[go: up one dir, main page]

LU92353A1 - Antibody-mediated delivery of RNAI - Google Patents

Antibody-mediated delivery of RNAI Download PDF

Info

Publication number
LU92353A1
LU92353A1 LU92353A LU92353A LU92353A1 LU 92353 A1 LU92353 A1 LU 92353A1 LU 92353 A LU92353 A LU 92353A LU 92353 A LU92353 A LU 92353A LU 92353 A1 LU92353 A1 LU 92353A1
Authority
LU
Luxembourg
Prior art keywords
molecule
sirna
peptide
positively charged
antibody
Prior art date
Application number
LU92353A
Inventor
Sebastian Baeumer
Wolfgang Berdel
Julia Fremerey
Carsten Mueller-Tidow
Original Assignee
Univ Muenster Wilhelms
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Univ Muenster Wilhelms filed Critical Univ Muenster Wilhelms
Priority to LU92353A priority Critical patent/LU92353A1/en
Publication of LU92353A1 publication Critical patent/LU92353A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6883—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/85—Fusion polypeptide containing an RNA binding domain
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Oncology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Description

Figure LU92353A1D00021
New LU-patent application
Westfaelische Wilhelms-Universitaet Muenster
Our ref: UKM14885LU ANTIBODY-MEDIATED DELIVERY OF RNAi
Description
FIELD OF THE PRESENT INVENTION
The present invention relates to a method for producing antibody-siRNA molecules of the present invention as well as to the molecules of the present invention, or molecules obtainable by the method of present invention. In addition, the present invention relates to the molecules of the present invention for use in a method of treatment e.g. cancer as well as pharmaceutical compositions, preparations or kits comprising the molecules of the present invention.
BACKGROUND OF THE PRESENT INVENTION
The number of known driver mutations in cancer has dramatically increased in recent years. Multiple driver mutations co-exist in single tumors with a significant number leading to activation of oncogenes. The main challenge for targeted drug delivery is either to repress oncogenes or to replace the ineffective tumor suppressor gene. According to the identification of several oncogenes like Ras and Myc, tumor suppressor genes like p53, signalling cascades and effectors of pathways that initiate cell growth and suppress apoptosis targeted drug therapies become a prospective possibility in anti-cancer therapy (Johnston et al., 2006).
Unfortunately, the majority of these oncogenes are not targetable by current therapeutic approaches. KRAS mutations are a prominent example: KRAS ranks high among the most frequently mutated oncogenes in human carcinomas (Jemal et al., 2010). Mutations in the codons 12,13 and 61 transform the intrinsic GTPase activity of KRAS to a constitutively active conformation and induce a KRAS gain of function (Rajagopalan et al., 2002). Despite intensive research, none of the tested inhibitors have been clinically successful (Karp et al., 2012). KRAS mutations are often also associated with drug resistance. Especially for solid tumors, which contain a large number of genetic mutations, the targeting of single driver mutations might not be sufficient to induce long lasting remission or eventually to cure. Given the multitude and variety of genomic aberrations in cancer it appears that several drivers need to be targeted simultaneously for effective therapy.
Small interfering RNA (siRNA) constitutes a class of molecules that allows effective and specific targeting of oncogenic proteins. Indeed, siRNA has emerged as a major tool in molecular biology techniques and is an important tool to identify suitable therapy targets in cancer (Fire et al., 1998). However, siRNA therapy approaches are scarce. One of the most urgent problems that hinder siRNA development as therapeutic tools concerns the delivery.
To deliver siRNA into cells, the siRNA has to gain access to the cytoplasm where the siRNA can enter the RNAi pathway; negatively charged siRNA has to cross the phospholipid bilayer of the cell membrane which is negatively charged as well. The entrance to the intracellular environment of the cell is restricted by size and the strong negative charge of the nucleic acids (Meade and Dowdy 2007; Peer and Lieberman 2011). The average molecular weight (MW) of a siRNA is ~13-kDa. Naked siRNA is not stable and would be degraded immediately by endogenous enzymes (Peer and Lieberman 2011). Half-life of a naked or unmodified siRNA in the cytosol is less than 10 minutes (Wang et al., 2010).
To overcome this barrier, most tissues of the body need a carrier system for siRNA delivery. Several possibilities of siRNA delivery in vitro have been established. siRNA can be delivered with transfection reagents into the cell. The siRNA is then complexed with a carrier of cationic lipids. The siRNA-cationic lipid complex protects the siRNA from degradation and offers an uptake by the cell. Transfections of siRNA in vivo, in primary as well as in non-dividing cells result in a relatively low efficiency rate. Another method for siRNA delivery is based on electroporation of the cell. Due to a powerful electric pulse the membrane temporarily loses its semipermeability and ensures the uptake of siRNA. This method, however, results in relatively high cell death and requires parameter optimization for every cell type. The delivery of siRNA can also be maintained by viral gene transfer with several recombinant viral vectors based on a retrovirus like adeno-associated virus or lentivirus. Some of the viral vectors are able to integrate the siRNA into the genome and provide a stable RNA expression and a long term gene knockdown. Possible mutagenic and immunogenic effects can arise from viral gene transfer (Lee et al., 2010). Due to severe side effects which may occur according to these transfection mechanisms, none of the above mentioned methods provides a sufficient delivery of siRNA in vivo. Delivery of a potent drug should be cell specific and effective. Neither of which is currently achievable in clinical situations for siRNA. Methods of siRNA delivery include nanoparticles and cationic liposomes (Christie et al., 2012), cationic peptides such as protamin (Hauser et al. 2010; Choi et al. 2009) or poly-arginin (Kumar et al., 2008). Yet, it still remains a challenge to deliver siRNA directly to the cell in a therapeutically acceptable way (Peer and Lieberman 2011; Rossi 2005).
Monoclonal antibodies (mAb) were discovered in the early 1970s and have gained success as therapeutic targets in the last 20 years (Beck et al., 2010). They are used for example in the treatment of osteoporosis, transplant rejection, autoimmune diseases and several malignancies. Today a large number of mAbs in clinical trials are focused on oncogenes. MAb that target specific cell surface antigens in order to inhibit cell signalling could be a promising addition to conventional chemotherapy (Ochsenbein 2008). Additionally, antibodies which can be internalized for example via receptor-mediated endocytosis can be utilized for antibody mediated drug therapy. A promising alternative and a great advantage to conventional chemotherapy would be the combination of antibodies or peptides with the molecular mechanism of RNAi. The antibodies can be used as a carrier to deliver siRNA into the cell. According to this approach the complex has to be internalized and delivered across the cell membrane to the cytoplasm where the specific siRNA can enter the RNAi pathway (Lyer and Kadambi 2011; Van der Velden et al., 2001).
Along this line, Rothdiener et al. established a liposomal carrier system for antibody dependent delivery of anti-leukemia siRNA into CD33-positive myeloid tumor cells. An anti-CD33 single-chain Fv fragment was used to deliver therapeutic siRNA (Rothdiener et al., 2010).
Song and his colleagues delivered siRNA into tumor cells using heavy-chain antibody fragments (Fab) fused to a highly basic cellular protein called protamine (Song et al., 2005).
Similarly, US2012/0258534 describes a fusion protein comprising a cell targeting moiety and protamine.
Also other studies attempted to provide a combination of antibodies or peptides with the molecular mechanism of RNAi:
Gao et al. describes siRNA delivery through an anti-EGFR antibody conjugation by liposomes (Gao et al., 2011).
Kumar et al. describes a sCFvCD7 with an additional Cys residue at its C-terminal end conjugated to a 9R peptide (Kumar et al., 2008).
Hauser et al. describes a monovalent antibody coupled via biotin to protamine (Hauser et al., 2010).
