ , X ₆ , X10, Xll, X12, X13, X14, X15 And X _i6 are A, D, E, F, G, Η, I, K, L, N, P, Q, R, S, T,

V, W or Y; And x7, X ₈ , X ₉ , X17, X18 and X ₁₉ are A, D, E, F, G h, I , TO, L N, P, Q, R, S, T, V, W, Y or removed. SEQ ID NO: 6=TCL9 (loop EG)

LPAPKNLWSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLT

GLKPGTEYTVSIYGV X ₁ X ₂ XzX4X ₅ XbX7X8X9 X10X11X12SNPLSAI FTT;

Where x5, X ₆ and X ₇ represent yourself A, D, E, F, G XL, x2, ^x3 , ^x4 , h, I, K L N, P, Q, R, S, T, V, W or Y; And X ₈ , X ₉ , X ₁₀ , Xu and X12 represent A, D E, F, g, n, I K, L, N P, Q, R, s T, V, W, Y or removed.

TCL14 Library (SEQ ID NO: 7)

LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX ₁ IX ₂ YX ₃ EX4X ₅ X ₆ X ₇ GEAIVLTVPGSERS YDLTGLKPGTEYX ₈ VX ₉ IXioGVKGGXiiXi ₂ SXi ₃ PLSAI FTT ;

Where

Xi, X ₂ , Xs, X ₄ , X ₅ , Xe, X ₇ , Xs, X9, X10, X11, X12 and X13 are A, D, E, F, G, Η, I, K, L, Ν , P, Q, R, S, T, V, W, Y, C, or M.

TCL24 Library (SEQ ID NO: 8)

LPAPKNLWSRVTEDSARLSWTAPDAAFDSEXiIX ₂ YX3EX ₄ X ₅ X ₆ X7GEAIX ₈ LX ₉ VPGSE RSYDLTGLKPGTEYXi ₀ VXiiIXi ₂ GVKGGXi3Xi4SXi5PLXi ₆ AXi ₇ FTT ;

Where

- 42 042392

Xi, X ₂ , Xs, Xo X ₅ , Xe, Xu, Xu X12, Xi Xi Xi Xie and X _i7 are A, D, E, F, G, Η, I, K, L, N, P, Q , R, S, T, V, Y or W.

SEQ ID NO: 9=Sloning-FOR

GTGACACGGCGGTTAGAAC

SEQ ID NO: 10=Sloning-REV

GCCTTTGGGAAGCTTCTAAG

SEQ ID NO: 11=POP2250

CGGCGGTTAGAACGCGGCTACAATTAATAC

SEQ ID NO: 12=DigLigRev

CATGATTACGCCAAGCTCAGAA

SEQ ID NO: 13=BC9

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACA ATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG GATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCG TCTGTCTTG GNNNNNNNNNNNNNNNNNNNNNNNNNNN TTY GAC TCTTTCCTGATC GAG TAG GAG GAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACC TGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCC AAAGGC

SEQ ID NO: 14=BC8

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACA ATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG GATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCG TCTGTCTTG GNNNNNNNNNNNNNNNNNNNNNNNN TTY GAC TCTTTCCTGATC CAG ТАС CAG GAA TCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGA CCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAA GGC

SEQ ID NO: 15=BC7

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACA ATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG GATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCG TCTGTCTTG GNNNNNNNNNNNNNNNNNNNNN TTY GAC TCTTTCCTGATC CAG ТАС CAG GAAT С T GAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCG GTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC

SEQ ID NO: 16=BC6

- 43 042392

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACA ATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG GATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCG TCTGTCTTG GNNNNNNNNNNNNNNNNNN TTY GAG TCTTTCCTGATC CAG TAG CAG GAAT С T GAA AAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTC TGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC

SEQ ID NO: 17=130 mer-L17A

CGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCG GCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATG CTG

