Compositions and methods for diagnosis and treatment of cancer
10370433 · 2019-08-06
Assignee
Inventors
- Ugur Sahin (Mainz, DE)
- Joycelyn Wüstehube-Lausch (Worms, DE)
- Markus Fiedler (Halle an der Saale, DE)
- Matin Daneschdar (Budenheim, DE)
- Hans-Ulrich Schmoldt (Klein-Winternheim, DE)
Cpc classification
C12Y304/21026
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to the diagnosis and treatment of cancerous diseases, in particular cancerous diseases expressing Seprase (Fap-alpha; fibroblast activation protein alpha). More particularly, the invention concerns peptides targeting Seprase.
Claims
1. A Seprase binding peptide which comprises the amino acid sequence Gly Arg Gly Pro (SEQ ID NO: 5), wherein (a) the Seprase binding peptide forms and/or is part of a cystine knot structure containing at least three disulphide bridges formed from pairs of cysteine molecules, and (b) the amino acid sequence Gly Arg Gly Pro (SEQ ID NO: 5) is part of the cystine knot structure wherein the amino acid sequence is located between the first cysteine and the second cysteine of the cystine knot structure.
2. The Seprase binding peptide of claim 1, which comprises the amino acid sequence: TABLE-US-00037 (SEQIDNO:6) TyrXaa1Xaa2TrpXaa3Xaa4GlyArgGlyPro wherein Xaa1 is any amino acid, Xaa2 is any amino acid, Xaa3 is any amino acid, Xaa4 is any amino acid.
3. The Seprase binding peptide of claim 1, which comprises the amino acid sequence: TABLE-US-00038 (SEQIDNO:7) TyrXaa1AsnTrpThrProGlyArgGlyPro wherein Xaa1 is any amino acid.
4. The Seprase binding peptide of claim 1, which comprises the amino acid sequence: TABLE-US-00039 (SEQIDNO:8) TyrSerAsnTrpThrProGlyArgGlyPro.
5. The Seprase binding peptide of claim 1, which comprises the amino acid sequence: TABLE-US-00040 (Xaa)n1Cys(Xaa)n2GlyArgGlyPro(Xaa)n3Cys (Xaa)n4Cys(Xaa)n5Cys(Xaa)n6Cys(Xaa)n7Cys (Xaa)n8 wherein the Cys residues form a cystine knot structure, Xaa is independently from each other any amino acid and n1, n2, n3, n4, n5, n6, n7, and n8 are the respective numbers of amino acids, wherein the nature of the amino acids Xaa and/or the number of amino acids n1, n2, n3, n4, n5, n6, n7 and n8 are such that a cystine knot structure can form between the Cys residues, wherein n1 is 0 to 4, n2 is 3 to 10, n3 is 0 to 4, n4 is 3 to 7, n5 is 2 to 6, n6 is 1 to 3, n7 is 3 to 7, and n8 is 0 to 4.
6. The Seprase binding peptide of claim 1, which comprises the amino acid sequence: TABLE-US-00041 (SEQIDNO:17) CysXaa1TyrXaa2Xaa3TrpXaa4Xaa5GlyArgGly ProXaa6CysArgArgAspSerAspCysProGlyXaa7 CysIleCysArgGlyAsnGlyTyrCys wherein Xaa1 is any amino acid, Xaa2 is any amino acid, Xaa3 is any amino acid, Xaa4 is any amino acid, Xaa5 is any amino acid, Xaa6 is any amino acid, Xaa7 is any amino acid.
7. The Seprase binding peptide of claim 1, which comprises the amino acid sequence: TABLE-US-00042 (SEQIDNO:18) CysProTyrXaa1AsnTrpThrProGlyArgGlyPro Xaa2CysArgArgAspSerAspCysProGlyXaa3 CysIleCysArgGlyAsnGlyTyrCys wherein Xaa1 is any amino acid, Xaa2 is any amino acid, Xaa3 is any amino acid.
8. The Seprase binding peptide of claim 1, which comprises the amino acid sequence: TABLE-US-00043 (SEQIDNO:19) CysProTyrSerAsnTrpThrProGlyArgGlyPro AspCysArgArgAspSerAspCysProGlyArgCys IleCysArgGlyAsnGlyTyrCys.
9. The Seprase binding peptide of claim 1, which comprises the amino acid sequence: TABLE-US-00044 (SEQIDNO:12) Xaa1Xaa2CysXaa3TyrXaa4Xaa5TrpXaa6Xaa7Gly ArgGlyProXaa8CysArgArgAspSerAspCysPro GlyXaa9CysIleCysArgGlyAsnGlyTyrCysGly wherein Xaa1 is any amino acid, Xaa2 is any amino acid, Xaa3 is any amino acid, Xaa4 is any amino acid, Xaa5 is any amino acid, Xaa6 is any amino acid, Xaa7 is any amino acid, Xaa8 is any amino acid, Xaa9 is any amino acid.
10. The Seprase binding peptide of claim 1, which comprises the amino acid sequence: TABLE-US-00045 (SEQIDNO:21) Xaa1Xaa2CysProTyrXaa3AsnTrpThrProGlyArg GlyProXaa4CysArgArgAspSerAspCysProGly Xaa5CysIleCysArgGlyAsnGlyTyrCysGly wherein Xaa1 is any amino acid, Xaa2 is any amino acid, Xaa3 is any amino acid, Xaa4 is any amino acid, Xaa5 is any amino acid.
11. The Seprase binding peptide claim 1, which comprises the amino acid sequence: TABLE-US-00046 (SEQIDNO:22) GlyLysCysProTyrSerAsnTrpThrProGlyArg GlyProAspCysArgArgAspSerAspCysProGly ArgCysIleCysArgGlyAsnGlyTyrCysGly.
12. A Seprase binding peptide comprising the amino acid sequence: TABLE-US-00047 (SEQIDNO:23) GlyAlaCysProTyrSerAsnTrpThrProGlyArg GlyProAspCysArgArgAspSerAspCysProGly ArgCysIleCysArgGlyAsnGlyTyrCysGly.
13. The Seprase binding peptide of claim 1, wherein the cystine knot structure is based on the open chain trypsin inhibitor II from Momordica cochinchinensis (MCoTI-II).
14. A Seprase binding peptide comprising the amino acid sequence Gly Arg Gly Pro (SEQ ID NO: 5) and at least one fusion partner.
15. The Seprase binding peptide of claim 14, wherein the fusion partner comprises a heterologous amino acid sequence.
16. A Seprase binding agent comprising the amino acid sequence Gly Arg Gly Pro (SEQ ID NO: 5) covalently and/or non-covalently associated with at least one further moiety.
Description
FIGURES
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EXAMPLES
(30) The techniques and methods used herein are described herein or carried out in a manner known per se and as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. All methods including the use of kits and reagents are carried out according to the manufacturers' information unless specifically indicated.
Example 1
Materials and Methods
(31) Plasmid Constructions
(32) CHO codon optimized full length human Seprase (NCBI accession number NP_004451) was synthesized by Geneart and subcloned into pENTRcht vector (Invitrogen). The plasmid was verified by DNA sequencing and named pENTR-huSeprase. Afterwards, the respective insert was shuttled into a piggy Bac transposon vector (PB53x EF1 Series) by Gateway cloning (Invitrogen) to generate transposon expression plasmids. These plasmids contain an EF1 alpha promoter to drive expression of the cDNA, an IRES-EGFP cassette and hygromycin as a selection marker. The plasmid was used for the generation of a stable Seprase expressing cell line.
(33) Utilizing pENTR-huSeprase plasmid DNA as a PCR template, a cDNA fragment encoding the Seprase extracellular domain (amino acids 29-760) was amplified with a forward primer (5-GCGCAAGCTTGCTGCGGCCCTCCCGGGTGCAC-3) and a reverse primer (5-GCGCAGCGGCCGCGTCGGACAGGGAGAAGCACTGC-3) The PCR product, excluding the coding sequences of both the short cytoplasmic (amino acids 1-6) and hydrophobic transmembrane domains of seprase (amino acids 7-29), was inserted into a modified pCEP4 vector (Invitrogen). Compared to pCEP4, the modified vector additionally contains a Kozak consensus sequence, a hexahistidine (H6) fusion tag and the coding sequence of a secretion signal from the V-J2-C region of the mouse Ig kappa-chain for efficient secretion of the recombinant protein. The cDNA sequence of the Seprase extracellular domain was inserted, in frame, along with the N-terminal secretion signal and the C-terminal H6 tag, allowing efficient secretion, easy detection (by anti-His Antibody; Invitrogen) and rapid purification (by Ni-chelate affinity chromatography) of recombinant Seprase. The final construct was verified by DNA sequencing, named pCEP4-IgKappa-huSeprase-coCHO_26-760aa-H6 and used for the generation of recombinant soluble human Seprase (rhuSeprase).
(34) Cell Lines
(35) Chinese hamster ovarian cells (CHO-K1) were obtained from ATCC and grown in DMEM/F-12 medium supplemented with penicillin (100 units/ml), streptomycin (100 mg/ml) and 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Invitrogen). Cells were maintained at 37 C. in 5% CO.sub.2-humidified air atmosphere and passaged every 48-72 hours. MOCK or human Seprase expressing CHO-K1 cells (CHO-K1-MOCK and CHO-K1-huSeprase) were grown under the same conditions as the wild type cells with addition of 200 g/mL hyromycin B (Invitrogen). For production of rhuSeprase the Freestyle CHO-S cell line from Invitrogen was used. This suspension cell line has been distinguished as a separate sub-clone from the common CHO-K1 cell line (D'Anna, 1996; D'Anna et al., 1997; Deaven & Petersen, 1973). Cells were cultured in polycarbonate, disposable, sterile Erlenmeyer flask with vented cap (125 mL or 500 mL) using 15-25% of the nominal volume at 120-135 rpm (Minitron Incubator shaker, Infors-HT) under standard humidified conditions (37 C. and 8% CO.sub.2). Cells were sub-cultured when the density was approximately 1-1.510.sup.6 viable cells/ml, typically every 48-72 hours in protein free chemically defined medium for CHO cells (CD CHO medium, Invitrogen) supplemented with 1HT supplement, and 4 mM glutamine (Invitrogen).