An object of the present was therefore to provide an alternative method for cell specific drug delivery, which should be applicable also in vivo.
The present invention solves that problem by providing a peptide comprising a binding domain capable of binding to a cell surface molecule, which is internalized, coupled via a hetero-or homobifunctional linker to a positively charged molecule.
The method of the present invention is a simple and fast way to generate functioning siRNA-antibody molecules, wherein the antibody can be used as it is, without being dependent on modifications prior to antibody siRNA conjugation such as addition of a Cys residue as shown by Kumar et al or the preparation of a fusion protein (Song et al., 2005; US2012/0258534).
The present invention relates to a method for producing a molecule comprising: i) a peptide comprising a binding domain capable of binding to a cell surface molecule, which is internalized; ii) a nucleic acid molecule; iii) a hetero- or homobifunctional linker; iv) a positively charged molecule wherein the production comprises: a. coupling of the positively charged molecule to the hetero- or homobifunctional linker; b. coupling of at least one thiol, amino or carboxyl group of the peptide with the hetero-or homobifunctional linker coupled to the positively charged molecule as obtained in a; c. non-covalent binding of the nucleic acid molecule to the positively charged molecule.
In one embodiment, the method of the present invention includes a coupling to the heterobifunctional linker in a), which is achieved via a NH2-group of the positively charged molecule.
In another embodiment, the method of the present invention includes a coupling to the homobifunctional linker in a), which is achieved via a thiol group of said positively charged molecule.
In another embodiment, the method of the present invention includes a coupling to the homobifunctional linker in a), which is achieved via a carboxyl group of said positively charged molecule.
In a further embodiment, the coupling in b) of the method of the present invention is a direct reaction.
In another embodiment, the method of the present invention includes hetero- or homobifunctional linker having no cleavable disulfide bond (S-S).
In yet another embodiment, the method of the present invention includes no purification after the coupling in a).
In yet another embodiment, the method of the present invention the peptide comprising the binding domain is a oligopeptide, polypeptide or protein.
In a further embodiment, the method of the present invention provides for a peptide which is an antibody, preferably a single-chain antibody or a Fab-fragment of an antibody. Most preferably the antibody is selected from the group consisting of anti-EGFR antibody, anti-CD44 antibody, anti-EpCAM antibody, anti-CD33 antibody, anti-CD117 antibody. In one embodiment, the the anti-EFR antibody is Cetuximab.
In another embodiment, the method of present invention includes a nucleic acid molecule, which is selected from siRNA or miRNA, preferably the nucleic acid molecule is selected from EZH2 siRNA, KRAS SiRNA, BRAF siRNA, MEK1 siRNA, CDK1 siRNA, CDK4 siRNA, CDK6 siRNA, FLT3 siRNA, MLL siRNA, CSF1R siRNA, hAES siRNA. in one embodiment, the method of the present invention includes KRAS esiRNA.
According to another embodiment, the method of the present invention includes a positively charged molecule that is selected from the group consisting of wherein the positively charged molecule is selected from the group consisting of protamine, Sso7d, histones, poly Lysine, poly Arginine, preferably (Arg)9, avidin, synthetic polypeptides, synthetic cationic polymer, carbon nanotubes modified to comprise a net positive charge.
In one embodiment of the method of the present invention, the positively charged molecule is protamine.
According to another embodiment, the method of the present invention the synthetic polypeptides is polyetheleneimin.
In a further embodiment of the method of the present invention includes that the quantity of free thiol groups of the peptide and/or the positively charged molecule is determined by an adequate assay.
In a further embodiment of the method of the present invention includes that the quantity of free NH2 and/or carboxyl groups of the peptide and/or the positively charged molecule is determined by an adequate assay.
The present invention also relates to a molecule obtainable by the method of the present invention. The present invention further relates to a molecule comprising: i) a peptide comprising a binding domain capable of binding to a cell surface molecule, which is internalized; ii) a positively charged molecule; iii) a heterobifunctional linker, with one portion coupled to the peptide and another portion coupled to the positively charged molecule, wherein at least one thiol, amino or carboxyl group of the peptide is coupled to a portion of the heterobifunctional linker; iv) a nucleic acid molecule; wherein the nucleic acid molecule is non-covalently bound to the positively charged molecule.
The present invention also relates to a molecule comprising: i) a peptide comprising a binding domain capable of binding to a cell surface molecule, which is internalized; ii) a positively charged molecule; iii) a homobifunctional linker, with one portion coupled to the peptide and another portion coupled to the positively charged molecule, wherein at least one thiol, amino or carboxyl group of the peptide is coupled to a portion of the homobifunctional linker; iv) a nucleic acid molecule; wherein the nucleic acid molecule is non-covalently bound to the positively charged moleculemolecule.
In one embodiment, the molecule obtainable by the method of the present invention comprises per lmol peptide lmol positively charged molecule and 2mol nucleic acid molecule.
In one embodiment, the molecule obtainable by the method of the present invention comprises per lmol peptide lmol positively charged molecule and 6mol nucleic acid molecule.
In one embodiment, the molecule obtainable by the method of the present invention comprises per lmol peptide 3mol positively charged molecule and 2mol nucleic acid molecule.
In one embodiment, the molecule obtainable by the method of the present invention comprises per lmol peptide 3mol positively charged molecule and 6mol nucleic acid molecule.
In one embodiment, the molecule obtainable by the method of the present invention comprises per lmol peptide 3mol positively charged molecule and lOmol nucleic acid molecule.
In one embodiment, the molecule obtainable by the method of the present invention comprises per lmol peptide 7mol positively charged molecule and 6mol nucleic acid molecule.
In one embodiment, the molecule obtainable by the method of the present invention comprises per lmol peptide 7mol positively charged molecule and lOmol nucleic acid molecule.
In one embodiment, the molecules of the present invention includes a coupling of the thiol group in iii) resulting in a thioether bond.
In another embodiment, the molecule of the present invention includes a coupling between the portion of the heterobifunctional linker and the positively charged molecule in iii), which is achieved via a NHrgroup of said positively charged molecule, preferably said coupling via the NH2-group results in an amine bond.
In yet another embodiment, the molecule of the present invention includes a coupling between the portion of the homobifunctional linker and the positively charged molecule in iii), which is achieved via a thiol-group of said positively charged molecule, preferably said coupling of the thiol group results in a thioether bond.
The present invention additionally relates to a pharmaceutical composition comprising the molecule of the present invention and/or the inventive molecule obtainable by a method of the present invention.
The present invention additionally relates to a molecule of the present invention or the inventive molecule obtainable by the method of the present invention or the pharmaceutical composition of the present invention for use in a method for treating cancer in a subject, preferably, the cancer is a therapy resistant cancer mediated by mutant KRAS.
Due to the feet that some patients with EGFR overexpressing tumors are resistant to EGFR inhibition it has to be mentioned that EGFR expression itself is not a good indicator of response to EGFR targeted therapy (Bianco et al., 2006). The influence of RAS mutations on Cetuximab resistance has to be taken into consideration if Cetuximab is used as a carrier for siRNA delivery into tumor cells. It is estimated that 30-40% of colon carcinoma patients carry a KRAS mutation. It was shown in several clinical trials that patients with a KRAS mutations are resistant towards Cetuximab and do not respond to the mAb, whereas patients with a KRAS wildtype are sensitive to Cetuximab (Dunn et al., 2011).
In one embodiment, the cancer is a carcinoma, sarcoma, lymphoma or leukemia, germ cell tumors, blastoma or metastasis.
In one embodiment, the molecule of the present invention or the inventive molecule obtainable by a method of the present invention or the pharmaceutical composition of the present invention are used in a method for inhibiting and/or controlling tumor growth in a subject.