SEQ ID NO: 18=POP222ext

OGG CGG TTA GAA GGC GGC TAC AAT TAA TAG

SEQ ID NO: 19=LS1114

CCA AGA CAG AGG GGC AGA GTC TTC GGT AAG GCG AGA AAG AAG CAG

GTT TTT CGG CGG CGG CAG CAT GGT AGA TCC TGT TTC

SEQ ID NO: 20=LS1115

COG AAG ACT CTG GCC GTC TGT GTT GG

SEQ ID NO: 21=LS1117

CAG TGG TGT CAG GGA TTC CTG GTA CTG GAT CAG GAA AGA GTC GAA

SEQ ID NO: 22=SDG10

CATGCGGTCTCTTCCGAAAAAGTTGGTGAAGCGATCGTCCTGACCGTTCCGGGT

SEQ ID NO: 23=SDG24

GGTGGTGAAGATCGCAGACAGCGGGTTAG

SEQ ID NO: 24=POP2222

CGGCGGTTAGAACGCGGCTAC

SEQ ID NO: 25=SDG28

AAGATCAGTTGCGGCCGCTAGACTAGAACCGCTGCCACCGCCGGTGGTGAAGATCGCAG AC

SEQ ID NO: 26=FG12

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACA ATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG GATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCG TCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAA AAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTC T GAAAC C G G G TAC C GAATACAC CGTTTCTATCTACGGTGTT NNNNNNNNNNNNNNNNNNNNNNN

- 44 042392

NNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCAC CATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC

SEQ ID NO: 27=FG11

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACA ATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG GATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCG TCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAA AAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTC T GAAAC C G G G TAC C GAATACAC CGTTTCTATCTACGGTGTT NNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCAT GGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC

SEQ ID NO: 28=FG10

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACA ATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG GATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCG TCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAA AAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTC T GAAAC C G G G TAC C GAATACAC CGTTTCTATCTACGGTGTT NNNNNNNNNNNNNNNNNNNNNNN NNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGC AGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC

SEQ ID NO: 29=FG9

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACA ATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG GATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCG TCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAA AAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTC T GAAAC C G G G TAC C GAATACAC CGTTTCTATCTACGGTGTT NNNNNNNNNNNNNNNNNNNNNNN NNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGC GGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC

SEQ ID NO: 30=FG8

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACA ATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG GATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCG TCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAA AAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTC T GAAAC C G G G TAC C GAATACAC CGTTTCTATCTACGGTGTT NNNNNNNNNNNNNNNNNNNNNNN

- 45 042392

NTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGT TCTAGTCTAGCGGCCGCAACTGATCTTGGC

SEQ ID NO: 31=FG7

GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACA ATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAG GATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCG TCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAA AAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTC TGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNTC TAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCT AGTCTAGCGGCCGCAACTGATCTTGGC

SEQ ID NO: 32=PSMW1 (N'-AviTag-HisTag-GS-Cyno PSMA_ECD)

KSSSEATNITPKHNMKAFLDELKAENIKKFLHNFTQIPHLAGTEQNFQLAKQIQSQWKE FGLDSVELTHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPAGYENVSDIVPPFSAFSP QGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGATGVILYSD PDDYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGMAEAVGLPSIPV HPIGYYDAQKLLEKMGGSASPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTSEVTRIYNV IGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAWHEIVRSFGMLKKEGWRPRRTILFASWD AEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVYNLTKELESPDE GFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSSYP LYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSWLPFDCRDYAWLRKYADKIYNI SMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLRDFDKSNPILLRMMNDQLMFLERAFIDP LGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSQAWGEVKRQISIATFTVQAA AETLSEVA

SEQ ID NO: 33=PSMW8 (N'-AviTag-HisTag-GS-Chimp PSMA_ECD)

KSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKE FGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVLDIVPPFSAFSP QGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSD PADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRHGIAEAVGLPSIPV HPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNV IGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAWHEIVRSFGTLKKEGWRPRRTILFASWD AEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVYNLTKELKSPDE GFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYP LYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAWLRKYADKIYNI SMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFTERLQDFDKSNPILLRMMNDQLMFLERAFIDP

