Patent Application
20200131227
The present invention relates to a polypeptide with tumor binding activity, and its use in the treatment and diagnosis of cancer.
Radiolabeled polypeptides for diagnosis and targeted therapy of tumors are characterized by efficient transport to the tumor cells and a fast clearance. Since linear peptides display poor in vivo stability peptides embedded in disulfide-stabilized miniproteins which are endowed with an excellent proteolytic stability and beneficial pharmacokinetic profile are of growing interest for the development of tumor-affine ligands.
Integrins (ITG) are a family of heterodimeric cell surface receptors that mediate cellular adhesion to extracellular matrix proteins and serve as bidirectional signal transducers to regulate differentiation, migration, proliferation and cell death. ITGαvβ6 has been shown to be highly expressed on HNSCC as well as lung, colon, breast and pancreas carcinoma and is often associated with poor prognosis (Bandyopadhayay et al., Curr Drug Targets, 2009, 10, 645-52). Thus, it is desirable to generate polypeptide with tumor binding activity targeting the receptor integrin αvβ6 (ITGαvβ6). A linear peptide comprising the integrin binding motives RGD and LXXL is known in the prior art. However, short linear peptides are not particularly stable in a physiological environment and often have low affinities.
The present inventors have overcome these problems by identifying the polypeptides of the present invention, which inter alia provide one or more of the following advantages: (i) good target affinity, (ii) increased stability under physiological conditions, and (iii) improved cancer therapy and diagnosis and evaluation.
In a first aspect, the present invention relates to a polypeptide, preferably with the ability of specifically binding to a tumour, which comprises, essentially consists or consists of the amino acid sequence of formula (I) or (II)
wherein
B4 is any amino acid, preferably an amino acid selected from the group consisting of Y, W, P, E, and D, p is an integer selected from 0, 1, 2 or 3, preferably 3, more preferably B4 in formula (I) is YPD, FPD, WPD, YPE, FPE and WPE and B4 in formula (II) is DPY, DPF, DPW, EPY, EPF, and EPW, most preferably YPD in formula (I) and DPY in formula (II);
In a second aspect, the present invention relates to the polypeptide of the first aspect of the invention for medical use, in particular for use in the treatment of cancer.
In a third aspect, the present invention relates to the polypeptide of the first aspect of the invention for use in the diagnosis and/or evaluation of cancer, preferably for in vivo use.
Employing a phage display library based on the molecular scaffold of SFTI-1 for alternate biopanning on HNO97 cells or selected membrane protein fractions of this cell line the present inventors identified a peptide containing the RGDKXXL motif (SEQ ID NO: 42). The amino acid substitution of K4 to L4 in the original peptide improved the binding and affinity of the RGDLXXL (SEQ ID NO: 43) containing SFTI-1 derivate (SFITGv6) for a variety of HNSCC and other tumor cell lines of epithelial origin. SFITGv6 demonstrated an outstanding stability in human serum over a period of 24 hours and high affinity (KD=14.8 nM) for ITGαvβb. In correlation to the ITGαvβ6 expression of different cell lines as measured by FACS analysis the 125I-labeled peptide displayed specific binding to HNO97 cells and other HNSCC cells as well as to further carcinoma cell lines of different origin including lung, bladder and colon with an average of 7% which was competed to more than 90% by addition of 10−6 M unlabeled analog. The experiments concerning the binding kinetics and internalization revealed a maximal binding of 125I-SFITGv6 to HNO97 cells after exposure for 60 min followed by a decrease to less than 15%. On the contrary, an increasing uptake and internalization of 177Lu-DOTA-SFITGv6 to values of up to 57.3% and up to 37.4%, respectively, as well as retention of more than 50% of the applied dose for at least 4 hours in HNO97 cells was measured. This indicates a time-dependent intracellular deionization of 125I-labeled peptide followed by the efflux of free radioiodine. The long-lasting accumulation of the 117Lu-DOTA labeled peptide in tumor cells, however, allows for late imaging and possibly for a therapeutic application.
Using 68Ga-DOTA-SFITGv6, the inventors were able to successfully and selectively image the ITGαvβ6-expressing HNO97 tumor of Balb/c mice within 20 min in a small animal PET. The rapid clearance of unbound and unspecifically bound activity from the blood and the surrounding tissues resulted in an excellent tumor-to-background ratio 40 min after the injection remaining for at least 140 min. In parallel to the in vitro competition experiments a complete inhibition of 68Ga-DOTA-SFITGv6 accumulation in the tumor was achieved by injecting a nonradioactive analog before. This is evidence for the selective in vivo imaging of a tumor that endogenously expresses ITGαvβ6. The biodistribution data revealed a significantly higher accumulation of 177Lu-DOTA-SFITGv6 in the HNO97 tumor as compared to healthy tissues even after 4 and 6 hours (Table 1). However, in contrast to the binding kinetics in culture a washout of the radioactivity from the HNO97 tumor with time was noticed which might be due to a fast clearance of the tracer from the blood within the first 30 min. Of particular interest is the high and constant level of radioactivity of almost 42% ID/g in the kidneys in HNO97 tumor bearing Balb/c nude mice with hardly any clearance.
Peptide-based histochemical staining of HNO97-xenotransplanted mouse tumors and different human HNSCC tumor tissues revealed a specific, strong and homogeneous binding of biotinylated SFITGv6. As expected from the binding to different squamous and adenocarcinoma cell lines in vitro, the peptide displayed a moderate to strong binding in brain metastases derived from breast cancer and NSCLC, respectively. Since tumor-free lymph nodes did not show any staining inflammation-associated binding of the peptide can be excluded. Accordingly, tumor-specific but not inflammation-associated binding of SFITGv6 was observed in PET/CT performed in two patients suffering from either recurrent hypopharynx carcinoma or NSCLC after application of 68Ga-DOTA-SFITGv6 and 18F-FDG, respectively. In line with the small animal PET images of HNO97 xenografts the SUV values of SFITGv6 in these patients persisted from 1 to 3 hours after injection. Interestingly in contrast to 68Ga-DOTA-SFITGv6 18F-FDG accumulation was detected in inflammatory lesions and reactive lymph nodes of both patients. The property of FDG accumulation in inflammatory or infectious processes is well-known and may occasionally lead to false positive evaluation and incorrect up-staging of tumor patients with a tremendous impact on therapy management. Thus, in ITGαvβ6-positive tumors the peptide-based tracer provided a clear advantage over FDG with respect to false positive lesions.
SFITGv6 also accumulated in the kidneys, the bowel, the stomach and in the thyroid of both patients. Interestingly, the localization of the radioactivity in the bowel changed with time indicating the excretion of the tracer. In fact, regions of interest drawn in jejunum, terminal ileum and cecum revealed a decrease of the activity in the jejunum and a time-dependent increase in terminal ileum and cecum. This is evidence for a secretion of SFITGv6 into the stomach or duodenum/jejunum followed by an intraluminal transport to the terminal ileum and the cecum. With regard to the identification of small tumor lesions in the abdomen or a peritonitis carcinomatosa the administration of laxatives might be necessary to reduce intra-bowel activity and avoid a high background.
In conclusion, the polypeptide of the present invention is a novel stable ITGαvβ6-specific polypeptide with high affinity for a variety of HNSCC and other tumors. Due to the accumulation of the peptide in different tumors but not in inflammatory lesions and normal tissues of tumor patients the peptide of the present invention represents a promising tracer for imaging and therapy of cancer, particularly of ITGαvβ6-positive carcinoma.