The present invention relates to a molecule of the present invention or the inventive molecule obtainable by the method of the present invention the pharmaceutical composition of the present invention, for delivering a nucleic acid molecule to the site of a tumor in a subject, preferably the nucleic acid is a siRNA targeting KRAS and/or EZH2. According to one embodiment of the present invention, the siRNA reduces KRAS expression of a cell. In another embodiment, the siRNA molecule reduces EZH2 expression of a cell. According to another embodiment of the present invention, the cell is present in a subject. According to another embodiment of the present invention the subject is mammal, preferably a human being.
The present invention also relates to a preparation obtainable by a method of the present invention.
The present invention also relates to a preparation comprising the molecule of the present invention and/or the inventive molecule obtainable by a method of the present invention.
The present invention additionally relates to a kit comprising one or more coupling buffer/reagents and protocol suitable for performing the method of the present invention.
The present invention also relates to a kit comprising the molecule of the present invention or the inventive molecule obtainable by the method of the present invention the pharmaceutical composition of the present invention.
The present invention furthermore relates to a use of the molecule of the present invention and/or the inventive molecule obtainable by a method of the present invention or the pharmaceutical composition of the present invention, in the treatment of cancer in a subject.
The present invention relates to a method of treating cancer in a subject, comprising administering a therapeutically effective amount of the molecule of the present invention and/or the inventive molecule obtainable by a method of the present invention or the pharmaceutical composition of the present invention to said subject.
The present invention relates to a use of the molecule of the present invention and/or the inventive molecule obtainable by a method of the present invention or the pharmaceutical composition of the present invention, for the preparation of a medicament.
What the present inventors did was the coupling of a peptide comprising a binding domain to a positively charged peptide. The positively charged peptide, here protaminsulfate, was amino* terminally coupled to the bifunctional crosslinker Sulfo-SMCC in a 1:12 molar ratio in amino-free PBS buffer, (left to react for Ih at RT) and then coupled to cysteine residues of α-EGFR monoclonal antibody (31 μΜ stock) in a 5:1 molar ratio.
Non-reacted educts and protamine doublets were separated from the high molecular weight a-EGFR mAB-protamin product by gel filtration chromatography in Zeba spin desalting columns (Pierce No. 89891). The α-EGFR mAB-protamin adduct was stored at 4eC and was stable for several weeks.
The coupling of siRNA to α-EGFR mAB-protamin was accomplished by sIRNA duplexes against KRAS (KRAS-Mission esiRNA EHU114431, Sigma-Aldrich; SEQ ID NO: 9) that were bound to α-EGFR mAB-protamin in a 10-fold molar excess at 4eC for 3 h. This complex was prepared freshly before use.
For the estimation of siRNA load capacity and serum stability of the complex, constant concentrations (2.5 μΜ) of control siRNA duplexes were pre-incubated with increasing amounts of a-EGFR mAB-protamin up to a 40 fold fold molar excess for 1 h at 4°C, subjected to agarose gel electrophoresis and stained by ethidium bromide. α-EGFR mAB-protamin complexed siRNA proved to be immobile in 2% agarose whereas the unbound 25 bp siRNA duplex band travelled at expected size.
For siRNA stability estimation, control siRNA coupled to α-EGFR mAB-protamin was exposed to filtered Hctll6 cell culture supernatant including FCS for indicated timespans, subjected to 0.4% agarose gel electrophoresis and stained by ethidium bromide. The α-EGFR mAB-protamin/siRNA adduct was detectable as a weakly mobile complex hardly leaving the gel pouch.
DESCRIPTION OF THE FIGURES
Figure 1 A: Neuropilin-1 expression in leukemic cells
The histogram plot shows cell surface levels of Neuropilin-l (NRP-1) in leukemic and human breast cancer (MDA) cells. The highest expression of NRP-1 was observed in Mv4-ll cells, indicated in grey line with border. HL-60 (black line) and Kasumi (light grey line) cell lines showed the same amount of NRP-1 expression. MDA cells indicated in dark grey demonstrated the lowest expression of NRP-1.
Figure IB: Propidium iodide staining of HL-60 cells with different peptide concentration
The diagram shows a propidium iodide (PI) staining of HL-60 cells with different anti-NRP-1 peptide concentration compared to two control peptides. Cells with a concentration of 0.01 μΜ and 0.1 μΜ of the anti-NRP-1 peptide showed less than 6% apoptotic cells, whereas a concentration of 100 μΜ resulted in almost complete cell death. 100 μΜ of both control peptides showed only 13% PI positive HL-60 cells.
Figure 2: Antibody binding of the NRP-1 receptor does not protect cells from peptide-mediated apoptosis
Mv4-ll cells were either incubated with (grey) or without (black) an anti-NRP-1 antibody to block the Neuropilin receptor. The cells were then incubated with 100 μΜ of the anti-NRP-1 peptide. This resulted in complete cell death, regardless if the receptor was blocked by an anti-NRP-1 antibody.
Figure 3: Human breast cancer cells as well as leukemic cells become apoptotic according to 100 μΜ of the anti-NRP-1 peptide
The figure shows a bar diagram of Mv4-ll, U937, HL-60 and MDA cells. The cells were treated with an anti-EGFR control peptide and the anti-NRP-1 peptide with either 100 nM or 100 μΜ. None of the peptides with a concentration of 100 nM induced apoptosis, whereas 100 μΜ of the anti-NRP-1 peptide resulted in nearly complete cell death of more than 90% apoptotic cells in all tested cell lines. 100 μΜ of the control peptide showed slightly increased levels of apoptosis in leukemic cells.
Figure 4: Neuropilin-1 cell surface levels of HL-60 and Kasumi cells with and without different RPARPAR anti-NRP-1 peptides
This figure shows anti-NRP-1 levels of untreated HL-60 and Kasumi cells and cells incubated with either one of the different anti-NRP-1 peptides with a concentration of 1.3 μΜ. HL-60 and Kasumi cells with the 9R-containing peptide (grey) and untreated cells (black) showed the same cell surface level of NRP-1, whereas cells incubated with the C-residue containing peptide (grey with boarder) demonstrated a right shift, indicating a possible cytotoxicity.
Figure 5: Purification and Characterization of the anti-EGFR-single chain antibody (scFv 225). a) Elutionprofile of the denatured 225-scFv purified from E.coli inclusion bodies on a HIS-Tag-specific TALON-Sepharose-Matrix. Bound antibody was washed with urea buffer and with 150 mM Imidazol in the washing puffer eluted in the column. Protein was measured with a photometer at 280 nM (grey), conductivity of the solution in mS (dark grey), fractions (light grey), b) Analysis of the purification with a silver staining of the SDS-PAGE; c) Westernblot of the eluted 225-scFv with HIS-tag antibodies. D) EGFR expressing MDA cells were stained with a polyclonal FITC-conjugated anti-EGFR-antibody (grey) and analyzed by FACS. In the treatment of the cells with lOOnM the scFv ka the epitope was lost from the cell surface, through internalization of the EGFR receptors through binding.
Figure 6: Cell surface levels of EGFR in A549 and HCT-116 cells and antibody-mediated receptor-internalization
The figure shows the cell surface levels of EGFR in A549 and HCT-116 cells with and without Cetuximab. A549 and HCT-116 cells without Cetuximab (black) showed high cell surface levels of EGFR, whereas incubation of the cells with Cetuximab resulted in reduced levels of EGFR. This is indicated by a strong left shift (grey line) in the histogram plot.