(36) Production of Recombinant Human Seprase
(37) For large-scale expression of soluble recombinant human Seprase protein (rhuSeprase), the FreeStyle MAX CHO expression system (Invitrogen) was used, following the manufacturer's instructions. In brief, CHO-S cells were passed at 5-610.sup.5 cells/ml, incubated under standard humidified conditions at 120 rpm-135 rpm overnight in a Minitron Incubator shaker (Infors-HT). On the following day the cells were diluted to 110.sup.6 cells/ml in 150 ml into a 500 ml-shake flask on the day of transfection. 187.5 g of pCEP4-IgKappa-huSeprase-coCHO_26-760aa-H6 plasmid DNA was added into 3 ml of OptiPro SFM and mixed. 187.5 l of the FreeStyle MAX transfection reagent was diluted in 3 ml of OptiProSFM (Invitrogen) and mixed gently. Diluted FreeStyle MAX transfection reagent was added to diluted DNA solution, mixed gently, and incubated for 10 min at room temperature. DNA-FreeStyle MAX reagent complex was added slowly into the 500 ml-flask containing cells while slowly swirling the flask. Afterwards, transfected cell culture was incubated under standard conditions. Five to seven days after transfection, the supernatant was harvested and purified via conventional Ni-chelate affinity chromatography and size-exclusion chromatography using a HisTrap HP (GE Healthcare) and HiLoad 26/600 Superdex 200 prep grade SEC column (GE Healthcare) respectively. A portion of the purified protein was biotinylated by incubation with a 10 fold molar excess of EZ-Link Sulfo-NHS-LC-Biotin (Pierce) in PBS pH 8.8 for 2 h on ice. Protein was stored at 20 C. after buffer exchange to PBS supplemented with 5% mannitol (Roth) and 5% trehalose (Applichem).
(38) Generation of Stable Seprase Expressing Cell Lines
(39) Polyethylenimine, linear, MW 25000 (PEI) (Polysciences. Inc) reagent was used as the transfection reagent. Stable cell lines were established with the PiggyBac transposon system. Briefly, this system consists of a donor vector carrying an artificial transposon with a mammalian expression cassette for the recombinant transgene and a helper vector driving transient expression of the PB transposase (PBase) (Invitrogen). One day before transfection, 310.sup.5 CHO-K1 cells were plated in 2 ml of growth medium per well in a six-well plate. CHO-K1 cells were co-transfected with 2 g transposon vector plasmid and 0.8 g of transposase vector plasmid. Three days post-transfection, cells were split and placed in media containing 200 g/ml hygromycin B (Invitrogen). After 2 weeks of hygromycin selection, the transfection efficiency and the target expression was analyzed by flow cytometry, western blot and immunofluorescence analysis. The functionality was tested by enzyme activity assay using Z-Gly-Pro-AMC substrate (Bachem).
(40) Flow Cytometry Analysis
(41) For flow cytometry, 110.sup.6 cells were collected, washed once with FACS buffer (PBS+0.5 M EDTA+5% FBS) and incubated with an antibody against human Seprase (clone 1E5, Abnova; 1:50 dilution) or with different concentrations of Thioredoxin-A (Trx) miniprotein fusions for 1 h on ice. To analyze the binding of tetramerized miniproteins, biotinylated miniproteins were pre-incubated with a fivefold molar excess of Streptavidin-APC (Affymetrix eBioscience) for 10 min at room temperature. After incubation cells were washed thrice with FACS buffer. Cell treated with tetramerized miniproteins were used directly for flow cytometry analysis. To analyze Seprase expression or monovalent miniprotein binding, cells were stained with a secondary Cy5-labeled anti-mouse antibody (Dianova) or with PE-labeled anti-H6 antibody (R&D Systems, detects internal H6 tag within the Trx-miniprotein fusion). After 30 min cells were washed again with FACS buffer and analyzed using a FACS Canto II device (Becton Dickinson). The analysis gate was set on viable cells identified according to forward scatter/side scatter characteristics. Data were analyzed using FlowJo software (Version 10, Tree Star Inc.).
(42) Immunohistochemistry
(43) Tumors were immediately excised, transferred into embedding cassettes and fixed overnight at 4 C. in 4% Roti-Histo-Fix (pH 7) (Roth). After fixation, the tumors were washed in 70% ethanol to remove excess fixation solution. Thereafter, tumors were dehydrated in an ascending alcohol series and paraffin embedded in a tissue processor. Serial section (3 m thick) of the embedded tumors was performed with a microtome. The sections were mounted on glass slides and deparaffinized and rehydrated in a descending alcohol series. Then, sections were boiled for 20 min with 10 mM citrate buffer (pH 6) in a microwave. After washing with 1PBST, unspecific binding was blocked with 3% BSA in PBST for 30 min. 1:500 polyclonal anti-SMA antibody (Abcam) or 1 M biotinylated miniprotein, which was preincubated with Streptavidin-Cy3 (Rockland) before for tetramerization, was added and incubated overnight at 4 C. After washing the sections three times with 1PBST anti-SMA antibody binding was detected with the secondary antibody IgG anti-rabbit-FITC diluted 1:200 (Dianova). Secondary antibodies were incubated for 1 h at room temperature in the dark. Finally, sections were washed three times with PBS and subsequently incubated with Hoechst dye (Sigma-Aldrich), diluted 1:5000 in 1PBS for 10 min and mounted in mounting medium (Darko).
(44) To evaluate the expression levels of Seprase in TNBC, lung- and pancreas carcinoma immunohistochemical analyses were performed. As positive control CHO-K1-huSeprase tissues with positive expression levels of Seprase and as negative control human colon sections were used.
(45) For paraffin embedding pancreas tissue 3 m thick sections were deparaffinized with xylene and graded ethanol. Antigen retrieval was performed by heating the sections in 10 mM sodium citrate buffer, pH 6.0+0.05% Tween-20 at 120 C., cooled down for 10 min. Samples were quenched for 15 min in PBS+0.3% H.sub.2O.sub.2. Frozen tissue sections (breast and lung tissue) were sectioned at 5-8 m in a cryostat. The sections were thawed for 10 min at room temperature, rehydrated for 5 min in PBS and quenched for 10 min in BLOXALL (Vectorlabs).
(46) All sections were incubated with 10% normal goat serum at room temperature for 30 min to block non-specific reactions. This was followed by incubation with polyclonal rabbit anti-human Seprase antibody (Sigma) diluted to 0.5 g/ml for 1 h at room temperature. After washing with PBS the sections were incubated for 30 min with the secondary antibody (Power-Vision HRP anti-rabbit). The localization of immunostaining was demonstrated by incubation with the Vector NovaRED system (Vector Laboratories). Counterstaining with Mayer's Haematoxylin and dehydration of the sections were done with a Multistainer ST5020 (Leica). Afterwards, slides were mounted with XTRA-Kitt Medite mounting medium.
(47) Phage Display Selections Vs. rhuSeprase
(48) A randomized knottin library comprising approximately 110.sup.10 individual variants was applied for phage display selections vs. rhuSeprase. The library is based on the open chain trypsin inhibitor II from Momordica cochinchinensis (oMCoTi-II, (Avrutina, Schmoldt et al. 2005)). Three selection rounds were carried out using rhuSeprase immobilized on Maxisorp immuno tubes (Thermo Scientific) or via Streptavidin coated magnetic beads (Dynabeads 280 Streptavidin, Life Technologies).
(49) Production of Miniprotein Variants
(50) Biotinylated miniprotein variants were purchased from Pepscan. In these cases the miniproteins were generated by conventional solid-phase peptide synthesis followed by thermodynamic folding to the native cystine-knot structure. In all other cases miniproteins were produced recombinantly using a Thioredoxin-A (Trx) based fusion system in combination with E. coli Shuffle T7 Express strain (NEB) that allows disulfide bond formation in the cytoplasm of the bacterial host (Lobstein, Emrich et al. 2012). For semi-preparative or analytical recombinant synthesis, the miniprotein variant encoding genes were cloned into pET-32-LibEx vector via unique Bam HI and Apa I restriction sites to yield a tetrapartite fusion consisting of thioredoxin-A, a His-tag (H6), an S-tag and the miniprotein gene. For semi-preparative production of miniproteins expression was performed in 1 l shake flasks using standard lysogeny broth (LB) medium, whereas analytical scale production (e.g. for hit identification or analysis of MC-FA-010 alanine mutants) was carried out in 96 well plates using autoinduction medium (MagicMedia, Life Technology). After expression, cells were harvested and lysed with lysozyme or by sonification in combination with a freeze/thaw cycle. In both cases cleared cell lysates were subjected to a heating step at 80 C. for 10 min to remove a large amount of host cell proteins. The resulting protein preparation was either directly used for ELISA binding analysis (e.g. for hit identification purposes) or further purified via Ni-chelate affinity chromatography using Ni-NTA spin columns (Qiagen, analytical scale) or 5 ml HisTrap HP columns (GE Healthcare, semi-preparative scale). For the generation of tag-free miniproteins, trx-miniprotein fusions were cleaved with thrombin (Sigma) by overnight incubation at 37 C. with 0.5 U Thrombin/mg fusion proteins. Miniproteins could then be isolated by HPLC using a TSKgel ODS-120T column (Tosoh Bioscience). The final miniprotein preparations, gained after freeze-drying of the respective HPLC fractions, were analyzed by mass spectrometry and analytical size exclusion chromatography using a BioSep-SEC-S2000 column (Phenomenex). Yields were calculated by weighing or OD (280 nm) measurements.
(51) Western Blotting
(52) 110.sup.5 cells were cultured on a culture-dish, washed once with cold 1PBS and lysed in 500 L 4SDS lysis buffer (250 mM Tris-HCl, 34% Glycerol, 8.2% SDS, 5% -mercaptoethanol). Cells were scrapped with a cell scraper and in order to remove cellular debris, lysates were centrifuged for few minutes at 14000g at 4 C. Thereafter the lysates were sonicated. An aliquot of the lysate was boiled with 4SDS-lysis buffer added with bromphenolblue and analyzed by SDS-PAGE and subsequent western blotting. Following antibodies were used for detection: as primary antibody: anti-Seprase (Abcam), anti-His (Abcam) or anti--Actin and as a secondary antibody anti-mouse-HRP (clone).
(53) Hit Identification
(54) After three rounds of phage display selection the resulting pools were sub-cloned in the pET-32-LibEx expression vector to allow for the identification of putative rhuSeprase binders independently from the phage background. To this end, the respective miniprotein gene pools were PCR amplified with specific oligonucleotides. The resulting PCR product was purified, cleaved with Bam HI and Apa I restriction enzymes and ligated with similarly digested expression vector. After transformation of E. coli Shuffle T7 Express individual clones were picked and Trx-fusion proteins were produced in a 96well format as described above. For analysis of binding an ELISA assay was performed. Therefore a MaxiSorp 96 well plate (Nunc/Thermo Fisher Scientific) was coated with target protein or BSA (each 100 l of 5 g/ml protein solution in 50 mM Na-Carbonat, pH 9.4). Binding to target protein corresponds to signal and binding to BSA corresponds to noise. For normalization of single plates binding of MC-Myc-010 (Myc-binding cystine knot miniprotein) and Anti-cMyc antibody (clone 9E10) was analyzed in triplicates. Coating was performed over night at 4 C. Wells were washed 3 times with 300 l phosphate buffered saline containing 0.1% Tween 20 (pH 7.4,PBS-T). Wells were then blocked for 2 h with Blocking Solution (Sigma Aldrich). After a washing step (3PBS-T) Trx fusion protein containing lysates were diluted 1:5 in and incubated for 1 h at 4 C. with coated proteins. Washing and incubation step was repeated using anti-S-tag antibody (1:2000 in PBS, abeam). Before detection washing step was performed twice (3PBS-T and 3PBS). Detection was carried out using 3, 3,5,5-Tetramethylbenzidine Liquid Substrate (TMB solution, Sigma-Aldrich) and increase of absorption at 450 nm (detected in Tecan M200 Pro ELISA reader). Expression of single clones was analyzed by 96-well E-PAGE electrophoresis (Life Technologies) and quantification of protein bands via ImageQuant TL software package (GE Healthcare). For ranking of proteins signal to noise ratios of ELISAs were calculated and correlated with expression values. Top 30 clones were then used for further analysis.