Accordingly, in a first aspect the present invention relates to a polypeptide, preferably with the ability of specifically binding to a tumour, which comprises, essentially consists or consists of the amino acid sequence of formula (I) or (II)
wherein
Preferably, the present invention relates to a polypeptide comprising, essentially consisting or consisting of the amino acid sequence of formula (III) or (IV)
wherein
GH, GK, or GR, and B1 in formula (II) is HG, KG, or KG, most preferably in formula (I) GR and in formula (IV) RG;
It is further preferred that the polypeptide of the present invention comprises, essentially consists or consists of the amino acid sequence of formula (V) or (VI)
wherein
It is further preferred that the polypeptide of the present invention comprises, essentially consists or consists of the amino acid sequence of formula (V) or (VI)
wherein
It is further preferred that the polypeptide of the present invention has the amino acid sequence
wherein
Alternatively, the polypeptide of the present invention has preferably the sequence
wherein
Preferably, the present invention relates to a polypeptide comprising, essentially consisting or consisting of the amino acid sequence of formula (IX) or (X)
wherein
R, m is an integer selected from 0, 1, 2 or 3, preferably 2, more preferably B1 in formula (IX) is GH, GK, or GR, and B1 in formula (X) is HG, KG, or RG, most preferably in formula (IX) GR and in formula (X) RG;
Preferably, the present invention further relates to a polypeptide comprising, essentially consisting or consisting of the amino acid sequence of formula (XI) or (XII)
C
2-(B4)p-COO-R2
C
1-(B1)m-COO-R2
wherein
Preferably, the present invention further relates to a polypeptide comprising, essentially consisting or consisting of the amino acid sequence of formula (XIII) or (XIV)
wherein
Preferably, the present invention further relates to a polypeptide comprising, essentially consisting or consisting of the amino acid sequence of formulas (XV) to (XXVI):
wherein
Alternatively, the polypeptide of the present invention has preferably the sequence
wherein
In the peptides of the present invention, preferably, Z1 is any amino acid, preferably an amino acid selected from the group consisting of F, M, A, V, I, L, H, Y, W, T, Q, S, and N, more preferably F, H, Y, W, T and S, most preferably H or T, or not present.
In the peptides of the present invention, preferably, Z2 is any amino acid, preferably an amino acid selected from the group consisting of F, M, A, V, I, L, H, Y and W, more preferably F, H, Y and W, most preferably H, or not present.
It is further preferred that the peptides of the present invention form multimers, more preferably dimers, trimers, tetramers, pentamers or hexamers, most preferably trimers. The peptides of the multimers may be linked covalently or non-covalently, preferably covalently. Preferably, the peptides are not directly linked to each other but are linked to each other but through a linker group. The multimers of the invention may be further linked to one, two or more therapeutic agents, or an imaging agents. It is further preferred that the peptides of the multimers are covalently linked via a peptide linker, which may be further linked to one, two or more therapeutic agents, or imaging agents. The peptide linker is preferably comprised of 3 to 6 amino acids, more preferably 4 or 5 amino acids, most preferably 5 amino acids. Preferably, the peptide linker is a S and C rich linker, preferably comprising up to 80% S and C. Preferably, the peptide linker has the sequence GSGSK (SEQ ID NO: 40). Preferably, the monomeric peptides of the invention are linked to form a linear multimer. Preferably, a linear trimeric peptide of the present invention has SEQ ID NO: 41 (R1-HN-G-R-C1-T-G-R-G-D-L-G-R-L-C2-Y-P-D-G-S-G-S-K-G -R-C1-T-G-R-G-D-L-G-R-L-C2-Y-P-D-G-S-G-S-K-G-R-C1-T-G-R-G-D-L-G-R-L-C2-Y-P-D-COO-R2).
It is further preferred that the monomeric peptides of the invention are linked to form a branched multimer. Preferably, a branched trimeric peptide of the present invention has the formula (XXVII). Preferably, C1 and C2 of each monomeric peptide unit of the branched multimer form disulfide bonds.
The terms “amino acid” or “any amino acid” as used in the present invention refer to the 20 proteinogenic amino acids encoded by the universal genetic code as well as non-naturally occurring amino acids that have similar charge and size as the proteinogenic amino acids and further to selenocysteine and pyrrolysine. Accordingly, the amino acids of the present invention may preferably be selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, selenocysteine and pyrrolysine. For the amino acids of the present invention three-letter symbols and one-letter symbols are used according to the IUPAC “Nomenclature and Symbolism for Amino Acids and Peptides”
Preferably, the polypeptide of the present invention has a total length of at most 24 amino acids, more preferably at most 22 amino acids, more preferably at most 20 amino acids, more preferably at most 18 amino acids, most preferably at most 16 amino acids.
Preferably the number of amino acids that are positioned between the two Cys-residues in formulas (I) to (IV) and further (IX) to (XIV) varies between 8 to 16, more preferably 9 to 15, more preferably 9 to 13 and most preferably 9 to 12.
The polypeptides of the present invention are characterized by their ability to bind to the integrin receptor ITGαvβ6. Preferably, the binding affinity of the polypeptides of the present invention to ITG av136 is at least 50% of the affinity of the polypeptide with the amino acid sequence according to SEQ ID NO: 1. Preferably, the binding affinity of the present invention to ITG av136 is at least 60%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% of the affinity of the polypeptide with the amino acid sequence according to SEQ ID NO: 1.
The polypeptides of the present invention preferably have a KD of binding to ITGαvβ6.of less than 1×10−6 M, more preferably of less than 1×10−7 M, more preferably of less than m 5×10−8 M, more preferably of less than m 4×10−8 M, more preferably of less than m 3×10−8 M, more preferably of less than m 2×10−8 M more preferably of less than m 1×10−8 M, more preferably of less than 5×10−9 M, more preferably of less than 1×10−9 M.
The polypeptide of the present invention comprises integrin binding motives RGD and LXXL included in a small rigid peptide scaffold. The peptide scaffold is stabilized by a cysteine bridge resulting in a constrained structure with high proteolytic stability. Due to the exposed nature of the binding motive the construct further shows a high affinity for ITGαvβ6, while being easily accessible and quickly synthesized at low costs.
When chemically synthesizing a polypeptide e.g. by solid phase techniques, generating extremely high yields in each step is of overwhelming importance. Nevertheless, if each coupling step were to have 98% yield, a 18-amino acid peptide would be synthesized in 70% final yield, while a 36-amino acid peptide would be synthesized in 48% final yield, and a 54-amino acid peptide would be synthesized in a 34% final yield.
The term “antibody” as used in the context of the present invention refers to a glycoprotein belonging to the immunoglobulin superfamily; the terms antibody and immunoglobulin are often used interchangeably. An antibody refers to a protein molecule produced by plasma cells and is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, its antigen.
The term “antibody fragment” as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term “antibody fragment” include a fragment antigen binding (Fab) fragment, a Fab′ fragment, a F(ab′)2 fragment, a heavy chain antibody, a single-domain antibody (sdAb), a single-chain fragment variable (scFv), a fragment variable (Fv), a VH domain, a VL domain, a single domain antibody, a nanobody, an IgNAR (immunoglobulin new antigen receptor), a di-scFv, a bispecific T-cell engager (BITEs), a dual affinity re-targeting (DART) molecule, a triple body, a diabody, a single-chain diabody, an alternative scaffold protein, and a fusion protein thereof.
The term “diabody” as used within this specification refers to a fusion protein or a bivalent antibody which can bind different antigens. A diabody is composed of two single protein chains which comprise fragments of an antibody, namely variable fragments. Diabodies comprise a heavy chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL, or VL-VH). By using a short peptide connecting the two variable domains, the domains are forced to pair with the complementary domain of another chain and thus, create two antigen-binding sites. Diabodies can target the same (monospecific) or different antigens (bispecific).
A “single domain antibody”, as used in the context of the present invention refers to antibody fragments consisting of a single, monomeric variable domain of an antibody. Simply, they only comprise the monomeric heavy chain variable regions of heavy chain antibodies produced by camelids or cartilaginous fish. Due to their different origins they are also referred to VHH or VNAR (variable new antigen receptor)-fragments. Alternatively, single-domain antibodies can be obtained by monomerization of variable domains of conventional mouse or human antibodies by the use of genetic engineering. They show a molecular mass of approximately 12-15 kDa and thus, are the smallest antibody fragments capable of antigen recognition. Further examples include nanobodies or nanoantibodies.
The term “antibody mimetic” as used within the context of the present invention refers to compounds which can specifically bind antigens, similar to an antibody, but are not structurally related to antibodies. Usually, antibody mimetics are artificial peptides or proteins with a molar mass of about 3 to 20 kDa which comprise one, two or more exposed domains specifically binding to an antigen. Examples include inter alia the LACI-D1 (lipoprotein-associated coagulation inhibitor); affilins, e.g. human-γ B crystalline or human ubiquitin; cystatin; Sac7D from Sulfolobus acidocaldarius; lipocalin and anticalins derived from lipocalins; DARPins (designed ankyrin repeat domains); SH3 domain of Fyn; Kunits domain of protease inhibitors; monobodies, e.g. the 10th type III domain of fibronectin; adnectins: knottins (cysteine knot miniproteins); atrimers; evibodies, e.g. CTLA4-based binders, affibodies, e.g. three-helix bundle from Z-domain of protein A from Staphylococcus aureus; Trans-bodies, e.g. human transferrin; tetranectins, e.g. monomeric or trimeric human C-type lectin domain; microbodies, e.g. trypsin-inhibitor-II; affilins; armadillo repeat proteins. Nucleic acids and small molecules are sometimes considered antibody mimetics as well (aptamers), but not artificial antibodies, antibody fragments and fusion proteins composed from these. Common advantages over antibodies are better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs.