Figure 7: Cell surface levels of EGFR in MDA and HL-60 cells with and without Cetuximab (A) and Protamine coupling has no impact on the EGFR internalization activity of Cetuximab (B) A shows cell surface levels of EGFR in MDA and HL-60 cells with and without Cetuximab. MDA cells without Cetuximab (grey) showed high levels of EGFR, whereas an incubation of MDA cells with Cetuximab (black) resulted in reduced levels of EGFR. This is indicated by the grey arrow and the strong left shift in the histogram plot. HL-60 cells, used as a negative control did not express EGFR and showed no effect to Cetuximab. B The histogram shows high levels of EGFR in MDA cells without Cetuximab (black), whereas the levels of EGFR were reduced in MDA cells according to Cetuximab, regardless if Cetuximab was linked to protamine (grey boarder) or not (grey).
Figure 8: The impact of Cetuximab on K-RAS wild type and K-RAS mutated cell lines
This figure shows the result of an Annexin-V staining of MDA, K-RAS wild type and HCT-116, K-RAS mutated cells. 60% of the MDA cells treated with 50 nM of Cetuximab became apoptotic in comparison to untreated MDA cells, whereas Cetuximab showed no apoptotic effect on HCT-116 cells.
Figure 9: Properties of the EGFR-targetlng siRNA carrier system. A. Simplified sketch of coupling of α-EGFR mAB Cetuximab-IgG to protamin by bivalent crosslinker sulfo-SMCC. siRNA binds to protamin by electrostatic interactions. B. Agarose gel electrophoretic analysis of α-EGFR mAB-protamine/siRNA complex stability. siRNA coupled to α-EGFR mAB-protamin is protected from enzymatic degradation during incubation in cell culture medium. C and D. Analysis of siRNA binding capacity. Rising amounts of α-EGFR mAB-protamin (C) or the a-EGFR mAB alone (D) were coupled to constant amounts of siRNA and unbound siRNA was visualized on an agarose gel. α-EGFR mAB-protamin binds up to 8 mol siRNA per mol of protein in contrast to uncoupled α-EGFR mAB (C). Non-modified α-EGFR mAB does not bind siRNA (D).
Figure 10: Anti-EGFR antibody-protamin shuttles Alexa Fluor 488-labeled siRNA into EGFR-expressing cells. A. Flow-cytometrical analysis of EGF receptor exposure on Hctll6 cells before and after incubation with α-EGFR mAB. EGFR surface expression was detected using a FITC labeled α-EGFR-antibody that bound to a different extracellular epitope of EGFR than Cetuximab. HCT116 cells showed high EGFR expression on their surface ("α-EGFR-FITC”, second panel) in comparison to a control antibody ("control AB", first panel). EGFR expression is no longer detectable due to internalization of the receptor after treatment with uncoupled Cetuximab ("Cetuximab + α-EGFR-FITC", third panel) or Cetuximab-protamin ("Cetuximab-protamin + α-EGFR-FITC", fourth panel) as shown by flow cytometry. B. Immunofluorescent analysis of the internalization of α-EGFR mAB-protamin/siRNA/EGFR complexes. MDA, LoVo and Hctll6 cells were incubated for 3 h with ct-EGFR mAB-protamin or a-EGFR mAB alone that were labeled with Alexa Fluor 488-scrambled control siRNA. Successfully internalized complexes are displayed as green fluorescent cytoplasmic vesicles (arrows). DAPI nuclear stain and phase contrast (pc) exposures serve for subcellular localization of Alexa fluor 488-siRNA loaded vesicles (left-hand panels). Uncoupled α-EGFR mAB is not capable to deliver Alexa Fluor 488-labeled siRNA to EGFR expressing cells (right-hand panels).
Figure 11: Anti-EGFR antibody-directed RNAi reduces target gene expression in EGFR-expressing carcinoma cell lines A. Hctll6 cells were treated with 50 nM of α-EGFR mAB-protamin ("mAB-P") coupled to KRAS siRNA or the scrambled control siRNA ("mAB-P/scrm siRNA") for 72 h at 37°, lysed and subjected to Western blots against KRAS and actin. The combination of α-EGFR mAB-protamin with KRAS siRNA reduced the KRAS expression to 60%. Bar graphs on the right refer to densitométrie analysis of KRAS Western blot, respectively. B. Hctll6 and A549 cells were treated with 50 nM of α-EGFR mAB-protamin ("mAB-P") coupled to the indicated siRNAs for 72 h at 37°, lysed and subjected to Western blots against EZH2 and actin. The combination of α-EGFR mAB-protamin with EZH2 siRNA reduced the EZH2 expression more than 90%. Bar graphs on the right refer to densitométrie analysis of EZH2 Western blot.
Figure 12: Overcoming anti-EGFR antibody resistance in KRAS-mutated colorectal cell lines in vitro
Cells were treated with α-EGFR mAB-protamin (mAB-P)/siRNA at 30 nM final concentration, resupended in 96-welis in soft agar and cultivated for donogenic growth. After 7 days, a second treatment was performed. Colonies were fixed, stained with crystal violet, photographed and counted after three weeks. A. Cetuximab-sensitive cell line MDA showed 50% reduction of donogenic growth regardless if treated with α-EGFR mAB-protamin (mAB-P) alone or α-EGFR mAB-protamin coupled to KRAS siRNA. B-D. In contrast, Cetuximab-resistant cell lines LoVo (B), Hctll6 (C) and A549 (D) showed marked reduction of donogenic growth in presence of o-EGFR mAB-protamin (mAB-P)/KRAS siRNA, whereas scrambled-siRNA coupled to α-EGFR mAB-protamin (mAB-P) had no effect on colony numbers. Significance: n.s., not significant, *p<0.003, **p<0.001, of mean values α-EGFR mAB-protamin/KRAS group versus α-EGFR mAB-protamin group, respectively. Graphs show one representative experiment out of four independent experiments. The depicted experiments were performed at least with four wells per group.
Figure 13: Reduction of tumor size in Hctll6 xenografts in CD1 nude mice A. Outline of the anti-EGFR antibody/siRNA treatment regime. B. 2xl06 Cetuximab-resistant Hctll6 cells were subcutaneously implanted in CD1 nude mice. After reaching average tumor size of 200 mm3, mice were separated into three groups (n=6 each) and treated with α-EGFR mAB-protamin (mAB-P), α-EGFR mAB -protamin/KRAS-siRNA, or α-EGFR mAB - protamin/scrambled-siRNA at 4 mg/kg twice a week. Tumors treated with α-EGFR mAB-P/KRAS siRNA complexes showed a markedly reduced growth compared to α-EGFR mAB-P and α-EGFR mAB-P/scrambled siRNA detected by caliper measurements in a blinded fashion. Error bars in Fig 24B represent SEM. C. At the end of the experiment, most mice had to be sacrificed except four mice of the α-EGFR mAB-P/KRAS siRNA group, which were treated and followed for additional five days and showed only minimal tumor growth rates. The graph shows the normalized tumor volume development of the individual mice. D. After euthanization of the α-EGFR mAB-P/KRAS siRNA group (26 days after first treatment, n=3) and control groups (21 days after first treatment, n=3 each), tumors were prepared and weighted. Bar represents 1 cm. E. Average weight of α-EGFR mAB-protamin/KRAS siRNA treated tumors was significantly lower than of the control groups. P values: * 0.04, ** 0.01.
Figure 14: α-EGFR mAB P/KRAS mode of action hypothesis A. Tumor cells with wild type KRAS status are sensitive to α-EGFR mAB treatment. B. The outgrowth of tumor cell clones with KRAS mutations leads to a resistance towards a-EGFR mAB treatment and tumor cell survival due to a gain of function of KRAS. C. Coupling of KRAS siRNA to α-EGFR mAB leads to a tumor-cell specific knockdown of KRAS and subsequently an increased sensitivity to α-EGFR mAB treatment.