(55) Binding Analysis Via ELISA
(56) ELISAs were performed to assess and compare the binding properties and specificity of miniprotein variants. To this end, either recombinant proteins or whole cells have been used. For whole cell ELISA analysis 510.sup.5 cells were seeded on each well of a 96-well flat bottom plate (Corning). Therefore, cells were incubated for 20 hours at 37 C. in 5% CO.sub.2-humidified air atmosphere. Afterwards, the wells were blocked with 5% milk powder/PBS for 1 hour at RT. After removing the blocking buffer Trx-miniprotein solution was added to each well, and incubated with the cells for 1 hour at RT. Subsequently, the wells were washed 6 times extensively with PBS-T (PBS+0.1% Tween-20) and the amount of bound miniproteins was detected with horseradish peroxidase (HRP)-conjungated anti-S-tag antibody (Abcam). 3,3,5,5-Tetramethylbenzidin (TMB) (Sigma) was used as chromogenic substrate. HRP enzyme reactions were stopped with 0.2 M HCl after approximately 20 min and the plate was measured in a Victor V3 plate reader (Perkin Elmer) at 450 nm. For competition studies 0.1 M Trx-MC-FA-010 fusion protein was pre-mixed with different concentrations of solitary MC-FA-010 miniprotein (1-200 M) before incubation with cells.
(57) For protein based ELISA analysis 5 g of the respective recombinant protein (rhuSeprase; streptavidin: Sigma; DPP-IV: R&D Systems, BSA: Eurobio) were immobilized per well of a MaxiSorp plate (Nunc) by overnight incubation in coating buffer at 4 C. After washing thrice with 300 l PBS-T/well on a Hydrospeed plate washer (Tecan) the wells were blocked with 1Casein solution (Sigma, diluted in PBS) for 2 h at RT. Subsequently, the wells were washed as indicated above. 100 l of the respective Trx-miniprotein fusion diluted in PBS-T was then added and incubated for 1 h at 4 C. Simply heat-step purified protein was diluted 1:5, affinity purified proteins were applied in defined concentrations ranging from 0.39-50 nM. For competition ELISAs a fixed concentration of 3 nM Trx-miniprotein fusion was mixed with varying concentration (0.64-3167 nM) of solitary miniprotein before incubation. Binding of the Trx-fusion was detected after a PBS-T washing procedure using HRP coupled anti S-tag antibody as described above. Apparent Kd was calculated using Sigmaplot 10 and an one site saturation binding model for fitting of the data.
(58) SPR Analysis
(59) To obtain insights of the kinetic binding properties surface plasmon resonance (SPR) analysis was performed on a Biacore T100 device (GE Healthcare). Therefore, rhuSeprase was immobilized onto a CM5 chip via NHS/EDC mediated coupling as described by manufacturer at 10 l/min for 420 sec. Binding of MC-FA-010 in varying concentrations (37; 111.1, 331.3, 1000 and 3000 nM) to immobilized rhuSeprase was measured over a time period of 90 seconds for association and dissociation. Kd values were calculated using the provided software.
(60) Kinetics and affinity of monomeric and oligomeric MC-FA-010 and variants thereof to recombinant human Seprase (rhuSeprase) were determined using surface plasmon resonance spectroscopy (Biacore T-100, GE Healthcare). RhuSeprase (20 g/ml in PBS, 5% mannitol, 5% trehalose) was immobilized on an amino reactive Series S Sensor Chip CM5 (GE Healthcare). For binding analysis of monomeric Microbodies rhuSeprase was loaded to a maximum of 7500 RU, for oligomeric Microbodies to a maximum of 700 RU. Monomeric Microbodies were measured using a multi cycle kinetic method in a concentration range of 3.125 to 1000 nM based on expected dissociation constant. Association step was measured over a time period of 60-90 seconds, dissociation over 420 seconds. Trimeric Mircobodies were measured using a single cycle kinetics method in a concentration range of 0.3125 to 5 nM (association for 90 seconds, dissociation 420 seconds). Binding kinetics and steady state analysis were calculated using a 1:1 binding model (Biacore T-100 Evaluation Software, GE Healthcare).
(61) Alanine Scan Mutagenesis of MC-FA-010
(62) In order to gain insights into the structure-activity relationship of the seprase binder MC-FA-010 an alanine scan mutagenesis was performed. Therefore, every single amino acid of the variable region was exchanged by alanine on the DNA level. The mutant genes were synthesized by Geneart as DNA Strings and directly cloned into pET-32-LibEx expression vector via unique Bam HI and Apa I restriction sites. Production of alanine variants was done in 96well microtiter plates using the Shuffle T7 Express E. coli strain as described above. After purification with Ni-NTA spin columns (Qiagen) the binding properties of the variants were analyzed via ELISA and compared to the MC-FA-010 wildtype miniprotein.
(63) Affinity Maturation
(64) Based on the obtained data from the alanine scan mutagenisis and the identified binding motif of MC-FA-010/-012 a second phage library was generated. In this library, the critical amino acid positions for seprase binding were kept constant (Y, W and the GRGP sequence) whereas all other positions of the binding loop were randomized using all possible amino acids except cysteine. This library was screened again against recombinant soluble human seprase, applying four different conditions that vary with respect to stringency (monovalent or polyvalent display, with or without competition with free MC-FA-012 miniprotein, different washing conditions). After three selection cycles all pools were cloned into the pET-32 expression vector. For each pool 96 clones were expressed and analyzed using the hit identification process described above. 26 of the top-ranked clones were selected, produced in higher amounts and analyzed in more detail.
(65) Biodistribution and Tumor Targeting Analysis of Microbody AlexaFluor-680 Conjugates Using In Vivo Near-infrared Optical Imaging
(66) For in vivo imaging assays female Fox n1 nu mice (6-8 weeks of age, Harlan, Envigo) were used. The experiments were performed according to national regulations and approved by the local animal experiments ethical committee. Subconfluent CHO-K1-huSeprase cells were harvested and resuspended in PBS to a density of 110.sup.7 cells/ml. Prior to inoculation, cell viability was tested by 0.4% trypan blue exclusion assay (viable cells>90%). For subcutaneous injection 110.sup.6 CHO-K1-huSeprase cells in 100 l PBS were mixed with 100 l Matrigel (Corning) and injected into the right side of the limb. When tumor volumes reached 600-800 mm.sup.3, animals were randomly separated into several groups for different treatments (n=3 per each group). Then, 1.67 nmol AF680-(MC-FA-012).sub.3 or the control Microbody AF680-(MC-FA-0116).sub.3 were injected intravenously. At different time points after the injection, mice were anaesthetized by inhalation of isoflurane. In vivo imaging was conducted using a Xenogen IVIS Spectrum imaging System (Perkin Elmer, USA). Maximal near infrared signals (NIRF) were quantified using Living Image 2.5 (Xenogen, Perkin Elmer) image analysis software. For ex vivo NIRF imaging, the mice were sacrificed, and the tumor and major organs of each mouse were excised, weighed and analyzed by Xenogen IVIS System.
(67) Immunofluorescence Analysis
(68) 1 M biotinylated MC-FA-012 and the control MC-FA-0116 were preincubated with Streptavidin-Cy3 (Rockland) (molar ratio 5:1) for 30 min at room temperature. Cryosections (6 m) of tissues were fixed with acetone and blocked with 3% BSA/PBS to prevent non-specific binding. Then tissues were stained with anti-SMA antibody (Abcam) to detect activated fibroblasts and with the Microbody-Streptavidin mix for 30 min at 37 C. Sections were rinsed afterwards and incubated for 30 min with Alexa 488 conjugated secondary antibodies (Abcam) at 37 C. Finally, sections were washed again, incubated with Hoechst 33258 (Sigma-Aldrich) to detect nuclei, washed again twice and mounted in fluorescence mounting medium (Dako). Sections were then examined with an inverted fluorescence microscope (Zeiss AxioObserver.Z1).
(69) Small Animal PET-Imaging and Organ Distribution
(70) .sup.177Lu-Labeling of DOTA-(MC-FA-012).sub.3
(71) 2.5 nmol MC-FA-012 trimer was dissolved in 50 l 0.1 M sodium acetate buffer pH 5.0 and mixed with 1 l of an aqueous solution of 20% ascorbic acid. 2.5 l of .sup.177LuCl.sub.3 in 0.4 M sodium acetate buffer pH 5.0 (25 MBq) were added. The mixture was heated for 15 min at 95 C. and diluted to a total volume of 2.5 ml using 0.9% saline. Radiolabeling was performed without any separation of labeled and unlabeled compound. The radiochemical yield was determined by analytic RP-HPLC. .sup.177Lu-(MC-FA-012).sub.3 corresponds to .sup.177Lu-labeled DOTA-(MC-FA-012).sub.3.
(72) .sup.68Ga-Labeling of DOTA-(MC-FA-012).sub.3
(73) .sup.68Ga was gained from a .sup.68Ge/.sup.68Ga generator as [.sup.68Ga]GaCl.sub.3 in 0.6 M HCl. 5 nmol MC-FA-012 trimer and 10 l of an aqueous solution of 20% ascorbic acid were mixed with 550 l of the .sup.68Ga-eluate and neutralized with 160 l 2.5 M sodium acetate buffer (pH 8) to a final pH of 3.5. The mixture was heated for 13 min at 95 C., purified using a solid phase extraction cartridge (Agilent Varian Bond Elut Plexa) and diluted in 0.9% saline. Radiolabeling was performed without any separation of labeled and unlabeled compound. The radiochemical yield was determined by analytic RP-HPLC. .sup.68Ga-(MC-FA-012).sub.3 corresponds to .sup.68Ga-labeled DOTA-(MC-FA-012).sub.3.
(74) In Vivo Testing of Radiolabeled DOTA-(MC-FA-012).sub.3
(75) For in vivo experiments, 8 week old BALB/c nu/nu mice (Charles River) were subcutaneously inoculated into the right trunk with 510.sup.6 CT26-huSeprase cells, respectively. For imaging experiments (n=2), 510.sup.6 CT26 wildtype cells were additionally injected into the left trunk as a control. When the size of the tumor reached approximately 1 cm.sup.3, the radiolabeled compound was injected via the tail vein (10 MBq for small-animal PET imaging; 1 MBq for organ distribution).
(76) Organ Distribution of .sup.177Lu-(MC-FA-012).sub.3
(77) For organ distribution, the animals (n=3 for each time point) were sacrificed after indicated time points (from 30 min to 24 h). The distributed radioactivity was measured in all dissected organs and in blood using a -counter. The values are expressed as percentage injected dose per gram (% ID/g).