Preferably, the polypeptide of the present invention further comprises at least one compound selected from the group consisting of an imaging agent, a therapeutic agent, a linker group or a linker group linked to a therapeutic agent or an imaging agent, wherein the compound is preferably covalently bound outside the sequence comprised by C1 and C2 of the polypeptide, preferably to the N-terminus of the polypeptide.
Preferably, the therapeutic agent comprised in the polypeptide of the present invention is selected from the group consisting of small molecules, biopharmaceuticals, and combinations thereof.
The term “small molecules” as used in the present invention relates to a molecule with a molecular weight of about 1000 Dalton or less, preferably a molecular weight of about 900 Dalton or less, usually derived from total chemical synthesis. Suitably the small molecules of the present invention have a sufficient bioavailability and are capable of binding to a specific biological target.
Preferably, the small molecule is preferably an anti-cancer agent, more preferably selected from the group consisting of precyclophosphamide, methotrexate, 5-fluorouracil, mustine, vincristine, procarbazine, prednisolone, doxorubicin, bleomycin, vinblastine, dacarbazine, vincristine, etoposide, cisplatin, epirubicin, capecitabine, folinic acid, oxaliplatin, and combinations thereof.
The term “biopharmaceutical” as used in the present invention refers to pharmaceutical drug products manufactured in, extracted from, or semisynthesized from biological sources. Different from totally synthesized pharmaceuticals, they include vaccines, blood, blood components, allergenics, somatic cells, gene therapies, tissues, recombinant therapeutic protein, and living cells used in cell therapy. Biologics can be composed of sugars, proteins, or nucleic acids or complex combinations of these substances, or may be living cells or tissues. They or their precursors or components are isolated from living sources, such as human, animal, plant, fungal, or microbial.
Preferably, the biopharmaceutical used in the present invention is a protein, preferably a protein selected from the group consisting of an antibody, an antibody fragment, an antibody-mimetic, a cytokine, in particular an interferon, or an interleukin, and combinations thereof.
It is preferred that the antibody is suitable for the treatment of cancer, preferably selected from the group consisting of alemtuzumab, bevacizumab, ibritumomab tiuxetan, ipilimumab, nivolumab, ofatumumab, rituximab, tositumomab, and combinations thereof.
Preferably, the therapeutic agent further comprises a radioisotope selected from the group consisting of alpha radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, such as 18F, 51Cr, 67Ga, 68Ga, 111In, 99mTc, 140La, 175Yb, 153Sm, 166Ho, 88Y, 90Y, 149Pm, 177Lu, 47Sc, 142Pr, 159Gd, 212Bi, 72As, 72Se, 97Ru, 109Pd, 105Rh, 101m15Rh, 119Sb, 128Ba, 123I, 124I, 125I, 131I, 197Hg, 211At, 169Eu, 203Pb, 212Pb, 64Cu, 67Cu, 188Re, 186Re, 198Au and 199Ag.
More preferably, the therapeutic agent further comprises a radioisotope selected from the group consisting of 67Cu, 90Y, 131Lu, 186Re, 188Re, 211At, 225Ac, 212Bi, 213Bi, and combinations thereof.
Preferably, the imaging agent is a polydentate chelating agent. A chelating agent is capable of forming two or more separate coordinate bonds with a single central metal atom. Preferably, the chelating agent is selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), 1,4,7-triazacyclononane -1,4,7-triacetic acid (NOTA), hydrazine-nicotinic acid (HYNIC), mercaptoacetylglycyltriglycine (MAG3), ethylenediaminetetraacetic acid (EDTA), triethylenetetramine (TETA), iminodiacetic acid, Diethylenetriamine-N,N,N′,N′,N″-pentaacetic acid (DTPA) and combinations thereof, wherein 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is particularly preferred.
Preferably, the imaging agent further comprises a radioisotope selected from the group consisting of alpha radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, such as 18F, 51Cr, 67Ga, 68Ga, 111In, 99mTc, 140La, 175Yb, 153Sm, 166Ho, 88Y, 90Y, 149Pm, 177Lu, 47Sc, 142Pr, 159Gd, 212Bi, 72As, 72Se, 97Ru, 109Pd, 105Rh, 101m15Rh, 119Sb, 128Ba, 123I, 124I, 125I, 131I, 197Hg, 211At, 169Eu, 203Pb, 212Pb, 64Cu, 67Cu, 188Re, 186Re, 198Au and 199Ag.
It is further preferred that the polydentate chelating agent further comprises a radioisotope selected from the group consisting of 68Ga, 67Ga, 99mTc, 111In, 123I, 124I, 131I, 177Lu, and combinations thereof.
Particularly preferred polydentante chelating agents comprising a radioisotope are selected from the group consisting of 68Ga-DOTA, 177Lu-DOTA and combinations thereof. It if further preferred that these polydentante chelating agents comprising a radioisotope are coupled to the N-terminus of the peptide of the present invention.
In the alternative, it is preferred that the imaging agent is a molecule comprising a radioisotope selected from the group consisting of 18F, 11C, and combinations thereof.
Preferably, the imaging agent is a molecule comprising a 18F radioisotope selected from the group consisting of 18F-nucleosides, 18F-fluoroarenes, 18F-monosaccharides, and combinations thereof.
Examples of 18F-nucleosides include 18F-fluorothymidine (18F-FLT), 5-8F-fluorouracil (5-8F-FU), 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine (18F-FHBG), 2′-deoxy-2′-[18F]fluoro-1-D -arabinofuranosyl-adenine, 18F-FAA, and the like.
Examples of 18F-fluoroarenes include 18F-fluorobenzoic acid (18F-FBA), 4-azidophenacyl-18F -fluoride (18F-APF), and the like.
Examples of, 18F-monosaccharides include 2-deoxy-2-18F-fluoro-β-D-glucose (18F-FDG) and the like.
Preferably, the imaging agent is a molecule comprising a 11C radioisotope selected from the group consisting of 11C-metomidate, N-methyl-11C-vorozole, 11C-glucose, and combinations thereof
It is further preferably, that a radioisotope is directly introduced in one or more of the amino acids, preferably the sidechains of the amino acids, of the polypeptide of the invention. Accordingly, the polypeptide of the invention may comprise a radioisotope selected from the group consisting of 11C, 18F, 125I, 131I, and combinations thereof. It is particularly preferred that the radionuclides 125I and/or 131I are introduced into the side chain of a tyrosine comprised in the polypeptide of the present invention.
It is further preferred that imaging agent is a monodentate complex-forming molecule. The monodentate complex-forming molecule is capable of binding to a central atom, preferably a metal atom, in a coordination complex. The monodentate complex-forming molecule preferably selected from the group consisting of 5-fluorouracil, diacetyl-bis(N4-methyl-tiosemicarbazone (ATSM), pyrovaldehyde -bis(N4-methyltiosemicarbazone (PTSM), citrate, and combinations thereof.
Preferably, the monodentate complex-forming molecule further comprises a radioisotope selected from the group consisting of 64Cu, 68Ga, and combinations thereof.
The linker group is preferably selected from the group consisting of alkyl, cycloalkyl, heteroalkyl, arylalkyl, alkoxy, aryloxy, amino, amide, silyl, alkenyl, cycloalkenyl, heteroalkenyl, alkynyl, aryl, heteroaryl, acyl, carbonyl, carboxylic acid, ether, ester, nitrile, isonitrile, sulfanyl, sulfinyl, sulfonyl, phosphino, and combinations thereof. The person skilled in the art is able to select the suitable linker(s) depending on the respective application.
Preferably, the therapeutic agent and/or imaging agent are coupled to the respective amino acid of the polypeptide of the present invention by using an activated ester. In particular, in case of therapeutic agent and/or imaging agent to the amino acid having an amino group in a side chain this method can be used. Alternatively, the following coupling methods can be used in order to couple therapeutic agent and/or imaging agent to the respective amino acid, which are shortly summarized. The specific reaction conditions for achieving a coupling of a therapeutic agent and/or imaging agent to an amino acid with or without a linker can be easily determined by the person skilled in the art:
Formation of amides by the reaction of an amine and activated carboxylic acids, preferably NHS-esters or carbodiimides; a carbodiimide is a complete crosslinker that facilitates the direct conjugation of carboxyls to primary amine NHS esters are reactive groups formed by carbodiimide-activation of molecules containing carboxylate groups
Disulfide linkage using two thiols or one thiol that specifically reacts with pyridyl disulfides; Pyridyl disulfides react with sulfhydryl groups over a broad pH range (the optimum is pH 4-5) to form disulfide bonds. During the reaction, a disulfide exchange occurs between the molecule's SH-group and the 2-pyridyldithiol group. As a result, pyridine-2-thione is released.