- 46 042392

LGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGDVKRQISVAAFTVQAA

AETLSEVA

SEQ ID NO: 34: hexahistidine tag

HHHHH

SEQ ID NO: 35=P258AR6P1071_G03

LPAPKNLWSRVTEDSARLSWDIDEQRDWFDSFLIQYQESEKVGEAIVLTVPGSERSYD

LTGLKPGTEYTVSIYGVYHVYRSNPLSAIFTT

SEQ ID NO: 36=P258AR6P1070_A05

LPAPKNLWSRVTEDSARLSWTIDEQRDWFDSFLIQYQESEKVGEAIVLTVPGSERSYD

LTGLKPGTEYTVSIYGVYHVYRSNPLSAIFTT

SEQ ID NO: 37=P258AR6P1071_F04

LPAPKNLWSRVTEDSARLSWVIDEQRDWFDSFLIQYQESEKVGEAIVLTVPGSERSYD

LTGLKPGTEYTVSIYGVYHVYRSNPLSAIFTT

SEQ ID NO: 38=P258AR6P1070_F09

LPAPKNLWSRVTEDSARLSWTIDEQRDWFESFLIQYQESEKVGEAIVLTVPGSERSYD

LTGLKPGTEYTVSIYGVYHVYRSNPLSAIFTT

SEQ ID NO: 39=P258AR6P1071_D02

LPAPKNLWSRVTEDSARLSWAIDEQRDWFESFLIQYQESEKVGEAIVLTVPGSERSYD

LTGLKPGTEYTVSIYGVYHVYRSNPLSAIFTT

SEQ ID NO: 40=P229CR5P819_H11

LPAPKNLWSRVTEDSARLSWDIDEQRDWFDSFLIQYQESEKVGEAIVLTVPGSERSYD

LTGLKPGTEYTVSIYGVYHVYRSSNPLSAIFTT

SEQ ID NO: 41=P233FR9_H10

LPAPKNLWSRVTEDSARLSWTAPDAAFDSFAIGYWEWDDDGEAIVLTVPGSERSYDLT

GLKPGTEYPVYIAGVKGGQWSFPLSAIFTT

SEQ ID NO: 42=P233FR9P1001_B5-5

LPAPKNLWSRVTEDSARLSWTAPDAAFDSFTIGYWEWDDDGEAIVLTVPGSERSYDLT

GLKPGTEYPVYIAGVKGGQWSFPLSAIFTT

SEQ ID NO: 43=P233FR9P1001_H3-l

LPAPKNLWSRVTEDSARLSWTAPDAAFDSFPIGYWEWDDDGEAIVLTVPGSERSYDLT

GLKPGTEYHVYIAGVKGGQWSFPLSAIFTT

SEQ ID NO: 44=P233FR9P1001_D9

LPAPKNLWSRVTEDSARLSWTAPDAAFDSFPIGYWEWDDDGEAIVLTVPGSERSYDLT

GLKPGTEYWVYIAGVKGGQWSFPLSAIFTT

SEQ ID NO:45=P234CR9_A07

- 47 042392

LPAPKNLWSRVTEDSARLSWGEQFTIFDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLKPGTEYTVSIYGASGYEWFHAFGSSNPLSAIFTT

SEQ ID NO: 46=P234CR9_H01

LPAPKNLWSRVTEDSARLSWEWWVIPGDFDSFLIQYQESEKVGEAIVLTVPGSERSYD LTGLKPGTEYTVSIYGWNSGQWNDTSNPLSAI FTT

SEQ ID NO: 47=P233FR9_H10 (cterm cys)

LPAPKNLWSRVTEDSARLSWTAPDAAFDSFAIGYWEWDDDGEAIVLTVPGSERSYDLT GLKPGTEYPVYIAGVKGGQWSFPLSAIFTTC

SEQ ID NO: 48=P233FR9_H10 (K62C)

LPAPKNLWSRVTEDSARLSWTAPDAAFDSFAIGYWEWDDDGEAIVLTVPGSERSYDLT GLCPGTEYPVYIAGVKGGQWSFPLSAIFTT

SEQ ID NO: 49=P233FR9_H10 (E53C)

LPAPKNLWSRVTEDSARLSWTAPDAAFDSFAIGYWEWDDDGEAIVLTVPGSCRSYDLT GLKPGTEYPVYIAGVKGGQWSFPLSAIFTT

SEQ ID NO: 50=P233FR9_H10 (R11C)

LPAPKNLWSCVTEDSARLSWTAPDAAFDSFAIGYWEWDDDGEAIVLTVPGSERSYDLT GLKPGTEYPVYIAGVKGGQWSFPLSAIFTT

SEQ ID NO: 51=untargeted centirin (K62C)

LPAPKNLWSEVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLCPGTEYTVSIYGVKGGHRSNPLSAIFTTGGHHHHHH

SEQ ID NO: 52=Sortase A

MSHHHHHHSSGENLYFQSKPHIDNYLHDKDKDEKIEOYDKNVKEOASKDKKOOAKPQIP KDKSKVAGYIEIPDADIKEPVYPGPATREQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQF TNLKAAKKGSMVYFKVGNETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDYNEETGVVKET

SEQ ID NO: 53=Unlabeled Sortase A

SKPHIDNYLHDKDKDEKIEQYDKNVKEQASKDKKQQAKPQIPKDKSKVAGYIEIPDADI KEPVYPGPATREQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVG NETRKYKMTSIRNVKPTAVEVLDEQKGKDKQLTLITCDDYNEETGVWETKIFVATEVK

SEQ ID NO: 54 TEV protease cleavage site

ENLYFQS

SEQ ID NO: 55 EG Tencon hinge

KGGHRSN

SEQ ID NO: 56 Loop BC

DIDEQRDW

SEQ ID NO: 57 Loop BC

- 48 042392

TIDEQRDW

SEQ ID NO: 58 Loop BC

VIDEQRDW

SEQ ID NO: 59 Loop BO

AIDEQRDW

SEQ ID NO: 60 Loop BO

EWWVIPGD

SEQ ID NO: 61 Loop BC

GEQFTI

SEQ ID NO: 62 Loop BC

TAPDAA

SEQ ID NO: 63 Loop C

FDSFLIQYQE

SEQ ID NO: 64 Loop C

FESFLIQYQE

SEQ ID NO: 65 Loop C

FDSFAIGYWE

SEQ ID NO: 66 Loop C

FDSFPIGYWE

SEQ ID NO: 67 Loop C

FDSFTIGYWE

SEQ ID NO: 68 CD loop

SEKVGE

SEQ ID NO: 69 CD loop

WDDDGE

SEQ ID NO: 70 Loop F

TEYTVSIYGV

SEQ ID NO: 71 Loop F

TEYTVSIYG

SEQ ID NO: 72 Loop F

TEYPVYIAGV

SEQ ID NO: 73 Loop F

TEYWVYIAGV

SEQ ID NO: 74 Loop F

TEYHVYIAGV

SEQ ID NO: 75-140 are shown in the tables above

- 49 042392

SEQ ID NO: 141 full-length cynomolgus monkey PSMA

MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSSEATNITPKHNMK AFLDELKAENIKKFLHNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELTHYDVLLSYPNKT HPNYISIINEDGNEIFNTSLFEPPPAGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFK LERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGATGVILYSDPDDYFAPGVKSYPDGWNLPGG GVQRGNILNLNGAGDPLTPGYPANEYAYRRGMAEAVGLPSIPVHPIGYYDAQKLLEKMGGSASP DSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTSEVTRIYNVIGTLRGAVEPDRYVILGGHRD SWVFGGIDPQSGAAWHEIVRSFGMLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQ ERGVAYINADSSIEGNYTLRVDCTPLMYSLVYNLTKELESPDEGFEGKSLYESWTKKSPSPEFS GMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSSYPLYHSVYETYELVEKFYDPMFK YHLTVAQVRGGMVFELANSWLPFDCRDYAWLRKYADKIYNISMKHPQEMKTYSVSFDSLFSA VKNFTEIASKFSERLRDFDKSNPILLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNK >142 линкер

AEAAAKEAAAKEAAAKEAAAKEAAAKAAA >143 human PSMA ECD

KSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKE FGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSP QGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSD PADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPV HPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNV IGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAWHEIVRSFGTLKKEGWRPRRTILFASWD AEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDE GFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYP LYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAWLRKYADKIYSI SMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDP LGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAA AETLSEVA >144 FL человеческого PSMA с сигнальной последовательностью

MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMK AFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKT HPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFK LERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGG GVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPP DSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRD SWVFGGIDPQSGAAWHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQ

- 50 042392

ERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFS GMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFK YHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSA VKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNK YAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA >145 3-й домен FN3 тенасцина С

DAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGN LKPDTEYEVSLISRRGDMSSSNPAKETFTT >146 Fibcon

LDAPTDLQVTNVTDTSITVSWTPPSATITGYRITYTPSNGPGEPKELTVPPSSTSVTIT GLTPGVEYVVSLYALKDNQESPPLVGTQTT >147

VSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATI SGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT >148

GSGS >149

GGGSGGGS >150

GGGGSGGGGSGGGGSGGGGSGGGGS >151

APAP>152

APAPAPAPAP>153

APAPAPAPAPAPAPAPAPAP>154

APAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAP>155 albumin variant

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESA ENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM CTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRD EGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLL ECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDV CKNYAEAKDVFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKP LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA

- 51 042392

KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETF

TFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEE GKKLVAASQAALGL >156 cDNA H1O