Figure 15: α-EGFR mAB-protamin-siRNA coupling ratios and visualized targeting efficiencies on MDA cells
Varying amounts of protamin were coupled to α-EGFR antibody. These carrier complexes were then coupled to different amounts of siRNA. Culture of cells and picture acquisition was carried out as in, microscopic optical fields were counted for total cell number by DAPI stain and number of a-EGFR mAB-protamine/siRNA targeted cells by count of cells harboring green fluorescent vesicles.
Figure 16: Bacterial expression vector: pASK6-225 VLVH scFv
Bacterial secretion sequence- EGFR Binding region of Erbitux (VL> Linker>VH), 6 x His, termina-Cystein.
Figure 17: Antibody-dependent siRNA targeting in vivo
In mice injected intra-peritonealiy (A) or subcutaneously (B) with mAB-568-siRNA, tumors showed Alexa 555 fluorescent signals in the tumor rim. C and D depict parallel sections of A and B stained hematoxylin/Eosin (H/E). In mice injected with uncoupled $iRNA-Alexa555 (D and E), no fluorescent signals were detectable. F and G again show parallel sections of D and E stained H/E.
Figure 18: Treatment of mice bearing tumors with mAB-KRAS esiRNA A reduced expression of the general proliferation antigen Ki67 judged by red AEC (3-amino-9-ethylcarbazole) immunohistochemistry in low power and high power magnifications is shown (top left and right hand panel), whereas this effect was not detectable in tumors from mice treated with PBS control or mAB-control-esiRNA (bottom left and right hand panel).
Figure 19: CD1 mice bearing tumors were treated
Tumors were resected and prepared for quantitative KRAS expression RT-PCR analysis. Tumors treated with mAB-KRAS esiRNA consistently expressed reduced KRAS levels compared to mAB-control esiRNA treated tumors. Simultaneous analysis of human GAPDH expression by real-time RT-PCR was used for standardization.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.
Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or sometimes when used herein with the term "having".
When used herein "consisting of' excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.
In each instance herein any of the terms "comprising", "consisting essentially of and "consisting of' may be replaced with either of the other two terms.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
The present invention relates to a method for producing a molecule comprising: i) a peptide comprising a binding domain capable of binding to a cell surface molecule, which is internalized; ii) a nucleic acid molecule; iii) a hetero- or homobifunctional linker; iv) a positively charged molecule wherein the production comprises: a. coupling of the positively charged molecule to the hetero- or homobifunctional linker; b. coupling of at least one thiol, amino or carboxyl group of the peptide with the hetero- or homobifunctional linker coupled to the positively charged molecule obtained in a; c. non-covalent binding of the nucleic acid molecule to the positively charged molecule.
In this method, the steps a. and b. can be reversed. In such a case first the peptide would be coupled to the hetero- or homobifunctional linker and then this conjugate would be further reacted with the positively charged molecule. In some special cases, e.g. when a heterobifunctional linker is used, also steps a. and b. can take place at the same time.
When used herein, the term "peptide" or "polypeptide" or "protein" (all terms are used interchangeably herein) means a peptide, a protein, or a polypeptide which encompasses amino acid chains of a given length, wherein the amino acid residues are linked by covalent peptide bonds.
Peptides are chains of amino acid monomers linked by peptide (amide) bonds. The shortest peptides are dipeptides, consisting of 2 amino acids joined by a single peptide bond, followed by tripeptides, tetrapeptides, etc. A polypeptide is a long, continuous, peptide chain. One can differentiate between an oligopeptide, polypeptide, protein with regard to the length of the amino acid chains. The following table gives a raw idea on the different lengths of the peptide and the different nomenclature, however it is known to the skilled artesian that the borders are blurred. The present application envisages that oligopeptides, polypeptides as well as proteins are embraced by the term peptide. Thus, in one embodiment, the peptide is an oligopeptide, polypeptide or protein. In another embodiment, the peptide is an oligopeptide. In another embodiment, the peptide is a polypeptide or protein. In another embodiment, the peptide is a polypeptide. In another embodiment, the peptide is a protein.
Figure LU92353A1D00191
In one embodiment, the peptide comprises 5,10, 20, 30, 40, 50, 60, 70, 80, 90,100,150, 200, 250, 300,350,400,450,500,600, 700 or more amino acids.
However, peptidomimetics of such peptides/proteins/polypeptides wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs are also encompassed by the invention as well as other than the 20 gene-encoded amino acids, such as selenocysteine. Peptides, oligopeptides and proteins may be termed polypeptides. The term peptide also refers to, and does not exclude, modifications of the peptide, e.g., glycosylation, acetylation, phosphorylation and the like. Such modifications are well described in basic texts and in more detailed monographs, as well as in the research literature. The peptide that is preferably meant herein is an antibody.
The term "binding domain" means a peptide domain which binds to a specific atom or molecule, such as proteins at the cell surface of cells. Upon binding, proteins may undergo a conformational change. Any portion of a molecule which is capable of binding to a target is included. For example a binding domain is a peptide, an antibody-binding portion of an antibody or other protein, preferably an antibody. Further encompassed by the term binding domain by the present invention are peptides, preferably oligopeptides, polypeptides or proteins binding to a given target or naturally occurring ligands binding to a given target, preferably a receptor. In another embodiment, the naturally occurring ligands preferably only possess their binding capacity, while other physiological functions such as e.g. induction of signaling cascades initiated by binding to the receptor are not present any more. Thus, in one embodiment, the binding domain is a naturally occurring ligand. In another embodiment, the binding domain is a naturally occurring ligand, which has been modified such that mainly its binding capacity is still present. "Mainly" in this context again means that other physiologically functions of said naturally occurring ligand have been reduced or are preferably absent. In another embodiment, the binding domain is a naturally occurring ligand, which has been modified such that only its binding capacity is still present.
In one embodiment, the binding domain is a peptide. In another embodiment, the binding domain is an oligopeptide. In another embodiment, the binding domain is a polypeptide. In another embodiment, the binding domain is a protein. In another embodiment, the binding domain is a naturally occurring ligand. In another embodiment, the binding domain is a modified naturally occurring ligand, preferably the naturally occurring ligand has been modified such that it still comprises the binding capacity to its naturally occurring target, while other physiological functions are minimized, preferably absent. In one embodiment of the method of the present invention the peptide comprising a binding domain is an antibody.
The definition of the term "antibody" includes embodiments such as monoclonal, chimeric, single chain, humanized and human antibodies. In addition to full-length antibodies, the definition also includes antibody derivatives and antibody fragments, like, inter alia, Fab fragments. Antibody fragments or derivatives further comprise F(ab')2, Fv, scFv fragments or single domain antibodies such as domain antibodies or nanobodies, single variable domain antibodies or immunoglobulin single variable domain comprising merely one variable domain, which might be VHH, VH or VL, that specifically bind an antigen or epitope independently of other V regions or domains; see, for example, Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane "Using Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, 1999; Kontermann and Diibel, Antibody Engineering, Springer, 2nd ed. 2010 and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009. Said term also includes diabodies or Dual-Affinity Re-Targeting (DART) antibodies. Further envisaged are (bispecific) single chain diabody, tandem diabody (Tandab), „minibodies" exemplified by a structure which is as follows: (VH-VL-CH3)2, (scFv-CH3)2 or (scFv-CH3-scFv)2, „Fc DART" and „IgG DART", multibodies such as triabodies. Immunoglobulin single variable domains encompass not only an isolated antibody single variable domain polypeptide, but also larger polypeptides that comprise one or more monomers of an antibody single variable domain polypeptide sequence.