Thioether formation using maleimides or haloacetyls and a thiol component; Haloacetyls react with sulfhydryl groups at physiologic pH. The reaction of the iodoacetyl group proceeds by nucleophilic substitution of iodine with a sulfur atom from a sulfhydryl group to result a stable thioether linkage. The maleimide group reacts specifically with sulfhydryl groups when the pH of the reaction mixture is between pH 6.5 and 7.5 and forms a stable thioether linkage that is not reversible.
Amidine formation using an imidoester and an amine; Imidoester crosslinkers react rapidly with amines at alkaline pH but have short half-lives. As the pH becomes more alkaline, the half life and reactivity with amines increases; therefore, crosslinking is more efficient when performed at pH 10 than at pH 8. Reaction conditions below pH 10 may result in side reactions, although amidine formation is favoured between pH 8-10
Hydrazide linkage using carbonyls (e.g. aldehydes) and hydrazides; Carbonyls (aldehydes and ketones) react with hydrazides and amines at pH 5-7. Carbonyls do not readily exist in proteins; however, mild oxidation of sugar glycols using sodium meta-periodate converts vicinal hydroxyls to aldehydes or ketones. Subsequent reaction with hydrazides results in the formation of a hydrazone bond.
Amine linkage using carbonyls and amines under reductive conditions; Reductive amination (also known as reductive alkylation) is a form of amination that involves the conversion of a carbonyl group to an amine via an intermediate imine. The carbonyl group is most commonly a ketone or an aldehyde.
Copper-catalyzed triazole formation using nitriles and azides. The Huisgen-type 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne is used to give stable 1,2,3-triazoles.
Isothiourea formation using isothiocyanates and amines; the reaction between isothiocyanates and amines, i.e. the E-amino groups of lysine leads to a stable isothiourea bond.
Formation of esters by the reaction of an alcohol and activated carboxylic acids, preferably acid chlorides or carbodiimides; High temperature reaction allows direct reaction between alcohols and carboxylic acids to form stable esters, alternatively the carboxylic acids and be activated under acidic or base catalytic conditions.
Formation of ethers by the reaction of an alcohol and alkyl halides. Haloalkanes are reactive towards nucleophiles. They are polar molecules: the carbon to which the halogen is attached is slightly electropositive where the halogen is slightly electronegative. This results in an electron deficient (electrophilic) carbon which, inevitably, attracts nucleophiles.
The polypeptide of the present invention is suitable for use in medicine, preferably in the treatment of cancer. Preferred cancers to be treated are ITGαvβ6-positive carcinomas.
The polypeptide of the present invention is further suitable for use in the diagnosis and/or evaluation of cancer. The term “evaluation” includes the assessment of a cancer that is diagnosed and that is typically carried out by histologic or imaging methods to determine the treatment strategy, e.g. staging, size, assessment of cancer periphery, involvement of lymph nodes, and near or distant metastasis. Preferred cancers to be diagnosed and/or evaluated are ITGαvβ6-positive carcinomas.
Preferably, the cancer is selected from the group consisting of head and neck squamous cell carcinoma, hypopharynx carcinoma, collangiocarcinoma, breast cancer, brain cancer, pancreatic cancer, ovarian cancer, kidney cancer, bowel cancer, stomach cancer, cervical cancer, thyroid cancer, pancreatic cancer, gastric cancer, lung cancer, colorectal cancer, liver cancer.
Intraoperatively obtained tissues from HNSCC or brain metastasis were either snap frozen or in part used to establish tumor cell cultures HNO97, HNO407, HNO199, HNO210, HNO258 as described in the prior art. Cell line authentification was performed by the German Collection of Microorganisms and Cell Cultures (DMSZ). Written informed consent was obtained from all patients according to the research proposals approved by the Institutional Review Board at the Medical Faculty of the University of Heidelberg. The human carcinoma cell lines UM-UC-5 (bladder), LUDLU-1 (lung), and MCF-7 (breast), liposarcoma cell line SW872 were purchased from European Collection of Authenticated Cell Cultures (ECACC), the colorectal adenocarcinoma cell line HT29 was obtained from ATCC, and the authentification was performed by Multiplexion (Friedrichshafen). The breast carcinoma cell line T47D and the primary human gingival mucosa keratinocyte cells HPV16GM were kindly provided by Prof. Dr. Stefan Dithel (TU Braunschweig) and by Prof. Dr. Pascal Tomakidi (Freiburg), respectively. HPV16GM cells were cultured in Keratinocyte Growth Medium 2 (Promocell). All HNSCC cell lines as well as the cell lines T47D, MCF-7, LUDLU-1 and SW872 were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FCS. UM-UC-5 and HT29 were cultured in EDSS medium (Gibco) supplemented with 10% FCS, 1% L-glutamine and 1% non-essential amino acids and DMEM high Glucose medium supplemented with 1 mM sodium pyruvate and 10% FCS, respectively. All cell lines were negative for Mycoplasma and maintained at 37° C. and 5% CO2.
To harvest membrane proteins HNO97 and HPV16GM cells were grown to 90% confluence in multilayer flasks (HYPERFlask™, Corning) and detached by pre-incubation with PBS/0.5% EDTA and subsequent 0.025% trypsin treatment. Membrane proteins were extracted according to an ultracentrifugation-based protocol. Briefly, cells were washed, pelleted and lysed by mechanical dissociation using a dounce tissue grinder (Wheaton) with 4 mL of lysis buffer (50 nM TRIS (pH 7.3), 250 mM sucrose, 2 mM EDTA, 2 mM protease inhibitor). Lysates were centrifuged (2800×g, 4° C., 20 min) and supernatants including cytosolic and membrane proteins were collected. Next, ultracentrifugation (>100.000×g/1 hour/4° C.) (Sorvall™ Dicovery 90SE, Hitachi) was used to separate cytosolic and membrane proteins. Membrane fraction purity was verified by western blot assessing epidermal growth factor receptor (EGFR) expression (
Fractionation of membrane proteins was conducted by the liquid chromatography system ProteomeLab™ as described before (Beckhove et al., J Clin Investig, 2010, 120, 2230-429; Ménoret et al., J Nucl Med, 2013, 54, 2146-52). Briefly, a total of 2.5 mg protein was loaded on the first dimension (1D) chroma to focusing column according to manufacturer's protocol (Beckman Coulter). Proteins were separated by isoelectric focusing and fractions were collected at 0.3 pH intervals during the pH gradient. In the second dimension (2D) 200 μL of each 1D fraction was loaded onto the 2D reversed phase column (heated to 50° C.) for further fractionation. Column-bound proteins were resolved by a 30 min linear gradient from solvent A (0.1% aqueous trifluoracetic acid (TFA)) to solvent B (0.08% TFA acid in acetonitrile), respectively. Eluted proteins were detected at 214 nm and collected. UV absorbance data were further analyzed using the ProteoVue and DeltaVue software (Beckman Coulter). Protein profiles of tumor cells and human keratinocytes (HPV16GM) were compared regarding the appearance of distinct tumor-specific peaks. Corresponding fractions were selected and pooled (Billecke et al., Molecular & Cellular Proteomics, 2006, 5, 35-42) and depending on the protein concentration concentrated using a vacuum concentrator (Bachhofer Savant).
For Western blot analyses of the HNO97 protein fractions nucleus, cytosolic and membranous lysates were separated by SDS/PAGE and transferred to a PVDF Membrane (Roche). The membrane was blocked with 5% wt/vol milk powder and a rabbit anti-human EGFR antibody (Abcam, ab2430, 0.4 μg/mL) was applied. After washing, the membrane was incubated with either HPR-conjugated anti-rabbit secondary antibody (Abcam, ab97051, 0.05 μg/mL) or HRP-conjugated β-Actin antibody (Abcam, ab49900, 0.06 μg/mL). Detection of specific EGFR or β-actin bands was carried out by enhanced chemiluminescence (GE Healthcare).