(78) Small-animal PET Imaging with .sup.68Ga-(MC-FA-012).sub.3
(79) PET imaging was performed using the small-animal PET scanner Inveon PET (Siemens). After a 15 min transmission scan the anaesthetized mice were injected with approximately 2.5 nmol .sup.68Ga-(MC-FA-012).sub.3 (10 MBq). Within the first 60 min a dynamic scan was performed, followed by a static scan from 120 to 140 min after injection. Images were reconstructed iteratively using the 3D-OSEM+MAP method (Siemens) and were converted to standardized uptake value (SUV) images. Quantitation was done using a ROI technique and expressed as SUVmean.
(80) Diagnostic and Therapeutic Purposes
(81) .sup.68Ga-Labeling of DOTA-(MC-FA-012).sub.3
(82) 68 Ga (half-life 68 min; energy of positrons max. 1.9 MeV [.sup.+89%]) was gained from a .sup.68Ge/.sup.68Ga generator as [.sup.68Ga]GaCl.sub.3 in 0.6 M HCl. 2.5 nmol DOTA-(MC-FA-012).sub.3 and 10 l of an aqueous solution of 20% ascorbic acid were added to 1 ml of the .sup.68Ga-eluate (0.8-1 GBq) and diluted with 280 l 2.5 M sodium acetate buffer (pH 8) to a final pH of 3.5. The mixture was incubated at 95 C. for 15 min, purified using a solid phase extraction cartridge (Agilent Varian Bond Elut Plexa) and diluted in 0.9% saline. Radiolabeling was performed without any separation of labeled and unlabeled compound. The radiochemical yield was determined via analytical RP-HPLC.
(83) .sup.177Lu-Labeling of DOTA-(MC-FA-012).sub.3
(84) .sup.177Lu (half-life 6.71 d; energy of electrons max. 497 keV [.sup.79%]; energy of photons max. 113 keV [6%], 208 keV [11%]) was purchased from ITG GmbH Garching as [.sup.177Lu]LuCl.sub.3 in aqueous 0.04 M HCl solution. 15 nmol DOTA-(MC-FA-012).sub.3 were dissolved in 100 l 0.4 M sodium acetate buffer pH 5.0 and mixed with 10 l of an aqueous solution of 20% ascorbic acid. 70 l of .sup.177LuCl.sub.3 in 0.4 M sodium acetate buffer pH 5.0 (2.5 GBq) were added. The mixture was incubated at 95 C. for 15 min and diluted to a total volume of 5 ml using 0.9% saline. Radiolabeling was performed without any separation of labeled and unlabeled compound. The radiochemical yield was determined by analytic RP-HPLC and instant thin layer chromatography (ITLC-SG) with a solution of 0.5 M sodium citrate pH 5 with and without 10% methanol as solvent.
(85) PET Imaging
(86) Diagnostic imaging was performed using .sup.68Ga-(MC-FA-012).sub.3, which was applied intravenously (2.5 nmol, 63-359 MBq). Variation of injected radiotracer activity was caused by the short half-life of .sup.68Ga and variable elution efficiencies obtained during the lifetime of the .sup.68Ge/.sup.68Ga generator. The patients were investigated 1 and approx. 3 hours after administration of .sup.68Ga-(MC-FA-012).sub.3 using the PET/CT scanner Siemens Biograph-mCT Flow. After performing a CT scan for attenuation correction, static emission scans, corrected for dead time, scatter and decay, were acquired. Images were reconstructed iteratively and were converted to standardized uptake value (SUV) images.
(87) Medical imaging after administration of .sup.177Lu-(MC-FA-012).sub.3 was performed using the gamma camera GE Millenium VG5 Hawkeye one day after intravenous injection of approx. 2.5 GBq.
Example 2
Engineering of the Human FAP Binding oMCoTi-II Mutant MC-FA-010
(88) FAP binding cytine knot miniprotein MC-FA-010 was isolated from a highly diverse phage based oMCoTi-II library (containing 110.sup.10 individual variants). Sequence analysis of an enriched clone after three selection rounds revealed a miniprotein sequence with 35 amino acid (aa) length (
(89) To analyze the structure function relationship of the identified miniprotein an alanine scan mutagenesis was performed (
(90) The alanine scan mutagenesis revealed a binding motif consisting out of two aromatic and four aliphatic amino acids (YXXWXXGRGP,
Example 3
Target Binding of the Human FAP Binding oMCoTi-II Mutant MC-FA-010 and Variants Thereof
(91) MC-FA-010 Shows Affinity to Human Seprase in Nanomolar Range
(92) Affinity of MC-FA-010 binding to recombinant human Seprase was measured using concentration-dependent ELISA. Plates were coated with the soluble fraction of human Seprase and MC-FA-010 was added as a fusion protein to thioredoxin (Trx-MC-FA-010) in a concentration range of 0.39 to 50 nM. Detection of bound miniprotein was achieved via anti S-Tag-HRP conjugated antibody (S-tag is provided by thioredoxin fusion expression system,
(93) Those experiments show a specific binding of MC-FA-010 to human Seprase in general. As a next step a more detailed analysis of binding kinetic was performed using surface plasmon resonance technology (SPR). Therefore, recombinant human Seprase was immobilized on a CM5 chip (GE Healthcare) with an amino reactive surface. Association and dissociation of soluble monovalent MC-FA-010 was measured in a concentration series (37, 111.1, 333.3, 1000 and 3000 nM) using a Biacore T100 system. Association and dissociation data was fitted and corresponding dissociation constant and kinetic values (Kon, Koff and Kd) was calculated using the provided software of the system (
(94) MC-FA-010 Shows High Selectivity for Human Seprase
(95) To analyze the selectivity of MC-FA-010 for human Seprase, binding to the closely related dipeptidyl peptidase IV (DPP IV, CD26) was studied. DPP IV is a 88 kDa membrane bound glycoprotein which also can be proteolytically cleaved to a soluble form lacking 38 aa at the amino terminus.
(96) As expected MC-FA-010 binds strongly and selectively to Seprase. Only a very weak signal could be detected for MC-FA-010 binding to DPP IV in the highest concentration measured (
(97) MC-FA-010 Specifically Binds to Seprase-expressing Cells
(98) To investigate binding of MC-FA-010 to human Seprase-overexpressing CHO-K1-cells (CHO-K1-Seprase), an immunofluorescence staining was conducted. Target-negative CHO-K1-MOCK cells and a negative control Microbody, were used as controls to exclude unspecific binding of the Microbody to unrelated proteins on the cell surface. Before incubation with the cells MC-FA-010 and the control Microbody were biotinylated and preassembled on Cy3-conjugated streptavidin. In comparison to CHO-K1-MOCK, a specific binding of MC-FA-010-bio/SA-Cy3 was detected on CHO-K1-Seprase. As expected, the control Microbody does not bind to CHO-K1-Seprase cells (
(99) MC-FA-010 Specifically Binds to Seprase-expressing Tumors
(100) To analyze binding of MC-FA-010 (tetramerized via Streptavidin-Cy3) to murine Seprase expressing tumor cells, BALB/c mice were injected subcutaneously with CT26 cells. Mice were sacrificed 14 days after tumor cell implantation and the tumor was isolated for paraffin sections. Afterwards, 3 m sections were stained (
(101) Oligomerization of MC-FA-010 Increases its Affinity
(102) To study a possible avidity effect the binding activity of monovalent and tetravalent MC-FA-010 against human Seprase-overexpressing cells (CHO-K1-Seprase) was analyzed. Therefore, on the one hand monovalent Trx-MC-FA-010 fusion protein (consists of a Thioredoxin-His6-cassette) and on the other hand biotin-conjugated Microbody which was oligomerized using streptavidin-APC (MC-FA-010-bio/SA-APC) was used. Binding properties of the resulting MC-FA-010 constructs against human Seprase were determined by FACS and revealed a EC50 value of 177.6 nM of the monovalent MC-FA-010, while the tetravalent variant showed an even higher affinity with a EC50 of 2.367 nM (
(103) Chemical Oligomerization of MC-FA-012
(104) A DOTA conjugated trimerized version of the MC-FA-012 Microbody (DOTA-(MC-FA-012).sub.3) was purchased from Pepscan. The generation was based on an oxim ligation strategy. Therefore, a MC-FA-012 variant with an amino-terminal aminooxy group at the N-terminus was synthesized chemically. Besides, an anchor molecule was generated consisting of an amino-terminally attached DOTA moiety and three Lysine-Serine stretches separated by a GSGS linker sequence respectively. To form reactive aldehydes the terminal hydroxyl groups of the serine residues were oxidized with sodium-periodate. Finally, activated anchor and the aminooxy-MC-FA-012 variant were coupled in an oxim ligation reaction to form the DOTA-(MC-FA-012).sub.3 trimer (see
(105) The trimer was functionally analyzed in a FACS-based competition assay in comparison to the monomeric Microbody (see
(106) Beside the described DOTA conjugate DOTA-(MC-FA-012).sub.3 an AlexaFluor 680 conjugated variant was purchased from Pepscan for in vivo imaging use. AlexaFluor680-(MC-FA-012).sub.3 (AF680-(MC-FA-012).sub.3) anchor molecule was generated analog to DOTA-(MC-FA-012).sub.3. Coupling of the AlexaFluor680 moiety was done as activated ester in solution to the N-terminal amid.