Furthermore, the term "antibody" as employed herein also relates to derivatives or variants of the antibodies described herein which display the same specificity as the described antibodies. Examples of "antibody variants" include humanized variants of non-human antibodies, "affinity matured" antibodies (see, e.g. Hawkins et al. J. Mol. Biol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30,10832-10837 (1991)) and antibody mutants with altered effector function(s) (see, e.g., US Patent 5,648,260, Kontermann and Dübel (2010), loc. cit. and Little(2009), loc. cit.).
The term "antibody" also comprises immunoglobulins (Ig's) of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgGl, lgG2 etc.). Derivatives of antibodies, which also fall under the definition of the term antibody in the meaning of the invention, include modifications of such molecules as for example glycosylation, acetylation, phosphorylation, disulfide bond formation, farnésylation, hydroxylation, méthylation or esterification. A functional fragment of an antibody includes the domain of a F(ab')2 fragment, a Fab fragment, scFv or constructs comprising single immunoglobulin variable domains or single domain antibody polypeptides, e.g. single heavy chain variable domains or single light chain variable domains as well as other antibody fragments as described herein above. The F(ab')2 or Fab may be engineered to minimize or completely remove the intermolecular disulphide interactions that occur between the CHI and CL domains.
The term "human" antibody as used herein is to be understood as meaning that the antibody or its functional fragment, comprises (an) amino acid sequence(s) contained in the human germline antibody repertoire. For the purposes of definition herein, an antibody, or its fragment, may therefore be considered human if it consists of such (a) human germline amino acid sequence(s), i.e. if the amino acid sequence(s) of the antibody in question or functional fragment thereof is (are) identical to (an) expressed human germline amino acid sequence(s). An antibody or functional fragment thereof may also be regarded as human if it consists of (a) sequence(s) that deviate(s) from its (their) closest human germline sequence(s) by no more than would be expected due to the imprint of somatic hypermutation. Additionally, the antibodies of many non-human mammals, for example rodents such as mice and rats, comprise VH CDR3 amino acid sequences which one may expect to exist in the expressed human antibody repertoire as well. Any such sequence(s) of human or non-human origin which may be expected to exist in the expressed human repertoire would also be considered "human" for the purposes of the present invention. The term "human antibody" hence includes antibodies having variable and constant regions corresponding substantially to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al (Kabat et al., (1991) 'Sequences of Proteins of Immunological Interest, 5th Ed.1, National Institutes of Health).
The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in particular, CDR3. The human antibody can have at least one, two, three, four, five, or more positions replaced with an amino acid residue that is not encoded by the human germline immunoglobulin sequence.
The non-human and human antibodies or functional fragments thereof are preferably monoclonal. It is particularly difficult to prepare human antibodies which are monoclonal. In contrast to fusions of murine B cells with immortalized cell lines, fusions of human B cells with immortalized cell lines are not viable. Thus, the human monoclonal antibodies are the result of overcoming significant technical hurdles generally acknowledged to exist in the field of antibody technology. The monoclonal nature of the antibodies makes them particularly well suited for use as therapeutic agents, since such antibodies will exist as a single, homogeneous molecular species which can be well-characterized and reproducibly made and purified. These factors result in products whose biological activities can be predicted with a high level of precision, very important if such molecules are going to gain regulatory approval for therapeutic administration in humans. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post- translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U. S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.
It is especially preferred that the monoclonal antibodies or corresponding functional fragments be human antibodies or corresponding functional fragments. In contemplating antibody agents intended for therapeutic administration to humans, it is highly advantageous that the antibodies are of human origin. Following administration to a human patient, a human antibody or functional fragment thereof will most probably not elicit a strong immunogenic response by the patient's immune system, i.e. will not be recognized as being a foreign that is non-human protein. This means that no host, i.e. patient, antibodies will be generated against the therapeutic antibody which would otherwise block the therapeutic antibody's activity and/or accelerate the therapeutic antibody's elimination from the body of the patient, thus preventing it from exerting its desired therapeutic effect.
According to a further embodiment of the invention, the antibody may be an IgG antibody. An IgG isotype comprises not only the variable antibody regions of the heavy and light chains responsible for the highly discriminative antigen recognition and binding, but also the constant regions of the heavy and light antibody polypeptide chains normally present in "naturally" produced antibodies and, in some cases, even modification at one or more sites with carbohydrates. Such glycosylation is generally a hallmark of the IgG format, and located in the constant regions comprising the so called Fc region of a full antibody which is known to elicit various effector functions in vivo. In addition, the Fc region mediates binding of IgG to Fc receptor, as well as facilitating homing of the IgG to locations with increased Fc receptor presence - inflamed tissue, for example. Advantageously, the IgG antibody is an IgGl antibody or an lgG4 antibody, formats which are preferred since their mechanism of action in vivo is particularly well understood and characterized. This is especially the case for IgGl antibodies.
According to a further embodiment of the invention, the functional fragment of the antibody may preferably be an scFv, a single domain antibody, an Fv, a VHH antibody, a diabody, a tandem diabody, a Fab, a Fab' or a F(ab)2. These formats may generally be divided into two subclasses, namely those which consist of a single polypeptide chain, and those which comprise at least two polypeptide chains. Members of the former subclass include a scFv (comprising one VH region and one VI region joined into a single polypeptide chain via a polypeptide linker); a single domain antibody (comprising a single antibody variable region) such as a VHH antibody (comprising a single VH region). Members of the latter subclass include an Fv (comprising one VH region and one VL region as separate polypeptide chains which are non-covalently associated with one another); a diabody (comprising two non-covalently associated polypeptide chains, each of which comprises two antibody variable regions - normally one VH and one VL per polypeptide chain · the two polypeptide chains being arranged in a head-to-tail conformation so that a bivalent antibody molecule results); a tandem diabody (bispecific single-chain Fv antibodies comprising four covalently linked immunoglobulin variable - VH and VL - regions of two different specificities, forming a homodimer that is twice as large as the diabody described above); a Fab (comprising as one polypeptide chain an entire antibody light chain, itself comprising a VL region and the entire light chain constant region and, as another polypeptide chain, a part of an antibody heavy chain comprising a complete VH region and part of the heavy chain constant region, said two polypeptide chains being intermolecularly connected via an interchain disulfide bond); a Fab' (as a Fab, above, except with additional reduced disulfide bonds comprised on the antibody heavy chain); and a F(ab)2 (comprising two Fab' molecules, each Fab' molecule being linked to the respective other Fab' molecule via interchain disulfide bonds). In general, functional antibody fragments of the type described hereinabove allow great flexibility in tailoring, for example, the pharmacokinetic properties of an antibody desired for therapeutic administration to the particular exigencies at hand. For example, it may be desirable to reduce the size of the antibody administered in order to increase the degree of tissue penetration when treating tissues known to be poorly vascularized (for example, joints). Under some circumstances, it may also be desirable to increase the rate at which the therapeutic antibody is eliminated from the body, said rate generally being accelerable by decreasing the size of the antibody administered. An antibody fragment is defined as a functional antibody fragment in the context of the invention as long as the fragment maintains the specific binding characteristics for the epitope/target of the parent antibody, i.e. as long as it specifically binds to EGFR, CD33, CD 117, CD44 or EpCAM.
According to a further embodiment of the invention, said antibody or functional fragment thereof may be present in monovalent monospecific; multivalent monospecific, in particular bivalent monospecific; or multivalent multispecific, in particular bivalent bispecific forms. In general, a multivalent monospecific, in particular bivalent monospecific antibody such as a full human IgG as described hereinabove may bring with it the therapeutic advantage that the neutralization effected by such an antibody is potentiated by avidity effects, i.e. binding by the same antibody to multiple molecules of the same antigen, here EGFR, CD33, CD117, CD44 or EpCAM. Several monovalent monospecific forms of fragments of antibodies have been described above (for example, a scFv, an Fv, a VHH or a single domain antibody). Multivalent multi-specific, in particular bivalent bi-specific form may advantageously be a human single chain bi-specific antibody, i.e. a recombinant human antibody construct comprising two scFv entities as described above, connected into one contiguous polypeptide chain by a short interposed polypeptide spacer as generally known in the art (see for example WO 99/54440 for an anti-CD19 x anti-CD3 bi-specific single chain antibody). Here, one scFv portion of the bi-specific single chain antibody comprised within the bispecific single chain antibody will specifically bind EGFR, CD33, CD117, CD44 or EpCAM as set out above, while the respective other scFv portion of this bi-specific single chain antibody will bind another antigen determined to be of therapeutic benefit.