The combinatorial sunflower trypsin inhibitor 1-based phage display (SFTI8Ph) library based on the sunflower trypsin inhibitor (SFTI) 1 scaffold structure was constructed by PCR. To this end, 8 amino acids except cysteine were randomly inserted between Thr4 and Cysll in the binding loop of SFTI-1. The following oligonucleotides were used as templates for the PCR reaction: 5′-TTACTCGCTCCATGGGCGGCAGGTGTACTNNN NNN NNN NNN NNN NNN NNN NNN TGTTATCCCGAT-3′ (SFTI8Ph forward; NNN=timer encoding for one variable amino acid; Ella Biotech) (SEQ ID NO: 12) and 5′ -ATAATCTTGCGGCCGCACCGCCACCTGCTGCA TCGGGATAACA-3′ (SFTIPh reverse; Eurofins MWG Operon) (SEQ ID NO: 13). With regard to the 125I-labeling of peptides the triplet TTT was exchanged by TAT to achieve a substitution of phenylalanine12 to tyrosine. The PCR product was cloned in frame into the surface expression phagemid vector pSEX81 (Progen) and single clones were sequenced (GATC Biotech) to control the diversity of the library. For the preparation of phages presenting the peptides (theoretical diversity of 109) fused to the pIII coat protein on their surface the phagemid vector was transformed in XL1-Blue bacteria. After incubation of the bacteria and amplification of the phagemid in LB medium (20% Glucose, Ampicillin (100 μg/mL)) for 1 hour at 37° C. 100 μL M13KO7 helper phage (Invitrogen; 18311-019) was added to the suspension for packaging of the phagmid overnight in LB medium (Kanamycin (50 μg/mL)). The suspension was centrifuged for 20 min at 4° C. and 5000 rpm and the supernatant was added to ice-cold polyethylene-glycol solution for 30 min on ice for precipitation of the phages. After centrifugation for 15 min, at 4° C. and 13000 rpm the phages were resuspended in 1 mL PBS and stored at 4° C.
For the selection of HNSCC-specific peptides an alternating biopanning using the SFTI8Ph library (see above) against HNO97 cells and the corresponding protein fraction was performed. Initially, 1×109 phages were incubated for 1 hour with HNO97 grown to 90% confluence. Unbound phages were removed by washing steps and the cells were lysed with 1% Triton X-100 solution. Phages isolated from cell lysate were amplified and packaged in XL1 blue bacteria overnight and precipitated in polyethylenglycole solution. Subsequently, the phages were exposed alternatingly to HNO97 cells and HNO97 protein fractions (100 nM) in 96-well plates for 1 hour. After 5 PBS washing steps phages were eluted in 100 μL Glycin/HCl (pH 2.2) per well, neutralized by 15 μL Tris-HCl (pH 9.1) and amplified in XL1 blue bacteria. For titration, the phages were diluted (10-2, 10-4, 10-6) and grown on agar plates. Twelve selection rounds were performed followed by single-stranded DNA isolation of 16 clones (QIApreo Spin M13 Kit; Qiagen). DNA sequencing (GATC Biotech) allowed for the identification of the corresponding peptides which were synthesized using standard Fmoc/tBu chemistry (see below).
The peptides and their modifications were obtained by solid-phase peptide synthesis using standard Fmoc/tBu chemistry on an Applied Biosystems ABI 433A synthesizer. Cleavage of protection groups and resin was performed with 2.5% water and 2.5% triisopropylsilane in TFA for 1 hour. Deprotected peptides were precipitated in cold diethyl ether. For disulfide bridge formation peptides were dissolved in 80% acetic acid in water with a concentration of 1 mg/mL. Subsequently, the solution was poured into the same volume of a 0.25 mg/ml solution of iodine in acetic acid. The reaction was quenched after 5 min with a hundredth volume of a 20% aqueous ascorbic acid solution. Solvents were evaporated and the residuals dissolved in 50% acetonitrile in water supplemented with 0.1% TFA. Purification was achieved by semi preparative reversed-phase high-performance liquid chromatography (RP-HPLC) on a Waters XBridgeBEH130 PREP C18 column (5 μm, 19×150 mm). Analyses were performed on an Agilent 1100 HPLC system using a Chromolith Performance RP-C18e column (100×3 mm). As eluents, 0.1% TFA in water (eluent A) and 0.1% TFA in acetonitrile (eluent B) were used. Conditions: linear gradient from 0% to 100% B within 5 min with a flow rate of 2 mL/min; UV absorbance λ=214 nm. The identity of the peptides was verified by HPLC-MS analysis on an Agilent 1200 HPLC system equipped with a Waters Hypersil Gold™ aq (200×2.1 mm) coupled to an Exactive mass spectrometer (Thermo Fisher Scientific).
Radiolabeling with 125I and 131was conducted at the tyrosine moiety of the peptides. A 1 mM stock solution of the respective peptide in water was prepared. Labeling was performed by the chloramine-T method (35). Radiolabeled peptides were purified by analytical HPLC using a Chromolith Performance RP-18e-column, 100×4.6 mm. DOTA was coupled on solid phase via its mono-p-Nitrophenyol-ester in NMP and 4 equivalents DIPEA. Cleavage of protection groups and resin was performed with 2.5% water and 2.5% triisopropylsilane in TFA for 1.5 hour.
68Ga was eluted with 0.6 M aqueous HCl from a 68Ge/68Ga generator (IDB Holland). 5 μL of a 1 mM solution of the respective peptide in 300 μL aqueous 2.5 M sodium acetate, 10 μL saturated ascorbic acid, and 1 ml of 68Ga-generator eluate was heated at 95° C. for 15 min at pH 3.6. Purification was performed with a Chromabond C18 ec SPE cartridge (Macherey -Nagel). The reaction mixture was loaded onto the cartridge and free activity separated by washing the cartridge with 0.9% NaCl. Radiolabeled peptides were eluted with 70% aqueous ethanol. Solvents were evaporated and residual peptides dissolved in appropriate buffer.
50 MBq 177Lu chloride (ITG Isotope Technologies Garching) in 100 μL 0.4 M sodium acetate buffer pH 5 was added to 1 μL of a 1 mM peptide solution. The reaction mixture was heated at 90° C. for 15 min and purified with a SPE cartridge as described above.
For binding studies, 2.5×105 to 4×105 cells were seeded in 6-well plates and cultivated for 48 hours. Cells were incubated for 60 min with 1 mL serum-free medium containing the 125I-labeled peptide (see above). For competition experiments, cells were simultaneously exposed to unlabeled (10-4 M to 10-10 M) and 125I-labeled peptides. After three washing steps with PBS (pH 7.4) the cells were lysed with 0.5 mL of 0.3 M NaOH. Experiments concerning the internalization, kinetics and efflux were additionally performed with 177Lu-DOTA-SFITGv6. To evaluate kinetics and efflux HNO97 cells were incubated for different time intervals (10 min to 480 min) and 60 min, respectively, with 177Lu-DOTA-SFITGv6 and lysed as described. To continue with the efflux experiment radioactive medium was replaced by non-radioactive medium and cells were incubated again for 60 to 240 min before measuring radioactivity of the cell lysates and the medium. For internalization experiments the cells were exposed to 177Lu-DOTA-SFITGv6 for different time intervals (10 to 240 min) at 37° C. and 4° C. After three washing steps with PBS the cells were incubated with 1 mL of glycine-HCl 50 mmol/L in PBS (pH 2.8) for 10 min at RT to remove the surface bound activity. Then, cells were washed with 3 mL of ice-cold PBS and lysed as described. Radioactivity was determined in a gamma counter and calculated as percentage of the applied dose per 1×106 cells. Experiments were performed three times, and three repetitions per independent experiment were acquired.
Assessment of the binding affinity was performed on a BiaCore X100 (GE Healthcare). To avoid denaturation of the heterodimer during amide activation ITGαvβ6 and ITGαvβ3, respectively, were used as analytes and SFITGv6 was immobilized at its N-terminus on a C1 sensor Chip (BR-1005-35, GE Healthcare) using a manual amine coupling protocol. Briefly, after activation of the sensor chip surface with a solution of EDC 0.4 M/NHS 0.1 M for 5 min a 100 μM peptide dissolved in HBS-EP running buffer (0.1 M HEPES, 30 mM EDTA, 1.5 M NaCl, 0.5% surfactant P20, pH 7.2) was immobilized with a contact time of 30 sec until a loading level of 18 response units (RU) was obtained. After saturation of the sensor chip surface for 5 min with 1 M ethanolamine/HCl solution (pH 8.4) the binding affinity of the analyte dissolved in HBS-EP running buffer in appropriate concentrations to the ligand was measured (flow rate: 30 μL/min). The SPR-sensogram data were evaluated with the BiaCore evaluation software. The dissociation constant (KD) was determined by a 1:1 Langmuir model fit of the SPR-sensograms.