(107) Kinetic Analysis of Monomeric MC-FA-010 and Monomeric and Trimeric MC-FA-012 Binding to Recombinant Human Seprase
(108) The binding kinetics of monomeric and trimeric Seprase-binding Microbodies was determined using surface plasmon resonance spectroscopy on a Biacore T-100 system. For the monomeric MC-FA-010 Microbody a dissociation constant of 149 nM was measured. The MC-FA-012 variant with a single Lys2Ala exchange showed an affinity of 340 nM. Both trimeric variants DOTA-(MC-FA-012).sub.3 and AF680-(MC-FA-012).sub.3 showed a significantly higher affinity in sub-nanomolar range and slower offrate compared to the monomeric Microbodies. DOTA-(MC-FA-012).sub.3 has a dissociation constant of 12.4 M (steady state analysis, 249 pM). AF680-(MC-FA-012).sub.3 has a dissociation constant of 61.5 pM (steady state analysis, 669 pM). Compared to MC-FA-012 the offrate of DOTA-(MC-FA-012).sub.3 is around 530, and of AF680-(MC-FA-012).sub.3 around 134 times slower. Due to the small size (13000 Da) and slow offrate both trimeric constructs are predestined for in vivo imaging of tumors with a low overall background. Detailed kinetic data is summarized in table 3 and
(109) Scaffold Swapping
(110) In order to improve or to modulate the physicochemical properties of Microbody binders and characteristics based on them (e.g. net charge, stability, oral availability) sequence branches responsible for binding can be grafted to other alternative scaffolds. In this study the binding sequence of MC-FA-012 mainly located in the first loop was transferred into two scaffolds based on Trypsin inhibitor EETI-II of Ecballium elaterium (ET-FA-012) and an optimized McoTI-II scaffold (Momordica cochinchinensis, MO-FA-012). Corresponding sequence information is listed in table 4. DNA coding regions of said proteins were cloned in the vector backbone of pET32b-LibEx enabling an expression as fusion to thioredoxin. Expression and purification was performed as described above (Example 1). For SPR analysis thioredoxin was separated through Thrombin cleavage and additional purification with IMAC and RP-HPLC. To confirm the correct synthesis expected mass was verified via mass spectroscopy. The functionality of the newly constructed Microbodies was analyzed using SPR. Both Microbodies showed specific binding to immobilized rhuSeprase with a slightly weaker dissociation constant compared to MC-FA-012 (table 3 and
(111) MC-FA-012 Specifically Binds to Seprase-expressing Triple Negative Breast Cancer (TNBC)
(112) To analyze binding of MC-FA-012 to Seprase-expressing triple negative breast cancer, tumor sections were analyzed via immunofluorescence staining (
Example 4
Tumor Targeting in Seprase Expressing CHO-Xenograft with IRDye Conjungated MC-FA-012
(113) To analyze biodistribution and tumor targeting properties of the MC-FA-012 Microbody a xenograft was established in immunodeficient mice Foxn1(nu) using the CHO-K1-huSeprase and CHO-K1-MOCK cell lines. The huSeprase ligand (MC-FA-012) and a control Microbody (MC-CM-010) were conjugated to IRDye800CW using NHS chemistry and injected i.v. into tumor bearing mice. After 0.5 and 2 h mice were euthanized. Organs were taken out and IR signal was measured ex vivo on a Xenogen IVIS optical in vivo imaging system (
(114) Biodistribution and Tumor Targeting Analysis
(115) To analyze the pharmacokinetic and tumor targeting properties of the AF680-(MC-FA-012).sub.3 trimer biodistribution in female Fox n1 nu mice was monitored at six time points after injection (1, 2, 4, 6, 24 and 96 h). AF680-(MC-FA-0116).sub.3 and untreated mice served as controls. After each time point biodistibution was measured in vivo and ex vivo using a Xenogen Imaging system (
Example 5
Further Characterization of the Binding Loop and Initial Attempts for Affinity Maturation
(116) In order to analyse the interaction between the MC-FA-012 Microbody and seprase in greater detail and to identify more affine binders a focused library was generated on the basis of the alanine scan data and screened against soluble seprase. Therefore, four different selection conditions with varying stringency were applied. After three selection rounds all 4 pools were sub-cloned into an expression vector. 96 clones per pool were expressed and analysed with respect to expression rate as well as to target and unspecific binding properties. The s/n (signal to noise) value was calculated by division of the ELISA signal versus huSeprase and BSA and indicates for target specific binding. In addition to this s/n value a relative expression value (Ex) was calculated from SDS-PAGE data. Both values were taken into account for ranking of the clones. Ranking 1 and 2 values which differ with respect to weighting factors of the s/n value were calculated. The top-ranked clones of each pool (Pool 1: clones 1-4, Pool 2: 1-7, Pool 3: 1-4, Pool 4: 1-5 according to Ranking 2 values) or all clones with a ranking 2 value above 5 are shown in Tables 1 and 2 below.
(117) TABLE-US-00022 TABLE 1 Top-ranked clones of each pool >1 GACPYRNWMTGRGPLCRRDSDCPGRCICRGNGYCG >2 GACMYMNWTPGRGPDCRRDSDCPGRCICRGNGYCG >3 GACPYASWADGRGPHCRRDSDCPGRCICRGNGYCG >4 GACVYQHWQPGRGPSCRRDSDCPGRCICRGNGYCG >5 GACPYSRWAVGRGPSCRRDSDCPGRCICRGNGYCG >6 GACPYTRWQPGRGPSCRRDSDCPGRCICRGNGYCG >7 GACPYSNWAVGRGPSCRRDSDCPGRCICRGNGYCG >8 GACPYSRWAVGRGPDCRRDSDCPGRCICRGNGYCG >9 GACPYSNWAVGRGPSCRRDSDCPGRCICRGNGYCG >10 GACPYTNWRPGRGPACRRDSDCPGRCICRGNGYCG >11 GACPYSNWAVGRGPACRRDSDCPGRCICRGNGYCG >12 GACAYSSWSAGRGPMCRRDSDCPGRCICRGNGYCG >13 GACPYVNWAAGRGPVCRRDSDCPGRCICRGNGYCG >14 GACPYAVWASGRGPSCRRDSDCPGRCICRGNGYCG >15 GACEYSAWLAGRGPECRRDSDCPGRCICRGNGYCG >16 GACVYWQWIAGRGPVCRRDSDCPGRCICRGNGYCG >17 GACWYDPWWLGRGPVCRRDSDCPGRCICRGNGYCG >18 GACMYDTWAQGRGPNCRRDSDCPGRCICRGNGYCG >19 GACLYEVWPLGRGPQCRRDSDCPGRCICRGNGYCG >20 GACAYSNWQPGRGPHCRRDSDCPGRCICRGNGYCG
(118) TABLE-US-00023 TABLE 2 Clones with a ranking 2 value above 5 >1 GACPYRNWMTGRGPLCRRDSDCPGRCICRGNGYCG >2 GACMYMNWTPGRGPDCRRDSDCPGRCICRGNGYCG >3 GACPYASWADGRGPHCRRDSDCPGRCICRGNGYCG >4 GACVYQHWQPGRGPSCRRDSDCPGRCICRGNGYCG >5 GACPYSRWAVGRGPSCRRDSDCPGRCICRGNGYCG >6 GACPYTRWQPGRGPSCRRDSDCPGRCICRGNGYCG >7 GACPYSNWAVGRGPSCRRDSDCPGRCICRGNGYCG >8 GACPYSRWAVGRGPDCRRDSDCPGRCICRGNGYCG >9 GACPYSNWAVGRGPSCRRDSDCPGRCICRGNGYCG >10 GACPYTNWRPGRGPACRRDSDCPGRCICRGNGYCG >11 GACPYSNWAVGRGPACRRDSDCPGRCICRGNGYCG >12 GACPYSRWAVGRGPDCRRDSDCPGRCICRGNGYCG >13 GACPYANWAVGRGPNCRRDSDCPGRCICRGNGYCG >14 GACPYTYWHPGRGPGCRRDSDCPGRCICRGNGYCG >15 GACPYSNWRPGRGPECRRDSDCPGRCICRGNGYCG >16 GACPYANWMVGRGPSCRRDSDCPGRCICRGNGYCG >17 GACPYTRWAVGRGPDCRRDSDCPGRCICRGNGYCG >18 GACPYTRWAVGRGPDCRRDSDCPGRCICRGNGYCG >19 GACPYARWAAGRGPACRRDSDCPGRCICRGNGYCG >20 GACPYSTWQVGRGPSCRRDSDCPGRCICRGNGYCG >21 GACPYTRWTVGRGPSCRRDSDCPGRCICRGNGYCG >22 GACPYSRWAVGRGPDCRRDSDCPGRCICRGNGYCG >23 GACPYTNWQPGRGPACRRDSDCPGRCICRGNGYCG >24 GACPYTNWHPGRGPACRRDSDCPGRCICRGNGYCG >25 GACPYTNWQPGRGPACRRDSDCPGRCICRGNGYCG >26 GACPYTRWAVGRGPDCRRDSDCPGRCICRGNGYCG >27 GACPYARWVVGRGPSCRRDSDCPGRCICRGNGYCG >28 GACAYANWQVGRGPSCRRDSDCPGRCICRGNGYCG >29 GACPYTRWAVGRGPDCRRDSDCPGRCICRGNGYCG >30 GACPYARWVLGRGPDCRRDSDCPGRCICRGNGYCG >31 GACPYTNWHPGRGPDCRRDSDCPGRCICRGNGYCG >32 GACPYANWAVGRGPNCRRDSDCPGRCICRGNGYCG >33 GACPYTYWHAGRGPSCRRDSDCPGRCICRGNGYCG >34 GACPYSTWAVGRGPACRRDSDCPGRCICRGNGYCG >35 GACPYTNWQPGRGPACRRDSDCPGRCICRGNGYCG >36 GACPYTRWAVGRGPDCRRDSDCPGRCICRGNGYCG >37 GACPYRNWAVGRGPSCRRDSDCPGRCICRGNGYCG >38 GACPYATWQPGRGPSCRRDSDCPGRCICRGNGYCG >39 GACPYTNWHPGRGPACRRDSDCPGRCICRGNGYCG >40 GACPYTNWQPGRGPACRRDSDCPGRCICRGNGYCG >41 GACPYARWNVGRGPSCRRDSDCPGRCICRGNGYCG >42 GACPYTNWHPGRGPDCRRDSDCPGRCICRGNGYCG >43 GACPYANWTIGRGPACRRDSDCPGRCICRGNGYCG >44 GACPYARWHVGRGPSCRRDSDCPGRCICRGNGYCG >45 GACAYSNWAVGRGPSCRRDSDCPGRCICRGNGYCG >46 GACPYSTWAVGRGPDCRRDSDCPGRCICRGNGYCG >47 GACPYTNWAVGRGPSCRRDSDCPGRCICRGNGYCG >48 GACPYANWAVGRGPHCRRDSDCPGRCICRGNGYCG >49 GACPYRNWQPGRGPTCRRDSDCPGRCICRGNGYCG >50 GACPYSNWTVGRGPECRRDSDCPGRCICRGNGYCG >51 GACPYHTWAVGRGPGCRRDSDCPGRCICRGNGYCG >52 GACPYRNWSPGRGPHCRRDSDCPGRCICRGNGYCG >53 GACPYTFWRVGRGPACRRDSDCPGRCICRGNGYCG >54 GACPYSNWTVGRGPACRRDSDCPGRCICRGNGYCG >55 GACPYSRWAVGRGPDCRRDSDCPGRCICRGNGYCG >56 GACVYWQWIAGRGPVCRRDSDCPGRCICRGNGYCG >57 GACWYDPWWLGRGPVCRRDSDCPGRCICRGNGYCG >58 GACMYDTWAQGRGPNCRRDSDCPGRCICRGNGYCG >59 GACLYEVWPLGRGPQCRRDSDCPGRCICRGNGYCG >60 GACAYSNWQPGRGPHCRRDSDCPGRCICRGNGYCG >61 GACEYHVWMGGRGPHCRRDSDCPGRCICRGNGYCG
(119) A detailed characterization of the top-ranked clones especially concerning the kinetic parameters (K.sub.d, K.sub.on, K.sub.off) in comparison to the parental MC-FA-012 variant is on-going.