CTGCCAGCCCCGAAGAATTTGGTCGTTTCCCGTGTCACTGAGGACTCTGCACGTCTGAG CTGGACCGCACCGGACGCGGCGTTCGACAGCTTTGCAATCGGCTACTGGGAGTGGGATGATGAC GGCGAGGCCATTGTGCTGACCGTTCCGGGTAGCGAGCGCAGCTACGATCTGACCGGTCTGAAGC CGGGTACGGAATATCCGGTGTATATTGCGGGCGTGAAGGGTGGCCAGTGGAGCTTCCCGCTGAG CGCGATCTTTACCACC >157 кДНК P258AR6P1071_D02

CTGCCGGCCTCCGAAAAACCTGGTCGTTTCCCGTGTCACTGAAGATTCTGCACGCTTGAG

CTGGGCGATCGACGAGCAGCGTGACTGGTTTGAGAGCTTCCTGATTCAGTATCAAGAATCGGAA

AAAGTTGGCGAGGCCATCGTGCTGACCGTTCCGGGTAGCGAGCGCAGCTATGATCTGACGGGTC TGAAGCCAGGCACCGAGTATACGGTGAGCATTTACGGTGTCTACCATGTGTACCGTAGCAATCC GCTGAGCGCGATCTTCACCACC >158 cDNA

P233FR9P1001_H3-l

CTGCCAGCCCCGAAAAACTTAGTTGTCTCCCGCGTGACCGAAGATTCTGCTCGTCTGAG CTGGACTGCACCGGACGCGGCGTTCGACAGCTTTCCGATTGGCTACTGGGAGTGGGATGATGAC GGTGAAGCGATCGTGCTGACCGTTCCGGGTAGCGAGCGTAGCTATGACCTGACGGGTTTGAAAC CTGGTACCGAGTATCACGTTTACATTGCGGGCGTCAAGGGTGGCCAGTGGTCGTTCCCGCTGAG CGCAATCTTTACGACC

Claims (16)