Five MBq of the purified 131I-radiolabeled peptide was incubated in 300 μL human serum at 37° C. After different time intervals (15 min to 24 hours) 20 μL serum was precipitated with 40 μL acetonitrile. The stability of the labeled peptide in the supernatant was monitored by radio-HPLC at selected time points using a chromolith performance RP18ec column (3 mm×100 mm) equipped with a gamma detector (Packard COBRA™ Auto-Gamma, GMI). Separation condition was a gradient of 0% to 60% aqueous acetonitrile supplemented with 0.1% TFA over 10 min with a flow rate of 2 mL/min.
All experiments were conducted in compliance with the German animal protection laws. Eight week old Balb c/c nude mice (Charles River Laboratories) were inoculated subcutaneously at the right shoulder with 5×106 HNO97 cells in BD MatriGel™ (BD Bioscience). Xenografts were grown to a tumor diameter of 10-15 mm. For small animal-PET imaging mice were anesthetized using isoflurane inhalation and injected via tail-vein with 50 MBq (2 nmol) of the 68Ga-labeled DOTA-SFITGv6 peptide (see above) solution in 100 μL PBS. Images were recorded on an Inveon small-animal PET scanner (Siemens) using a 60 min emission scan in list mode and a 10 min transmission scan. Images were taken in 3-dimensional (3D) mode and reconstructed iteratively with a fully 3D algorithm from a 256×256 matrix for viewing transaxial, coronal, and sagittal slices of 0.9 mm thickness. Pixel size was 0.38×0.38×0.79 mm3 and transaxial resolution obtained was 0.9 mm. For blocking experiments 100 μL of a 1 mM aqueous solution of SFITGv6 was pre-administered intraperitoneally 30 min before injection of the radiolabeled peptide. Biodistribution studies were performed after administration of 100 μL of a 20 nM 177Lu-DOTA-SFITGv6 solution (1 MBq) as an intravenous bolus injection into the tail vein of the mice. After different time points (30 min to 6 hours) three animals, respectively, were sacrificed. Peripheral blood, heart, lung, spleen, liver, kidneys, muscle, brain, intestine, and injection site (tail, after intravenous injection only) were collected and weighted. Tissue-associated radioactivity was measured in a gamma counter (Berthold LB951G) and expressed as percentage of the injected dose per gram tissue (% ID/g).
Staining with the biotinylated PEG(12)-SFITGv6 peptide was performed on acetone-fixed cryosections (5 μm) of tumor tissues after blocking of unspecific binding using the Avidin/Biotin Blocking Kit (SP-2001, Vector Laboratories). A stock solution of the lyophilized peptide was prepared by dilution in 5% aqueous DMSO. Slices were incubated overnight at 4° C. with 10-5 M peptide concentration in antibody diluent (DAKO). Detection of bound peptide was carried out with the Vectastain Elite ABC Kit (PK-6100, Vector Laboratories) according to manufacturer's protocol. Peptide specificity was ensured by a scrambled (GRD) PEG(12)-SFITGv6 derivate, the somatostatin receptor ligand DOTATOC and negative controls (without peptide or primary antibody) Staining results were assessed by bright field microscopy (BX50) with the SC30 camera and the cell Sense software (all Olympus).
The PET/CT scan was performed 1 and 3 hours post tracer administration with a Biograph mCT Flow™ PET/CT-Scanner (Siemens Medical Solution) using the following parameters: slice thickness of 5 mm, increment of 3-4 mm, soft-tissue reconstruction kernel, care dose Immediately after CT scanning, a whole-body PET was acquired in 3D (matrix 200×200) in FlowMotion™ with 0.7 cm/min. The emission data were corrected for random, scatter and decay. Reconstruction was conducted with an ordered subset expectation maximisation (OSEM) algorithm with 2 iterations/21 subsets and Gauss-filtered to a transaxial resolution of 5 mm at full-width half-maximum (FWHM). Attenuation correction was performed using the low-dose non-enhanced CT data. The quantitative assessment of standardized uptake values (SUV) was done using a region of interest technique.
The tumor cell lines were analyzed for ITGAVB6 expression by flow cytometry (LSR II, BD Biosciences) using a primary rat monoclonal antibody against human ITGαvβ6 (Abcam, ab97588, 3 μg/mL, 1 hour, 4° C.) followed by incubation with the respective secondary antibody (Alexa Fluor 488 Abcam, ab150153, 1 μg/mL, 30 min, 37° C.). Antibody specificity was ensured by isotype-matched control. Data were analyzed with FlowJo software (TreeStar). Each experiment was repeated at least three times.
Immunohistochemical staining was performed on acetone-fixed serial cryosections (5 μm). Primary mouse anti-human ITGαvβ6 antibody (LSBio, LS-C24779, 0.1 mg/mL) was incubated for 1 hour at RT followed by an incubation with a biotin-conjugated anti-mouse secondary antibody (PK-6102, Vectastain) for 30 min at RT. Detection of binding of the secondary antibody was carried out with the Vectastain Elite ABC Kit (PK-6100, Vector Laboratories) according to manufacturer's protocol.
Employing the SFTI8Ph library for alternate selection rounds on the HNSCC cell line HNO97 and respective PF2D membrane protein fractions 7 out of 16 sequenced phages displaying the peptide sequence FRGDKMQL (SFPF-10) (SEQ ID NO: 15) were selected for further analysis. These 7 out of 16 sequenced clones showed the same motif (FRGDKMQL) (Table 1).
The sequence comprises a RGD and KXXL motif indicating ITGαvβ6-specificity of the peptide. ITGαvβ6-expression could be confirmed by flow cytometry analysis on several squamous cell carcinoma cell lines, including HNSCC (e.g. HNO97; up to 99.8%), bladder cancer (UM-UC-5; up to 88.7%), lung cancer (LUDLU-1; up to 90.9%), and breast cancer (MCF-2; up to 36.1%) (
In order to specify the amino acids contributing to the target-specific binding mutations were introduced either into the RGD motif or the adjacent KXXL sequence. As shown in
Compared to the originally identified peptide 125I-SFITGv6 displayed higher binding to the HNSCC cell lines HNO97 (24.5%), HNO210 (7.1%), HNO199 (6.6%) and HNO258 (3.5%) and to UM-UC-5 cells (10.4%). In addition, binding of 125I-SFITGv6 to the carcinoma-derived cell lines LUDLU-1 (4.3%) and to the adenocarcinoma cell line HT29 (2.8%) was measured (
Since time-dependent deionization of 1251 was expected experiments concerning the kinetics, internalization and efflux of SFITGv6 were additionally performed with the 177Lu-DOTA-labeled SFITGv6. In fact, binding of the 177Lu-DOTA-SFITGv6 to HNO97 cells continuously increased to 57.3% within 480 min, whereas the maximal uptake of 125I-SFITGv6 (37.3%) was measured after exposure for 60 min followed by a decrease to 14.5% (
The specificity of SFPF-10L for ITGαvβ6-expressing HNO97 cells was verified by incubation with the 125I-labeled peptide in presence of increasing concentrations of unlabeled peptide as competitor (10−10 M-10−4 M) and an inhibitory concentration of 50% (IC50) of 18 nM (
ITGαvβ6-specificity of SFITGv6 was further demonstrated by competition of SFITGv6 binding by already known ITGαvβ6-binding molecules TP H2009.1 (Elayadi et al., Cancer Research, 2007, 67, 5889-95), A2OFMDV2 (Saha et al., J Pathol, 2010, 222, 52-63) and HBP-1 (Nothelfer et al., J of Nucl Med, 2009, 50, 426-34) (
For the liposarcoma cell line SW872 and the breast carcinoma cell lines MCF-7 and T47D less than 1.5% of the peptide could be detected (
Small-animal PET imaging of Balb/c mice bearing ITGαvβ6-expressing HNO97 xenografts is shown in
To expand on the biodistribution of SFITGv6 the 177Lu-DOTA-linked peptide was injected intravenously into HNO97 tumor-bearing mice. Radioactivity in individual organs was measured after different time points following injection of the peptide and calculated as % injected dose (ID)/g (
In a next step, tumor cell affinity and intratumoral distribution of SFITGv6 was further assessed by histochemical peptide staining of different carcinomas (HNSCC, NSCLC, breast cancer) using biotin-labeled SFITGv6 (
PET/CT scans in a compassionate use setting were performed in two tumor patients after application of 68Ga-DOTA-SFITGv6 and 18F-FDG, respectively (
18F-FDG and the 68Ga-DOTA-SFITGv6 for one HNSCC patient
18F-FDG
68Ga-DOTA-SFITGv6
18F-FDG
68Ga-DOTA-SFITGv6
18F-FDG
68Ga-DOTA-SFITGv6
18F-FDG
68Ga-DOTA-SFITGv6
18F-FDG
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
18F-FDG = 314 MBq,
68Ga-DOTA-SFITGv6 = 321 MBq,
18F-FDG and the 68Ga-DOTA-SFITGv6 for one NSCLC patient
18F-FDG
68Ga-DOTA-SFITGv6
18F-FDG
68Ga-DOTA-SFITGv6
18F-FDG
58Ga-DOTA-SFITGv6
18F-FDG
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
58Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
68Ga-DOTA-SFITGv6
18F-FDG = 351 MBq,
58Ga-DOTA-SFITGv6 = 298 MBq,
HNSCC tumor tissues obtained intraoperatively were snap-frozen and stored at −80° C. or directly used to establish primary tumor cell cultures (HNO97, HNO399, HNO223, HNO210 and HNO199) as described in Example 1. Uniqueness of the established cell cultures was assessed by the German Collection of Microorganisms and Cell Culture (DMSZ). HNSCC cell lines were cultured in in RPMI 1640 medium (Gibco) supplemented with 10% FCS. All cell lines were negative for Mycoplasma and maintained at 37° C. and 5% CO2. Written informed consent was obtained from all patients according to the research proposals approved by the Institutional Review Board at the Medical Faculty of the University of Heidelberg.