(120) Binding Analysis of MC-FA-012 Variants with Altered Binding Sequence
(121) Seven selected clones of above mentioned ranking (Table 1 and 2, Example 5) had been further analyzed using SPR spectroscopy. The variants showed a dissociation constant in the same range (147-487 nM) as MC-FA-010 or MC-FA-012 (149-340 nM) with comparable offrate. Detailed kinetic data and sequence informations are summarized in table 3 and 4 and
Example 6
Biodistribution and Tumor Targeting Using 68Ga-(MC-FA-012)3 and 177Lu-(MC-FA-012)3
(122) Organ distribution of .sup.177Lu-(MC-FA-012).sub.3
(123) Organ distribution of .sup.177Lu-(MC-FA-012).sub.3 in CT26-huSeprase tumors bearing mice was monitored over 24 h. At six time points (0.5, 1, 2, 4, 6 and 24 h) mice were sacrificed and radioactivity in dissected organs had been measured. The measured dose is summarized in
(124) Small Animal PET Imaging Using .sup.68Ga-(MC-FA-012).sub.3
(125) Biodistribution and tumor targeting of .sup.68Ga-(MC-FA-012).sub.3 was measured during a time period of 140 minutes via PET scan. In both analyzed mice 68Ga-(MC-FA-012).sub.3 showed a specific tumor targeting (
Example 7
Diagnostic and Therapeutic Use of Microbody Trimers
(126) For diagnostic purpose 2.5 nmol .sup.68Ga-(MC-FA-012).sub.3 was injected i.v. Up to date five patients with advanced pancreatic cancer had been examined. Detailed information on applied dose is summarized in table 6. .sup.68Ga-(MC-FA-012).sub.3 showed high enrichment in primary tumors and metastasis (
(127) For therapeutic purpose 10-15 nmol .sup.177Lu-(MC-FA-012).sub.3 was injected i.v. (table 7). Two patients had been recently treated. The patients showed no acute nephrotoxicity. The treatment is still on going.
Example 8
Immunohistochemical Analysis of Seprase Expression in Different Tumor Entities
(128) To provide insights in Seprase expression in different tumor entities an immunohistochemical analysis had been performed.
(129) Pancreas Carcinoma
(130) Seprase expression in normal pancreas tissue is limited to langerhans islets, ducts and vessels. On pancreas carcinoma Seprase expression is additionally detected on tumor cells and fibroblasts (Table 8 and
(131) Triple Negative Breast Cancer (TNBC)
(132) On 39/39 TNBCs samples many weak till strong membranous and cytoplasmic stained fibroblasts (54%) were detected within fibrous tissue (sometimes with stronger signals around tumor cells). In one case signals on necrosis/pre-necrotic cells were found. On 3/3 normal human breast samples some medium stained fibroblasts (3%) were detected within fibrous tissue (Table 9 and
(133) Lung Carcinoma
(134) On 29/31 lung carcinomas many weak till strong membranous and cytoplasmic stained fibroblasts (55%) were detected within fibrous tissue. In five cases signals on necrosis/pre-necrotic cells were found. On 2/3 normal human lung samples some weak till strong stained fibroblasts (17%) were detected within fibrous tissue (Table 10 and
(135) Seprase seems to be an overexpressed target in pancreas cancer, TNBC and lung carcinoma. Therefore, the use of the identified Seprase ligand as diagnostic and therapeutic agent is not limited to the described clinical application.
(136) TABLE-US-00024 TABLE 3 Affinity determination of monomeric and trimeric Seprase binding Microbodies KD (M, Analyte ka (1/Ms) kd (1/s) KD (M) Chi.sup.2 (RU.sup.2) steady state) Chi.sup.2 (RU.sup.2) FIG. MC-FA-010 1.53E+06 0.2006 1.49E07 0.131 1.06E07 0.21 11A MC-FA-012 4.08E+05 0.1385 3.40E07 0.041 2.46E07 0.0416 11B DOTA-(MC- 2.11E+07 1.24E11 0.446 2.49E10 14.6 11C FA-012).sub.3 AF680-(MC- 1.68E+07
6.15E11 0.874 6.69E10 9.11 11D FA-012).sub.3 FA8-D06 3.46E+05 0.05303 1.53E07 1.2 1.13E07 1.42 12A FA7-A05 2.46E+05 0.05035 2.05E07 1.09 1.02E07 1.11 12B FA8-C09 9.92E+06 2.935 2.96E07 0.328 2.43E07 9.5 12C FA8-D03 9.39E+05 0.1381 1.47E07 0.824 1.51E07 0.137 12D FA8-D05 4.21E+06 0.8121 1.93E07 0.441 1.77E07 0.822 12E FA8-F04 6.24E+05 0.2003 3.21E07 0.218 3.32E07 0.21 12F FA8-G12 6.51E+05 0.3171 4.87E07 0.146 4.70E07 0.0436 12G ET-FA-012 6.08E+05 0.8528 1.40E06 0.271 4.54E06 0.488 13A MO-FA-012 3.82E+05 0.5128 1.34E06 1.43 1.16E06 0.561 13B Offrate highlighted in bold letters, trimeric constructs are highlighted in italic letters
(137) TABLE-US-00025 TABLE 4 Sequence listing Microbody Amino acid Sequence ET-FA-012 GSGACPYSNWTPGRGPDCSQDSDCLAGCVCGPNGFCG MO-FA-012 GSGACPYSNWTPGRGPDCSSDSDCPGACICLENGFCG FA7-A05 GSGACPYSRWMPGRGPSCRRDSDCPGRCICRGNGYCG FA8-C09 GSGACPYTNWRPGRGPACRRDSDCPGRCICRGNGYCG FA8-D03 GSGACPYTRWAVGRGPDCRRDSDCPGRCICRGNGYCG FA8-D05 GSGACPYTRWQPGRGPSCRRDSDCPGRCICRGNGYCG FA8-D06 GSGACPYSRWAVGRGPDCRRDSDCPGRCICRGNGYCG FA8-F04 GSGACPYSNWAVGRGPSCRRDSDCPGRCICRGNGYCG FA8-G12 GSGACPYTNWHPGRGPACRRDSDCPGRCICRGNGYCG
(138) TABLE-US-00026 TABLE 5 Organ distribution of .sup.177Lu-(MC-FA-012).sub.3 Mean % ID/g (n = 3) 30 min 1 h 2 h 4 h 6 h 24 h Blood 1.3512 0.3597 0.1353 0.042 0.0374 0.0429 Heart 0.6152 0.1869 0.1284 0.1015 0.0876 0.0641 Lungs 1.7729 0.6705 0.6067 0.4514 0.6476 0.189 Liver 0.6684 0.3983 0.4402 0.4101 0.431 0.3631 Spleen 0.9253 0.5493 0.6561 0.5931 0.6287 0.4561 Kidneys 286.7812 222.3177 272.3888 245.1877 259.4467 213.4107 Intestine 0.7435 0.5079 1.0902 0.2913 0.9309 0.3347 Brain 0.6895 0.2284 0.5818 0.3159 0.2159 0.2151 Muscle 0.0589 0.0423 0.0317 0.0241 0.1259 0.0253 Tumor 9.8546 6.7929 8.1504 7.7667 7.8057 5.8505 Injection site 4.2434 15.2418 2.8704 2.6095 1.5742 0.9827
(139) TABLE-US-00027 TABLE 6 .sup.68Ga-labeling of DOTA-(MC-FA-012).sub.3 for diagnostic purposes .sup.68Ga-(MC-FA-012).sub.3 MBq t.sub.R Patient Date (administered) nmol (HPLC) Surgery 1 29.10.15 359 2.5 2.22 No 2 02.11.15 351 2.5 2.22 Pancreatectomy (Whipple, part.) 3 24.11.15 63 2.5 2.24 No 4 14.01.16 166 2.5 2.20 Pancreatectomy/ splenectomy (part.) 5 02.02.16 355 2.5 2.21 No
(140) TABLE-US-00028 TABLE 7 .sup.177Lu-labeling of DOTA-(MC-FA-012).sub.3 for therapeutic purposes .sup.177Lu-(MC-FA-012).sub.3 t.sub.R Patient Date GBq (product) nmol (HPLC) 3 20.01.16 2.318 15 2.04 5 04.02.16 2.530 10 2.06
(141) TABLE-US-00029 TABLE 8 IHC study of Seprase expression in pancreatic cancer % pos. tumor cells Subcellular Normal epithelial Tissue Tissue ID 1 Antibody 0 +1 +2 +3 pattern cells % pos. Fibrous tissue Pancreas BT002857A_1F_1PEB Anti-Seprase X X X X X ++ 5 Pancreas BT002849A_1F_1PEB Anti-Seprase X X X X X ++ 5 Pancreas BT002850A_1F_1PEB Anti-Seprase X X X X X ++ 5 Pancreas BT002852A_1F_1PEB Anti-Seprase X X X X X + 55 + Pancreas BT002852A_1F_1PEB Anti-Seprase X X X X X + 55 + Pancreas BT002853A_1F_1PEB Anti-Seprase X X X X X ++ 5 Pancreas BT002854A_1F_1PEB Anti-Seprase X X X X X ++ 5 Pancreas BT002856A_1F_1PEB Anti-Seprase X X X X X + 45 Pancreas BT002858A_1F_1PEB Anti-Seprase X X X X X + 60 Pancreas BT002859A_1F_1PEB Anti-Seprase X X X X X + 60 Pancreas BT002873A_1F_1PEB Anti-Seprase X X X X X + 45 Pancreas BT002874A_1F_1PEB Anti-Seprase X X X X X + 35 Pancreas BT002875A_1F_1PEB Anti-Seprase X X X X X + 30 Pancreas BT002876A_1F_1PEB Anti-Seprase X X X X X + 20 Pancreas BT002877A_1F_1PEB Anti-Seprase X X X X X + 30 Pancreas Pancreas 4 Anti-Seprase X X X X X + 30 Pancreas Pancreas 2 Anti-Seprase X X X X X ++ 10 Colon Colon 2 Anti-Seprase X X X X X + 20 Pancreas_CA BT002813A_1F_1PEB Anti-Seprase 30 70 0 0 c/m + 5 ++ Pancreas_CA BT002829A_1F_1PEB Anti-Seprase 50 40 10 0 c/m X X + Pancreas_CA BT002830A_1F_1PEB Anti-Seprase 20 60 20 0 c/m ++ 5 ++ Pancreas_CA BT002831A_1F_1PEB Anti-Seprase 20 20 50 10 c/m + 5 ++ Pancreas_CA BT002845A_1F_1PEB Anti-Seprase 30 50 20 0 c/m + 5 ++ Pancreas_CA BT002846A_1F_1PEB Anti-Seprase 20 70 10 0 c/m 0 Pancreas_CA BT002847A_1F_1PEB Anti-Seprase 30 70 0 0 c/m + 5 Pancreas_CA BT002848A_1F_1PEB Anti-Seprase 90 8 2 0 c 0 Pancreas_CA