According to a further embodiment, the antibodies or functional fragments thereof may be derivatized, for example with an organic polymer, for example with one or more molecules of polyethylene glycol ("PEG") and/or polyvinyl pyrrolidone ("PVP"). As is known in the art, such derivatization can be advantageous in modulating the pharmacodynamic properties of antibodies or functional fragments thereof. Especially preferred are PEG molecules derivatized as PEG-maleimide, enabling conjugation with the antibody or functional fragment thereof in a site-specific manner via the sulfhydry! group of a cysteine amino acid. Of these, especially preferred are 20kD and/or 40 kD PEG-maleimide, in either branched or straight-chain form. It may be especially advantageous to increase the effective molecular weight of smaller human anti- EGFR, CD33, CD117, CD44 or EpCAM antibody fragments such as scFv fragments by coupling the latter to one or more molecules of PEG, especially PEG-maleimide.
The antibodies of the present invention also include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U. S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include "primitized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc.) and human constant region sequences. "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) of mostly human sequences, which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (also CDR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, "humanized antibodies" as used herein may also comprise residues which are found neither in the recipient antibody nor the donor antibody. These modifications are made to further refine and optimize antibody performance. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525 (1986); Reichmann et al.. Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).
Preferred antibodies are those which bind cell surface domains and are internalized in a receptor-dependent manner, e.g. anti-CD52 antibodies, anti-CD3 antibodies, anti-CD33 antibodies, anti-CD20 antibodies, anti-ErbB2 antibodies, anti-EGFR antibodies, anti-CD99 antibodies, anti-CD33 antibodies, anti-CD34 antibodies, anti-CD44 antibodies, anti-CD117 antibodies, anti-CA15-3 antibodies, anti-CA-125 antibodies, anti-CA27-29 antibodies, anti-EpCAM antibodies, anti-Carcinoembryonic antigen antibodies, anti- MARTI antibodies, anti- TPBG antibodies. In a particular embodiment of the method of the present invention, the antibody is a single-chain antibody or a Fab-fragment of an antibody. Preferably, the antibody is selected from the group consisting of anti-EGFR antibody, anti-CD44 antibody, anti-EpCAM antibody, anti-CD33 antibody, anti-CD117 antibody. In a preferred embodiment the method of the present invention includes an anti-EGFR antibody that is Cetuximab.
Preferably, the antibody is a monoclonal antibody, more preferably a monoclonal IgGl antibody, a single chain antibody or a Fab fragment of an antibody. Antibodies, which can be used in the method or for the molecules of the present invention include the Antibody fragments of Orthoclone® (anti-CD3), ReoPro™ (anti-GPIIb, Ilia), Rituxan™ (anti-CD20), Remicade® (anti- TNF-a), Simulet®(anti-CD25), Synagis™ (anti-RSV F protein), Zenapax® (anti-CD25), Herceptin® (anti-HER-2), Campath® (anti-CD52), Zevalin® (anti-CD20), Humira™ (anti-TNF a), Bexxar® (anti-CD20), Xolair® (anti-lgE), Avastin™ (anti-VEGF), Tysabri® (anti-a4 (subunit of a4ßl)), Erbitux™ (anti-EGFR), Vectibix™ (anti-EGFR), Lucentis™ (anti-VEGF-A) Soliris® (anti-CD59), Cimzia® (anti-TNF-a) and/or Simponi™ (anti-TNF-a). A "cell surface domain" as used herein means any protein on the cell surface. The cell surface domain also includes a cell surface antigen. It additionally includes any epitope that can be recognized on the cell surface of a cell. Preferably, the epitope or protein is cell type-specific as it only is present in a certain cell type. In one embodiment, the cell surface domain is present on a cancer cell. Interesting cell surface targets include CD19, CD20, CD22, C025, CD30, CD33, CD40, CD56, CD64, CD70, CD74, CD79, CD105, CD138, CD174, CD205, CD227, CD326, CD340, MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphB2, EphA2, FAP Av, integrin, Mesothelin, E6FR, TAG-72, GD2, CAIX and/or 5T4. Other interesting cell surface domains include CD52, CD3, CD117, CD99, CD34, CD44, CD117, CA15-3, CA-125, CA27-29, EpCAM, Carcinoembryonic antigen, melanoma antigen recognized by T-cells 1 (MARTI), trophoblast glycoprotein (TPBG).
Preferably such a cell surface domain is CD44, EpCAM (Epithelial cell adhesion molecule), CD33, CD117 or EGFR (epidermal growth factor receptor), more preferably the cell surface domain is EGFR. The cell surface domain can also provide for an epitope to which a binding domain in accordance with the present invention can bind.
In line with the above, the term "epitope" defines an antigenic determinant, which is specifically bound/identified by a binding domain as defined herein. The binding domain may specifically bind to/interact with conformational or continuous epitopes, which are unique for the target structure.
The term "cell surface antigen" as used herein denotes a molecule, which is displayed on the surface of a cell and which can serve as an epitope. In most cases, this molecule will be located in or on the plasma membrane of the cell such that at least part of this molecule remains accessible from outside the cell in tertiary form. A non-limiting example of a cell surface molecule, which is located in the plasma membrane is a transmembrane protein comprising, in its tertiary conformation, regions of hydrophilicity and hydrophobicity. Here, at least one hydrophobic region allows the cell surface molecule to be embedded, or inserted in the hydrophobic plasma membrane of the cell while the hydrophilic regions extend on either side of the plasma membrane into the cytoplasm and extracellular space, respectively. Non-limiting examples of cell surface molecules which are located on the plasma membrane are proteins which have been modified at a cysteine residue to bear a palmitoyl group, proteins modified at a C-terminal cysteine residue to bear a farnesyl group or proteins which have been modified at the C-terminus to bear a glycosyl phosphatidyl inositol ("GPI") anchor. These groups allow covalent attachment of proteins to the outer surface of the plasma membrane, where they remain accessible for recognition by extracellular molecules such as antibodies. Examples of cell surface antigens CD19, CD20, C022, CD25, CD30, CD33, CD40, CD56, CD64, CD70, CD74, CD79, CD105, CD138, CD174, CD205, CD227, CD326, CD340, MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphB2, EphA2, FAP Av, integrin, Mesothelin, EGFR, TAG-72, GD2, CAIX and/or 5T4. Other interesting cell surface domains include CD52, CD3, CD117, CD99, CD34, CD44, CD117, CA15-3, CA-125, CA27-29, EpCAM, Carcinoembryonic antigen, melanoma antigen recognized by T-cells 1 (MARTI), trophoblast glycoprotein (TPBG), preferably EGFR, CD44, EpCAM, CD33, CD117, most preferably EGFR.