For the assessment of the peptide binding capacity in vitro, 2.5-4×105 cells were seeded into 6-well plates and cultivated for 48 h. 125I-labeled peptide resuspended in 1 mL serum-free medium was added and incubated for either 10 min or 60 min with or without different concentrations of unlabeled peptide (10−4-10−11 M). Afterwards, cell monolayers were washed three times with 1 mL phosphate-buffered saline (pH 7.4) to remove unbound peptide and the cells were harvested with 1.4 mL lysis buffer (0.3 M NaOH). To evaluate kinetic properties the cells were exposed to 177Lu-DOTA-labeled peptides for 10, 30, 60, 120, and 240 min, respectively, and lysed as described. For the efflux experiments the cells were incubated with 177Lu-DOTA-labeled peptides for 60 min. Thereafter, radioactive medium was replaced by non-radioactive medium and cell monolayers were incubated for additional 60, 120, and 240 min and afterwards lysed as described earlier. The internalization was assessed after exposure of the cells to 177Lu-DOTA-labeled peptides for 10, 30, 60, 120, and 240 min at 37° C. Unspecifically bound peptide was removed from the surface by incubation with 1 M glycine-HCl solution (pH 2.2) for 10 min. The monolayer cell was rinsed with PBS and cells were lysed as described before. Radioactivity was determined in a y-counter and calculated as percentage of the applied dose per 1×106 cells. Experiments were performed three times, and three repetitions per independent experiment were acquired.
Assessment of the binding affinity was performed on a BiaCore X100 (GE Healthcare). To avoid denaturation of the heterodimer during amide activation ITGαvβ6 and ITGαvβ3, respectively, were used as analytes and SFITGv6 was immobilized at its N-terminus on a C1 sensor Chip (BR-1005-35, GE Healthcare) using a manual amine coupling protocol. Briefly, after activation of the sensor chip surface with a solution of EDC 0.4 M/NHS 0.1 M for 5 min a 100 μM SFLAP3 dissolved in HBS-EP running buffer (0.1 M HEPES, 30 mM EDTA, 1.5 M NaCl, 0.5% surfactant P20, pH 7.2) was immobilized with a contact time of 30 sec to obtain a loading level of 12 response units (RU). After saturation of the sensor chip surface for 5 min with 1 M ethanolamine/HCl solution (pH 8.4) the binding affinity of the ITGαvβ6 and ITGαvβ3, respectively, dissolved in HBS-EP running buffer in appropriate concentrations (1-50 μg/mL) to the ligand was measured (flow rate: 30 μL/min). The SPR sensogram data were evaluated with the BiaCore evaluation software. The dissociation constant (KD) was determined by a 1:1 Langmuir model fit of the SPR sensograms.
All animal experiments were conducted in compliance with the German animal protection laws. For in vivo small animal PET imaging and organ distribution studies eight-week old Balb/c nude mice (Charles River Laboratories) were inoculated subcutaneously at the right shoulder with a total of 5×106 tumor cells in MatriGel (BD Bioscience). Xenografts were grown until a tumor diameter of 10-15 mm was reached.
For imaging, tumor-bearing mice were anesthetized using isofluorane inhalation and injected into the tail-vein with a 100 μl PBS solution containing 68Ga-DOTA-SFLAP3 (HNO97: 37.9 MBq; HNO399: 26 MBq; HNO233: 27 MBq) and 68Ga-DOTA-SFITGv6 (HNO97: 30 MBq; HNO399: 34 MBq; HNO 223: 34 MBq). 3-dimensional (3D) PET images of whole mice were captured by the Siemens Inveon PET scanner as described in Example 1. To assess the biodistribution 100 μL of a 20 nM solution (1 MBq) of 177Lu-DOTA-SFLAP3 and 177Lu-DOTA-SFITGv6 were administered as an intravenous bolus injection into the tail vein of mice. For each time point (30 min to 6 hours) we sacrificed a total of three animals, and subsequently collected peripheral blood, heart, lung, spleen, liver, kidneys, muscle, brain, and intestine. Tissues were weighed and respective radioactivity was measured by a gamma counter (LB951G, Berthold Technologies). Values were expressed as percentage of the injected dose radioactivity (MBq) per gram tissue (% ID/g).
Immunohistochemical stainings were performed by use of the biotinylated PEG(12)-labled peptides (SFITGv6, SFLAP3) on 5 μm acetone-fixed tumor tissue cryosections. To prevent background staining we initially applied the Avidin/Biotin Blocking Kit (SP-2001, Vector Laboratories). The lyophilized peptides were diluted in 5% aqueous DMSO preparing a 1 mM Stock solution. The HNSCC cryosections were incubated with 10−5 M peptide concentration in antibody diluent (DAKO) at 4° C. overnight. To detect bound peptide the Vectastain Elite ABC Kit (PK-6100, Vector Laboratories) was applied according to manufacturer's protocol. Peptide binding specificity was ensured by use of a peptide derivate (scrambled control) and negative controls (without peptide). Due to instability of the peptide binding staining results were assessed on the same day by bright field microscopy (BX50) with the SC30 camera and the cell Sense software (all Olympus).
A non-contrast-enhanced PET/CT scan was performed 60 and 180 min after intravenous injection of the 68Ga-DOTA-labeled peptide using a SIEMENS-BIOGRAPH mCt Flow™ PET/CT-Scanner (Siemens Medical Solution) and the following parameters: slice thickness of 5 mm, increment of 3-4 mm, soft-tissue reconstruction kernel, Care dose Immediately after CT scanning, a whole body PET was acquired in 3-D (matrix 200×200) in FlowMotion with 0.7 cm/min. The emission data were corrected for randoms, scatter and decay. Reconstruction was conducted with an ordered subset expectation maximisation (OSEM) algorithm with 2 iterations/21 subsets and Gauss-filtered to a transaxial resolution of 5 mm at full-width half-maximum (FWHM). Attenuation correction was performed using the low-dose non-enhanced CT data. The quantitative assessment of tracer accumulation was done using a region of interest technique and using standardized uptake values (SUV).