BT002860A_1F_1PEB Anti-Seprase 15 70 15 0 c/m 0 +++ Pancreas_CA BT002861A_1F_1PEB Anti-Seprase 30 50 20 0 c/m X X ++ Pancreas_CA BT002868A_1F_1PEB Anti-Seprase 90 8 2 0 c/m + 5 + Pancreas_CA BT002869A_1F_1PEB Anti-Seprase 20 20 60 0 c/m ++ 5 + Pancreas_CA BT002870A_1F_1PEB Anti-Seprase 35 25 40 0 c/m ++ 5 Pancreas_CA BT002871A_1F_1PEB Anti-Seprase 20 55 25 0 c/m ++ 5 + Pancreas_CA BT002872A_1F_1PEB Anti-Seprase 25 45 30 0 c/m ++ 5 + Pancreas_CA 15B02208 Anti-Seprase 30 30 20 20 c/m ++ 5 Pancreas_CA 12B15338.1 Anti-Seprase 90 1 0 9 c/m ++ 5 Pancreas_CA 12B15338.2 Anti-Seprase 90 3 0 8 c/m ++ 5 CHO-K1- 15_503 Anti-Seprase 15 40 30 15 m 0 huSeprase CHO-K1- 15_503 Anti-Seprase 15 30 35 20 m 0 huSeprase CHO-K1- 15_503 Anti-Seprase 15 35 35 15 m 0 huSeprase Tissue % pos. Vessels % pos. Necrosis % necrotic area Comment Pancreas 0 + 75 X X +/++/+++ c langerhans islets, +/++ m vessels and ducts Pancreas 0 + 65 85 +/++/+++ c (prenecrotic) langerhans islets, +/++ m vessels and ducts Pancreas 0 + 70 75 +/++/+++ c (prenecrotic) langerhans islets, +/++ m vessels and ducts Pancreas 60 ++ 80 X X +/++/+++ c langerhans islets, +/++/+++ c/m fibroblasts, +/++/+++ m vessels and ducts Pancreas 60 ++ 80 X X +/++/+++ c langerhans islets, +/++/+++ c/m fibroblasts, +/++/+++ m vessels and ducts Pancreas 0 + 80 X X +/++/+++ c langerhans islets, +/++ m vessels and ducts; BG secretion Pancreas 0 + 75 X X +/++/+++ c langerhans islets, +/++ m vessels and ducts; BG secretion Pancreas 0 + 60 X X +/++/+++ c langerhans islets, +/++ m vessels and ducts Pancreas 0 ++ 75 X X +/++/+++ c langerhans islets, +/++/+++ m vessels and ducts Pancreas 0 + 65 X X +/++ c langerhans islets, +/++ m vessels and ducts Pancreas 0 ++ 75 X X +/++/+++ c langerhans islets, +/++ m vessels and ducts Pancreas 0 + 70 X X +/++/+++ c langerhans islets, +/++ m vessels and ducts Pancreas 0 ++ 75 X X +/++/+++ c langerhans islets, +/++ m vessels and ducts Pancreas 0 + 70 X X +/++/+++ c langerhans islets, +/++ m vessels and ducts Pancreas 0 + 70 X X +/++/+++ c langerhans islets, +/++ m vessels and ducts Pancreas 0 ++ 65 X X +/++/+++ c langerhans islets, +/++ m vessels and ducts Pancreas 0 ++ 65 X X +/++/+++ c langerhans islets, +/++ m vessels and ducts Colon 0 0 X X BG mucosa Pancreas_CA 10 + 65 X X +/++/+++ c/m langerhans islets, +/++/+++ c/m fibroblasts, +/++ m vessels and ducts Pancreas_CA 5 + 60 X X +/++ c/m fibroblasts, + m vessels, +/++ c/m mucosa (intestine); BG muscle Pancreas_CA 20 ++ 75 X X +/++/+++ c/m langerhans islets, +/++/+++ fibroblasts, +/++/+++ vessels and ducts; BG secretion Pancreas_CA 2 + 65 X X +/++/+++ c/m langerhans islets, +/++/+++ fibroblasts, +/++ vessels and ducts Pancreas_CA 30 ++ 75 X X +/++/+++ fibroblasts, +/++ vessels and ducts Pancreas_CA 0 0 5 /+/++ c/m mucosa (intestine) Pancreas_CA 0 + 65 5 +/++/+++ c/m langerhans islets, +/++/+++ fibroblasts, +/++ vessels and ducts; BG secretion Pancreas_CA 0 0 35 X Pancreas_CA 50 ++ 75 15 +/++/+++ c/m fibroblasts, +/++ m vessels, +/++/+++ m prenecrotic cells Pancreas_CA 45 + 65 X X +/++/+++ c/m fibroblasts, +/++ m vessels Pancreas_CA 2 0 25 +/++ c/m langerhans islets, +/++ fibroblasts Pancreas_CA 20 0 X X +/++/+++ c langerhans islets, +/++/+++ c/m fibroblasts Pancreas_CA 0 0 X X +/++/+++ c langerhans islets Pancreas_CA 15 0 X X +/++/+++ c langerhans islets, +/++ c/m fibroblasts Pancreas_CA 5 + 65 X X +/++/+++ c/m langerhans islets, + fibroblasts, + /++ vessels Pancreas_CA 0 + 75 X X +/++/+++ c/m langerhans islets, +/++ vessels and ducts; BG muscle Pancreas_CA 0 0 X X +/++/+++ c/m langerhans islets Pancreas_CA 0 0 X X +/++/+++ c/m langerhans islets CHO-K1- 0 0 ++ 50 BG necrosis; small stained dots huSeprase CHO-K1- 0 0 ++ 50 BG necrosis; small stained dots huSeprase CHO-K1- 0 0 ++ 50 BG necrosis; small stained dots huSeprase
(142) TABLE-US-00030 TABLE 9 IHC study of Seprase expression In triple negative breast cancer. Tumor/normal Tissue Tissue ID antibody Fibrous tissue % pos. Necrosis % pos. comment Tumor Breast IN000422A_T_121FEB anti-Seprase ++ 70 0 +/++ m/c fibroblasts Tumor Breast IN000423A_T_122FEB anti-Seprase ++ 75 10 +/++/+++ m/c fibroblasts Tumor Breast IN000424A_T_123FEB anti-Seprase + 80 20 +/++ m/c fibroblasts Tumor Breast IN000425A_T_124FEB anti-Seprase + 40 0 +/++/m/c fibroblasts Tumor Breast IN000426A_T_125FEB anti-Seprase ++ 30 10 +/++ m/c fibroblasts, tissue partly disrupted and not analyzable Tumor Breast IN000428A_T_127FEB anti-Seprase ++ 70 5 +/++/+++ m/c fibroblasts Tumor Breast IN000430A_T_129FEB anti-Seprase ++ 35 0 +/++ m/c fibroblasts Tumor Breast IN000431A_T_130FEB anti-Seprase ++ 40 0 +/++ m/c fibroblasts Tumor Breast IN000432A_T_131FEB anti-Seprase ++ 60 0 +/++/+++ m/c fibroblasts Tumor Breast IN000389A_T_88FFB anti-Seprase ++ 90 +++ 10 ++ m/c fibroblasts, ++/+++ necrosis, stained dots within necrosis Tumor Breast IN00407A_T_106FFB anti-Seprase ++ 60 5 +/++ m/c fibroblasts Tumor Breast IN000383A_T_82FFB anti-Seprase ++ 80 10 +/++ m/c fibroblasts Tumor Breast IN000384A_T_83FFB anti-Seprase ++ 90 0 +/++ m/c fibroblasts Tumor Breast IN000385A_T_84FFB anti-Seprase ++ 65 0 +/++ m/c fibroblasts, tissue partly missing and not analyzable Tumor Breast IN000386A_T_85FFB anti-Seprase + 30 20 +/+++ m/c fibroblasts, tissue partly disrupted and not analyzable Tumor Breast IN000387A_T_86FFB anti-Seprase ++ 45 0 +/++ m/c fibroblasts, tissue partly missing and not analyzable Tumor Breast IN000388A_T_87FFB anti-Seprase + 10 0 + m/c fibroblasts Tumor Breast IN000389A_T_88FFB anti-Seprase ++ 90 +++ 10 ++ m/c fibroblasts, ++/+++ necrosis, stained dots within necrosis Tumor Breast IN000390A_T_89FFB anti-Seprase ++ 85 0 +/++ m/c fibroblasts Tumor Breast IN000391A_T_90FFB anti-Seprase + 40 0 + m/c fibroblasts, ++/+++ fibroblasts around tumor cells, tissue partly not analyzable Tumor Breast IN000392A_T_91FFB anti-Seprase ++ 45 0 +/++ m/c fibroblasts Tumor Breast IN000393A_T_92FFB anti-Seprase ++ 90 5 +/++ m/c fibroblasts, tissue partly missing and not analyzable Tumor Breast IN000394A_T_93FFB anti-Seprase ++ 50 0 +/++ m/c fibroblasts, tissue partly disrupted and not analyzable Tumor Breast IN000399A_T_98FFB anti-Seprase ++ 95 1 +/++ m/c fibroblasts, ++/+++ fibroblasts around tumor cells, tissue partly disrupted and not analyzable Tumor Breast IN000401A_T_100FFB anti-Seprase ++ 35 0 +/++ m/c fibroblasts Tumor Breast IN000403A_T_102FFB anti-Seprase + 90 0 +/++ m/c fibroblasts, tissue partly disrupted and not analyzable Tumor Breast IN000404A_T_103FFB anti-Seprase + 1 0 Rarely + m/c fibroblasts, tissue partly disrupted and not analyzable Tumor Breast IN000405A_T_104FFB anti-Seprase +++ 90 25 +/++ m/c fibroblasts Tumor Breast IN000406A_T_105FFB anti-Seprase + 40 70 +/++ m/c fibroblasts Tumor Breast IN000407A_T_106FFB anti-Seprase ++ 60 5 +/++ m/c fibroblasts Tumor Breast IN000408A_T_107FFB anti-Seprase ++ 50 10 +/++ m/c fibroblasts Tumor Breast IN000409A_T_108FFB anti-Seprase + 30 0 +/++ m/c fibroblasts Tumor Breast IN000410A_T_109FFB anti-Seprase ++ 90 5 +/++/+++ m/c fibroblasts Tumor Breast IN000411A_T_110FFB anti-Seprase ++ 20 0 +/++ m/c fibroblasts Tumor Breast IN000412A_T_111FFB anti-Seprase ++ 35 0 +/++ m/c fibroblasts, tissue partly disrupted or missing and not analyzable Tumor Breast IN000413A_T_112FFB anti-Seprase + 20 10 + m/c fibroblasts Tumor Breast IN000414A_T_113FFB anti-Seprase ++ 35 0 +/++/+++ m/c fibroblasts Tumor Breast IN000415A_T_114FFB aati-Seprase ++ 50 0 +/++ m/c fibroblasts Tumor Breast IN000416A_T_115FFB anti-Seprase ++ 80 15 +/++ m/c fibroblasts Tumor Breast IN000420A_T_119FFB anti-Seprase ++ 30 0 +/++ m/c fibroblasts Tumor Breast IN000421A_T_120FFB anti-Seprase ++ 40 0 +/++ m/c fibroblasts, tissue partly disrupted and not analyzable Tumor Breast IN000389A_T_88FFB 2 Ab only 0 X X 2 AB only control Tumor Breast IN000407A_T_106FFB 2 Ab only 0 X X 2 AB only control Normal Breast 3947 anti-Seprase ++ 1 X X ++ fibroblasts, unspecific signals on secretion Normal Breast 3008 anti-Seprase ++ 1 X X ++ fibroblasts, unspecific signal and secretion Normal Breast 2587 anti-Seprase ++ 10 X X ++ fibroblasts, unspecific signal Normal Breast IN00804A_N_7FFB anti-Seprase ++ 1 X X ++ fibroblasts, unspecific signals on secretion Normal Breast IN00805_N_8FFB anti-Seprase ++ 1 X X ++ fibroblasts Normal Breast 3008 2 Ab only 0 X X 2 AB only control Normal Breast 2587 2 Ab only 0 X X 2 AB only control