CLAIM 1. A polypeptide that binds to human prostate-specific membrane antigen (PSMA) containing an amino acid sequence 89% identical to the amino acid sequence of SEQ ID NO: 41; amino acid sequence SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 or 140, or the amino acid sequence of SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46, 47, 48, 49, 50, 51, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 , 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 , 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 or 140 containing 1, 2, 3, 4, 5, 6, 7, or 8 substitutions, wherein the substitution is a cysteine residue at at least one residue position corresponding to n positions of residues 6, 11, 22, 25, 26, 52, 53, 62 of SEQ ID NO: 41, or at the C-terminus of the peptide. 2. A human PSMA binding polypeptide according to claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 41. 3. A human PSMA-binding polypeptide according to claim 1, further containing methionine at the N-terminus of the polypeptide. 4. A polypeptide conjugate that binds to human PSMA, containing an amino acid sequence 89% identical to the amino acid sequence of SEQ ID NO: 41; an amino acid sequence of SEQ ID NO: 35-140 or an amino acid sequence of SEQ ID NO: 35-140 containing 1, 2, 3, 4, 5, 6, 7 or 8 substitutions, wherein the substitution is a cysteine residue at at least one position residue corresponding to residue positions 6, 11, 22, 25, 26, 52, 53, 62 of SEQ ID NO: 41, or at the C-terminus of the peptide, and wherein the human PSMA-binding polypeptide conjugate is conjugated to a second molecule, which is a cytotoxic agent, a detectable label, or polyethylene glycol. - 52 042392 5. The human PSMA-binding polypeptide conjugate of claim 4, wherein the cytotoxic agent is auristatin, monomethylauristatin phenylalanine, dolostatin, a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin, or a radioactive isotope. 6. A human PSMA-binding polypeptide conjugate according to claim 4, wherein the detectable label is a radioactive isotope, magnetic beads, metal beads, colloidal particles, fluorescent dye, R-phycoerythrin, electron-dense reagent, enzyme, biotin, digoxigenin or hapten. 7. A polypeptide conjugate that binds to human PSMA, containing an amino acid sequence 89% identical to the amino acid sequence of SEQ ID NO: 41; an amino acid sequence of SEQ ID NO: 35-140 or an amino acid sequence of SEQ ID NO: 35-140 containing 1, 2, 3, 4, 5, 6, 7 or 8 substitutions, wherein the substitution is a cysteine residue at at least one position the residue corresponding to the position of residues 6, 11, 22, 25, 26, 52, 53, 62 of SEQ ID NO: 41, or at the C-terminus of the peptide; and wherein the human PSMA-binding polypeptide conjugate is conjugated to a functional group that increases half-life. 8. The human PSMA-binding polypeptide conjugate of claim 7, wherein the half-life increasing functional group is an albumin-binding molecule, polyethylene glycol (PEG), albumin, an albumin variant, or at least a portion of an immunoglobulin Fc region. 9. A pharmaceutical composition for the treatment of cancer comprising a human PSMA binding polypeptide according to claim 1 or 3 or a human PSMA binding polypeptide conjugate according to any one of claims 4 to 8 and a pharmaceutically acceptable carrier. 10. A method of treating a patient having a cancer characterized by PSMA overexpression, which comprises administering a therapeutically effective amount of a human PSMA binding polypeptide according to claim 1 or 3 or a human PSMA binding polypeptide conjugate according to any one of claims 4 to 8, and a pharmaceutically acceptable carrier to a patient for a period of time sufficient to treat the cancer. 11. The method of claim 10 wherein the cancer is a solid tumor, prostate cancer, colorectal cancer, gastric cancer, clear cell kidney cancer, bladder cancer, lung cancer, or kidney cancer. 12. Use of a human PSMA binding polypeptide according to claim 1 or 3 or a human PSMA binding polypeptide conjugate according to claims 4 to 8 as a cancer diagnostic agent. 13. The use of claim 12 wherein the human PSMA binding polypeptide further comprises a modified cysteine conjugated to R-phycoerythrin (PE). 14. A method for detecting PSMA-expressing cells in a biological sample, comprising treating the biological sample with a human PSMA-binding polypeptide conjugate according to claim 6 and determining whether the biological sample contains PSMA-expressing cells by detecting binding of the polypeptide to PSMA-expressing cells in the sample, wherein detection of polypeptide binding to PSMA-expressing cells in the sample detects PSMA-expressing cells in the biological sample. 15. The method of claim 14, wherein the PSMA-expressing cells are PSMA-expressing tumor cells. 16. A method for delivering a polypeptide to PSMA-expressing tumor cells, comprising administering a polypeptide according to claim 1 or 3 or a human PSMA-binding polypeptide conjugate according to any one of claims 4-8 to a patient having PSMA-expressing tumor cells. - 53 042392 Fig. 1 Fig. 2A Fig. 2B Fig. 2C - 54 042392 Fig. BEHIND Fig. ЗВ h KSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLA с KSSSEATNITPKHNMKAFLDELKAENIKKFLHNFTQIPHLAGTEQNFQLA h KQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSL с KQIQSQWKEFGLDSVELTHYDVLLSYPNKTHPNYISIINEDGNEIFNTSL h FEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMK C C INCSGKIVIARYGKVFRGNKVKNAQLAGATGVILYSDPDDYFAPGVKSYP h DGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIP c DGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGMAEAVGLPSIP h VHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVK c VHPIGYYDAQKLLEKMGGSASPDSSWRGSLKVPYNVGPGFTGNFSTQKVK h _{MHIHSTNEVTRIYNVIGTLRGAVEpDRYVILGGHRDSWVFGGIDpQSGAA} c MHIHSTSEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAA h VVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQ C VVHEIVRS FGMLKKEGWRPRRTILFASWDAEE FGLLGSTEWAEE NS RLLQ h ERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSL c ERGVAYINADSSIEGNYTLRVDCTPLMYSLVYNLTKELESPDEGFEGKSL h YESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWET C ΥΕ3ΜΤΚΚ3Ρ3ΡΕΕ3ΟΜΡΚΙ5ΚΕΟ36ΝΡΕΕνΡΕΰΕΕ6ΙΑ5ΟΡΑΡΥΤΚΝΜΕΤ h NKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVL C I^SSYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSWL h PFDCRDYAVVLRKYADKIYSTSMKHPQEMKTYSVSFDSLFSAVKNFTEIA C PFDCRDYAWLRKYADKIYNISMKHPQEMKTYSVSFDSLFSAVKNFTEIA h SKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYA c SKFSERLRDFDKSNPILLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYA h PS3HNKYAGESFPGIYDALFDIESКVDPSKAWGEVKRQIYVAAFTVQAAA c PSSHNKYAGESFPGIYDALFDIESKVDPSQAWGEVKRQISIATFTVQAAA h ETLSEVA C ETLSEVA