The RGD motif-containing octamers of the natural ITGαvβ6-ligands FN1, TNC, VTN, LAP1 and LAP3 were grafted between Thr4 and Cys11 into the binding loop of the SFTI scaffold (Table 5). First, the binding properties of the 125I-labeled SFTI derivates SFFN1, SFTNC, SFVTN, SFLAP1 SFLAP3, and SFITGv6 were compared as described in Example 1 using five primary HNSCC cell lines that differ in their ITGαvβ6-expression as assessed by FACS analysis (
Next, competition experiments were performed to assess the specificity of the peptide binding. In all tested HNSCC cell lines, binding of both peptides 125I-SFLAP3 and 125I-SFITGv6 could be almost completely competed by adding 10−6 M non-radioactively labeled counterparts as competitor (FIG. 14B, C). However, SFLAP3 exhibited a higher affinity (mean IC50=3.5 nM) when compared to SFITGv6 (mean IC50=14.11 nM). Concordantly, the SPR spectroscopy analysis revealed a higher affinity of SFLAP3 for ITGαvβ6 (KD=7.4±0.9 nM) (
Considering time-dependent deionization of iodine 125 the determination of kinetic, internalization, and efflux were performed using 177Lu-DOTA-labeled peptides. The kinetic experiment revealed a continuous increase in peptide uptake over the whole time period of measurement for both 177Lu-DOTA-labeled peptides. Admittedly, 177Lu-DOTA-SFLAP3 (
To compare the targeting properties of SFLAP3 and SFITGv6 in vivo 68Ga-DOTA-labeled peptides were administered to HNO97-, HNO399-, or HNO223-xenografted mice and visualized by small animal PET imaging. A fast and continuous accumulation in HNO97 tumor lesions 60 min post injection was observed for both 68Ga-DOTA-SFLAP3 [SUV mean=0.63] (
In analogy to the previous experiments, we compared the tumor cell affinity of SFLAP3 and SFITGv6 in sections of HNO97 xenograft tumors and patient-derived HNO399 and HNO210 tumors by peptide histochemical staining using biotin-labeled SFLAP3 and SFITGv6. Both peptides revealed binding to the epithelial tumor cells of the tissue sections, whereas rather low binding was observed in the stromal compartment, indicating tumor cell specificity (
Finally, we investigated the therapeutic and/or diagnostic suitability of SFLAP3 exemplarily in one 79 year old, male carcinoma patient presented with lymph node swelling in the right cervical area assuming the diagnosis cancer of unknown primary. In order to clarify the diagnosis 322 MBq 68Ga-DOTA-labled SFLAP3 was administered followed by a PET/CT scan (
Peptides were synthesized using standard Fmoc/tBu solid phase peptide synthesis (SPPS) and an Applied Biosystem ABI 433A Synthesizer. Rink amid aminomethyl polystyrene resin was used as solid phase. Amino acids were thereby applied in 5 fold access. To obtain DOTA conjugated peptides, 1.2 eq. tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate was coupled to the N-terminus of the side protected peptide on solid resin. Further details as described in Example 1.
For the final oxime ligation of the trimer, the core peptide and the aminooxy-modified SFLAP3 was heated over night at pH 2-3.
For in vitro binding Studies, 4*105-6*105 cells were seeded in 6 well plates and cultivated for 24 to 48 hours. At the start of the experiment the medium was replaced with 1 ml serum free medium with a definite amount of radioactive labeled peptide per well. For normal binding assays the cells were incubated for 60 min at 37° C. and afterwards washed 3 times with PBS (pH 7.4). Cells were lysed with 2 times 0.5 ml of a 0.3 M NaOH +0.2% SDS lysis-buffer. Competition assays were performed with 125I-labeled and unlabelled (10−6 to 10−11M) peptide and incubated at the same time for 60 min.
Cells were incubated for 10 to 240 min with 177Lu-labelled peptide in internalization assays. After the washing steps, the cells were incubated with 1 ml glycine-HCl (1 M, pH 2.2) buffer for 10 min to remove surface bound peptide. Prior to the lyses the cells were washed again. Values are given as percentage of applied dose per 106 cells.
The efflux was tested with 177Lu-labeled peptides. After 60 min incubation the radioactive medium was removed and 1 ml fresh serum-free medium was added. After additional incubation of 60, 120 or 240 min, cell bound activity was measured.
All experiments were conducted in compliance with the German animal protection laws. 6 to 8 week old Balb c/c nude mice (Charles River Lab.) were inoculated with 5*106 Capan-2 cells in 100 μl Opti-MEM medium. After 4 to 6 weeks the tumor has grown 10-15 mm in diameter. For small animal PET imaging 25 MBq of the 68Ga-labeled peptide (2 nmol) was injected into the tail vein. Images were recorded on an Inveon small-animal PET scanner (Siemens) as described in Example 1.
For biodistribution assays 1 MBq of 177Lu-labeled peptide was injected into the tail vein of each mouse. After different time points each animal was sacrificed. Peripheral blood, spleen, liver, intestine, kidney, heart, lung, brain, muscle, and tumor were removed and weighted. Radioactivity was measured in a gamma counter and expressed as injected dose per gram (% ID/g).
PET/CT scans of tumor patients and body scintigraphy were performed as described in Example 1.
SFLAP3 is a integrin αVβ6 binding ligand in which the LAP3 binding sequence GRGDLGRL is embedded in the sunflower trypsin inhibitor-1 (SFTI) scaffold. The cysteins in the flanking amino acids GRCT and CYPD of the SFTI scaffold form disulfide bridges leading to a highly stable peptide (
Since the binding capacity of the peptide to cancer cells depends on the number of receptor molecules expressed at their surface, different cell lines were analyzed with regard to their possible use in further experiments. Uptake assays on ASPC-1, BXPC-3, Capan-1, Capan-2 and Mia PaCa-2 cells showed that Capan-2 cells had the highest 125I-SFLAP3 binding value with 14% (
Different variations of the SFLAP3 binding sequence GRGDLGRL were tested. In these variations the surrounding amino acids of the native LAP3 protein were added or removed and transferred into the SFTI scaffold (
Since multimerization may lead to additive binding effects, a trimerized version of the SFLAP3 was produced using a DOTA-(GSGSK)3 linker. The 177Lu-DOTA-SFLAP3-trimer (Formula (XXVII)), 177Lu-DOTA-SFLAP3K (SEQ ID NO: 34) and 177Lu-DOTA-SFLAP3 (SEQ ID NO: 28) were compared regarding the cell uptake on Capan-2 cells. The addition of lysine (177Lu-DOTA-SFLAP3K) did not result in an increased cellular uptake compared to the lysine-free version. This was in contrast to the DOTA free uptake results. The trimerized peptide nearly tripled the value (
Competition assays showed high affinities in the lower nanomolar level. Thereby only slight differences were detectable with IC50 values of 6,2 nM for 125I-DOTA-SFLAP3 and 2,1 nM for 1251-DOTA-SFLAP3K. 125I-DOTA-SFLAP3-trimer instead showed a high improvement in the affinity with an IC50 of 0.76 nM (
With all three peptides internalization rates between 25-50% were measured (
The efflux experiments showed that 50% of the 177Lu-DOTA-SFLAP3 and the 177Lu-DOTA-SFLAP3K-peptides are released into the culture medium. In contrast, the trimerized peptide showed a reduced efflux of around 20% during 4 hours following exposure of the tumor cells to the radiolabelled peptide (
The proteolytic stability of SFLAP3 was shown for up to 24 hours in human serum. Slight degradations of SFLAP3K could be determined by radio-HPLC analysis after 4 hours of incubation. The SFLAP3-trimer degradation even started after 2 hours (data not shown). The optimal biodistribution of peptides in vivo is normally at its highest rate within the first 2 hours. Thus, the peptides should be stable for in vivo assays within the most critical time period.
Capan-2 xenograftet balb/c nu/nu mice were injected with 68Ga-DOTA-SFLAP3 to obtain PET images. 20 min p.i. tracer accumulation was seen in the tumor, which lasted until the end of the experiment (
The accumulation of 68Ga-DOTA-SFLAP3K into the tumor was slightly better compared to the original peptide (
To obtain more precise pharmacokinetic data, Capan-2 bearing mice were injected with 177Lu-DOTA-SFLAP3. At 0.5, 1, 2, 4 and 6 hours p.i. the organs and tumors were removed, weighted and the radioactivity was measured. The results are presented as injected dose/gram (ID/g). After 1 hour the tumor had the highest activity with 4% ID/g (
Based on the preclinical data a patient with metastasized pancreatic cancer at final stage was selected for PET/CT imaging with 68Ga-DOTA-SFLAP3. PET imaging showed multiple metastatic sites throughout the body and especially the liver area with a high accumulation over 3 hours into the tumor lesions (
Thereafter, the patient was treated with 6.5 GBq 177Lu-DOTA-SFLAP3. Scintigraphic images 1 day after tracer administration showed an accumulation in the metastatic sites (
Similar to pancreatic cancer, ovarian cancer is often integrin αVβ6 positive as has been shown by immunohistochemistry studies. Tested cell lines showed low uptake of 125I-SFLAP3 (data not shown). The cell line OV433 and OV429 with the highest binding of 5% 125I-SFLAP3 were not tumorigenic in our mice model. Based on the positive reports of the above mentioned IHC studies PET imaging was done in 4 late stage ovarian cancer patients (2 patients shown). Metastases were clearly visible in the upper part of the upper body (
| Number | Date | Country | Kind |
|---|---|---|---|
| 17158242.2 | Feb 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/054852 | 2/27/2018 | WO | 00 |