(143) TABLE-US-00031 TABLE 10 IHC study of Seprase expression in lung cancer. Fibrous Tumor/normal Tissue Tissue ID antibody tissue % pos. Necrosis % pos. comment Tumor Lung 810 anti-Seprase +++ 30 5 ++/+++ m/c fibroblasts around tumor cells Tumor Lung 816 anti-Seprase ++ 75 0 +/++ m/c fibroblasts, tissue partly disrupted and not analyzable Tumor Lung 820 anti-Seprase ++ 90 0 +/++ m/c fibroblasts Tumor Lung 828 anti-Seprase + 60 0 +/++ m/c fibroblasts Tumor Lung 1021 anti-Seprase + 90 0 +/++ m/c fibroblasts, tissue partly disrupted and not analyzable Tumor Lung 1023 anti-Seprase ++ 45 30 +/++ m/c fibroblasts, tissue partly folded and not analyzable Tumor Lung 1025 anti-Seprase + 20 0 +/++ m/c fibroblasts, tissue partly disrupted and not analyzable Tumor Lung 1027 anti-Seprase ++ 75 50 +/++ m/c fibroblasts Tumor Lung 1031 anti-Seprase ++ 80 40 +/++/+++ m/c fibroblasts Tumor Lung 1041 anti-Seprase ++ 90 0 +/++ m/c fibroblasts, tissue partly disrupted and not analyzable Tumor Lung 1047 anti-Seprase + 60 0 +/++ m/c fibroblasts, tissue partly disrupted and not analyzable Tumor Lung 1049 anti-Seprase ++ 90 0 +/++ m/c fibroblasts, tissue partly disrupted and not analyzable Tumor Lung 1051 anti-Seprase ++ 75 0 +/++/+++ m/c fibroblasts, tissue partly disrupted and not analyzable Tumor Lung 1061 anti-Seprase + 10 0 +/++ m/c fibroblasts Tumor Lung 1082 anti-Seprase ++ 60 45 +/++ m/c fibroblasts Tumor Lung 1296 anti-Seprase ++ 95 0 +/++ m/c fibroblasts Tumor Lung 1299 anti-Seprase ++ 5 5 +/++ m/c fibroblasts Tumor Lung 1301 anti-Seprase ++ 5 0 +/++ m/c fibroblasts Tumor Lung 1304 anti-Seprase + 20 35 +/++ m/c fibroblasts Tumor Lung 1307 anti-Seprase ++ 95 0 +/++ m/c fibroblasts Tumor Lung 1308 anti-Seprase ++ 40 5 +/++/+++ m/c fibroblasts Tumor Lung 1309 anti-Seprase ++ 35 5 +/++ m/c fibroblasts, tissue partly disrupted and not analyzable Tumor Lung 1310 anti-Seprase + 95 25 +/++ m/c fibroblasts Tumor Lung 1314 anti-Seprase ++ 85 0 +/++ m/c fibroblasts Tumor Lung 2164 anti-Seprase ++ 80 +++ 15 +/++ m/c fibroblasts, ++/+++ necrosis, stained cells within necrosis Tumor Lung 2167 anti-Seprase ++ 70 +++ 10 +/++ m/c fibroblasts, ++/+++ necrosis, stained cells within necrosis Tumor Lung 2168 anti-Seprase ++ 65 +++ 5 +/++ m/c fibroblasts, ++/+++ necrosis, stained cells within necrosis, tissue partly disrupted and not analyzable Tumor Lung 2172 anti-Seprase + 10 +++ 5 +/++ m/c fibroblasts, ++/+++ necrosis, stained cells within necrosis Tumor Lung 2174 anti-Seprase + 65 +++ 20 +/++ m/c fibroblasts, ++/+++ necrosis, stained cells within necrosis Tumor Lung 2164 2 Ab only 0 X X 2 AB control Tumor Lung 1307 2 Ab only 0 X X 2 AB control Normal Lung 569 Anti-Seprase 0 X X No staining detectable Normal Lung 575 Anti-Seprase ++ 40 X X +/++/+++ m/c fibroblasts, tissue partly disrupted and not analyzable Normal Lung 581 Anti-Seprase + 2 X X + m/c fibroblasts Normal Lung 575 2 Ab only 0 X X 2 AB control
(144) TABLE-US-00032 TABLE 11 Quantification of PET-data: Organ distribution of .sup.68Ga-(MC-FA-012).sub.3 1 and 3 hours after administration (SUV max/SUV mean) in patient 1 (rt = right, lft = left). 3 h 1 h SUV Organs SUV max SUV mean SUV max mean Brain 0.36 0.06 0.37 0.19 Pancreas tail (tumor) 7.61 4.54 10.37 5.04 Pancreas head (tumor) 8.85 4.77 9.78 5.1 Lungs 0.78 0.41 0.81 0.45 Liver 6.29 4.51 8.41 4.18 Spleen 3.04 2.18 2.97 1.17 Intestine ROI1 1.58 0.76 1.55 0.57 Intestine ROI2 1.95 1.12 1.96 0.55 Kidneys rt 69.95 43.87 128.76 74.5 Kidneys lft 73.35 47.86 118 76.3 Aorta (Background) 3.04 2.09 2.46 1.26 Pulm. mestast. rt 2.04 1.36 1.54 0.97 Pulm. mestast. rt cranial 1.39 0.95 1.19 0.77 Pulm. mestast. rt (2) 2.13 0.75 1.09 0.41 Pulm. mestast. rt basal 1.36 1.07 1.27 0.75
(145) TABLE-US-00033 TABLE 12 Quantification of PET-data: Organ distribution of .sup.68Ga-(MC-FA-012).sub.3 1 and 3 hours after administration (SUV max/SUV mean) in patient 2 (rt = right, lft = left). 3 h 1 h SUV Organs SUV max SUV mean SUV max mean Brain 0.51 0.08 0.46 0.21 Pancreas 2.76 2.67 5.07 2.47 Lungs 0.58 0.43 0.51 0.26 Liver 2.29 1.56 1.97 1.08 Spleen 2.19 1.51 1.56 0.81 Intestine ROI1 2.13 1.42 1.5 0.4 Intestine ROI2 1.13 0.73 1.5 0.6 Intestine ROI3 1.71 0.9 Kidneys rt 88.69 55 122.19 78.1 Kidneys lft 90.25 56.99 115.36 72.37 Aorta (Background) 2.65 1.86 1.37 0.8 Liver metast. 8.93 5.09 8.23 4.37 Liver metast. medial 4.6 2.53 Liver metast. rt lateral 3.71 2.44 4.23 2.27 Liver metast. cranial 8.66 4.94 Pulm. metast. rt 1.77 1.14 1.7 1 2.11 1.23 Pulm. metast. lft 1.51 0.79 1.47 0.86
(146) TABLE-US-00034 TABLE 13 Quantification of PET-data: Organ distribution of .sup.68Ga-(MC-FA-012).sub.3 1 and 3 hours after administration (SUV max/SUV mean) in patient 3 (rt = right, lft = left). 1 h 2 h Organs SUV max SUV mean SUV max SUV mean Brain rt 0.59 0.32 0.54 0.28 Brain lft 0.28 0.14 0.58 0.36 Pancreas 2.32 1.04 0.76 0.31 Lungs rt 1.63 0.78 1.6 0.72 Lungs lft 1.83 0.92 1.44 0.86 Liver 1.87 1.07 2.27 1.22 Spleen 2.29 1.55 3.13 1.21 Intestine 2.32 1.16 0.76 0.31 Kidneys rt 77.1 46.67 102.47 63.11 Kidneys lft 78.34 46.13 106.37 59.7 Aorta (Background) 1.99 1.4 2.27 0.93 Saliv. glands rt 1.4 0.7 1.46 0.58 Saliv. glands lft 1.4 0.7 1.17 0.59 Liver metast. rt (1) 9.65 5.26 9.39 5.11 Lymph node metast. 11.91 6.26 16.34 8.21 Liver metast. rt (2) 8.53 5.28 9.59 5.58 Liver metast. central 8.51 4.79 9.7 5.35 Liver metast. lft 9.27 5.26 10.97 5.77
(147) TABLE-US-00035 TABLE 14 Quantification of PET-data: Organ distribution of .sup.68Ga-(MC-FA-012).sub.3 1 and 3 hours after administration (SUV max/SUV mean) in patient 4 (rt = right, lft = left). 2.5 h 1 h SUV Organs SUV max SUV mean SUV max mean Brain rt 0.17 0.1 0.2 0.11 Brain lft 0.26 0.14 0.72 0.41 Pancreas Lungs 2.14 1.04 2.24 1.21 Liver 3.95 2.38 5.41 2.48 Spleen 2.35 0.44 1.99 0.42 Intestine 1.21 0.56 3.45 0.73 Kidneys rt 47.87 26.16 70.49 40.9 Kidneys lft 50.86 34.71 76.42 46.22 Aorta (Background) 4.21 3.48 2.43 1.52 Saliv. glands rt 1.69 0.88 1.38 0.78 Saliv. glands lft 1.58 1.02 1.55 0.74 Peritonitis carcinomatosa 4.66 2.51 6.93 3.74 Pulm. metast. (1) 3.58 2.01 2.83 1.65 Pulm. metast. (2) 2.75 1.66 2.42 1.47 Pulm. metast. (3) 4.08 2.37 3.39 2.09
(148) TABLE-US-00036 TABLE 15 Quantification of PET-data: Organ distribution of .sup.68Ga-(MC-FA-012).sub.3 1 and 3 hours after administration (SUV max/SUV mean) in patient 5 (rt = right, lft = left). 3 h 1 h SUV Organs SUV max SUV mean SUV max mean Brain rt 0.18 0.1 0.2 0.09 Brain lft 0.1 0.06 0.26 0.12 Parotis rt 1.08 0.72 1.01 0.44 Parotis lft 1.21 0.96 1.05 0.64 Kidneys rt 111.38 70.54 159.98 101.18 Kidneys lft 106.01 63.23 138.8 88 Gluteal muscle rt 0.93 0.53 0.98 0.34 Gluteal muscle lft 0.9 0.54 0.89 0.28 Liver 1.66 1.05 2.32 0.67 Pancreas tail (tumor) 4.56 2.43 3.82 1.91 Pancreas head 2.43 1.76 1.63 0.97 Lungs rt 0.64 0.38 0.52 0.22 Lungs lft 1.11 0.56 1.36 0.76 Aorta (Background) 1.76 1.15 1.22 0.54 Intestine 1.47 0.95 1.62 0.45 Liver metast. (main) 9.27 4.84 7.82 4 Liver metast. caud. med 4.85 3.04 5.59 3.42 Liver metast. caud. lat 4.47 2.58 4.67 2.48
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