The term "internalized" as used in the present invention means endocytosis, in which molecules such as proteins are engulfed by the cell membrane and drawn into the cell. In particular, the cell surface domain to which the binding domain binds is internalized. A method of how this internalization can be measured is disclosed in the examples of the present application. Otherwise, for example such a process may be observed by time-laps microscopy, where the receptor of interest and the cell membrane are double stained. Preferably, the peptide comprising a binding domain capable of binding to a cell surface molecule is internalized upon binding to the cell surface molecule. Preferably, a molecule of the present invention is internalized. A "linker" means a crosslinker or cross-linking agent containing at least two functional groups. In one embodiment, the method of the present invention includes cross-linking agents that are homo- or heterobifunctional having functional groups including but not limited to carbodiimide, carbonyl, imidoester, isocynate, maleimide, N-hydroxysuccinimide (NHS)-ester, sulfo-NHS-ester, PFP-ester, hydroxymethyl phosphine, arylazide, pyridyl disulfite, vinyl sulfone. The bonds they produce include, but are not limited to, amide, disulfide, hydrazine, thioether and ester bonds. Functional groups that can be targeted by the linker or cross linking agent for crosslinking include primary amines, sulhydryls, carbonyls, hydroxyls, carbohydrates, carboxyls and the like, preferably amines and sulfhydryl, most preferably sulfhydryl. Preferably a linker comprises no cleavable disulfide bond (S-S). In one embodiment, the linker comprises a pH-depended cleavable side. A linker may be water soluble, cell membrane permeable, of various spacer arm length, be spontaneously reactive or comprise photoreactive groups. Furthermore, the linker may be labeled or tagged.
In one embodiment, the linker can directly be conjugated with the peptide (comprising the binding domain) and the positively charged molecule, an example for that is the Sulfo-SMCC linker. In another embodiment, the linker can be conjugated with the peptide (comprising the binding domain) and the positively charged molecule in a two-step mechanism, an example for that is the EDC together with the Sulfo-NHS linker. This reaction needs an extra reaction step in comparison to the sulfo-SMCC direct linking (so called two-step linkers). Such two-step linkers are also envisaged by the present invention; preferably these are homobifunctional linkers such as EDC and Sulfo-NHS.
Examples of cross-linking agents include, but are not limited to /V-hydroxysulfosuccinimide (Sulfo-NHS), Sulfosuccinimidyl(perfluoro-zidobenzamido)ethyl-l,3'-dithiopropionate (Sulfo-SFAD), succinimidyl 4-formylbenzoate (SFB), succinimidyl 4-hydrazinonicotinate acetone hydrazone (SANH), l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), 2-lminothiolane (Traut's Reagent), N-Succinimidyl (acetylthio)acetate (SATA) and 3[2-py- ridyldithio]propionyl hydrazide (PDPH) or any other linker which is publicly available and known to the person skilled in the art. A "heterofunctional" linker means a linker, wherein the targeted functional groups are different from each other. In one embodiment, the linker comprises an amine and a sulfhydryl, or an amine and a hydroxyl as functional groups that are targeted for cross-linking, preferably amine and a sulfhydryl. In another embodiment, the method of the present invention includes a linker that comprises an amine and a sulfhydryl as functional groups that are targeted for cross-linking. In another embodiment, the method of the present invention includes a heterobifunctional linker that does not comprise a cleavage site e.g. has no cleavable disulfide bond (S-S). Preferred heterofunctional linkers are e.g. a-Maleimidoacetoxy-succinimide ester (AMAS), N(4-[p-Azidosalicylamido]butyl)- 3'-(2‘-pyridYldithio) propionamide (APDP*), (ß-Maleimidopropionic acid)hydrazide»TFA (BMPH), (ß-Maleimidopropyloxy)succinimide ester (BMPS), ε-Maleimidocaproic acid (EMCA), (ε-Maleimidocaproyloxy)succinimide ester (EMCS), (y-Maleimidobutyryloxy)succinimide ester (GMBS), K-Maleimidoundecanoic acid (KMUA), Succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxy-(6-amidocaproate) (LC-SMCC), Succinimidyl-6-(3'-[2-pyridyl-dithio]propionamido)hexanoate (LC-SPDP), m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), Succinimdyl-3-(bromoacetamido)propionate (SBAP), Succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl iodoacetate (SIA), Succinimidyl 4-(p-maleimido-phenyl)butyrate (SMPB), NHS-PEG24-Maleimide SM(PEG24), NHS-PEG12-Maliemide (SM[PEG]12), NHS-PEG8-Maliemide (SM(PEG]8), NHS-PEG6-Maleimide (SM(PEG)6), NHS-PEG4-Maliemide (SM[PEG]4), NHS-PEG2-Maliemide (SM[PEG]2), Succinimidyl 4-(N-maleimido-methyi)cyclohexane-carboxylate (SMCC), succinimidyl iodoacetate (SIA), Succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), (s-Maleimidocaproyloxy)sulfosuccinimide ester (Sulfo-EMCS),-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), Succinimidyl-6-(ß-maleimidopropionamido)hexanoate (SMPH), N-(y-Maleimidobutryloxy)sulfosuccinimide ester (Sulfo-GMBS),-(K-Maleimidoundecanoyloxy)sulfosuccinimide ester (Sulfo-KMUS), Sulfosuccinimidyl 6-(a-methyl-a-[2-pyridyldithio]-toluamido)hexanoate (Sulfo-LC-SMPT), Sulfosuccinimidyl 6-(3'-(2-pyridyl-dithio]propionamido)hexanoate (Sulfo-LC-SPDP), Maleimidobenzoyl-hydroxysulfosuccinimide ester (Sulfo-MBS), Sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (Sulfo-SIAB), Sulfosuccinimidyl 4-(N-maleimido methyl)cydohexane-l-carboxylate (Sulfo-SMCC), Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (Sulfo-SMPB) Ν,Ν-Dicyclohexylcarbodiimide (DCC), l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), Sulfo-NHS-(2-6-[Biotinamido]-2-(p-azidobezamido) (Sulfo-SBED).
In one embodiment, the method of the present invention includes a linker that is (l-ethyl-3 [3-dimethylaminopropyl]carboiimide hydrochloride (EDC) linked to Sulfo NHS. In another embodiment, the method of the present invention includes a heterobifunctional linker that is SMCC, or sulfo SMCC; preferably sulfo-SMCC. A "homofunctional" linker means a linker, wherein the targeted functional groups are the same. In one embodiment, the method of the present invention includes linker comprising a sulfhydryl and a sulfhydryl as functional groups that are targeted for cross-linking. In another embodiment, the method of the present invention includes homobifunctional linker that do not comprise a cleavage side, e.g. have no cleavable disulfide bond (S-S).
Preferred homofunctional linkers are e.g. 1,4-bis-Maleimidobutan (BMB), Maleimidohexane (BMH), Maleimidoethane (BMOE), 1,8-bis-Maleimidodiethylene-glycol (BM(PEG)2), 1,11-bis-
Maleimidotriethyleneglycol (BM(PEG)3), Dimethyl suberimidate«2HCI (DMS), Dimethyl 3,3'-dithiobispropionimidate«2HC (DTBP), (2-Maleimidoethyl)amine (Trifunctional) (TMEA***).
The term "positively charged molecule" means a peptide having a net positive charge at or near physiological pH (e.g., in solutions having a pH between 4 to 10, between 5 to 9, or between 6 to 8) and bind nucleic acids through electrostatic interactions. Carriers of this class include, but are not limited to protamine, Sso7d, histones, poly Lysine, poly Arginine preferably (Arg)9, synthetic cationic polymers, avidin, synthetic polypeptides, carbon nanotubes modified to comprise a net positive charge. In one embodiment, the positively charged molecule is protamine. In another embodiment, the positively charged molecule is a synthetic polypeptide, preferably a synthetic cationic polymer, more preferably polyetheleneimin. "Protamine(s)" as used herein, refers to small, strongly basic proteins, the positively charged amino acid groups of which (especially arginines) are usually arranged in groups and neutralize the negative charges of nucleic acids because of their polycationic nature. Protamines may be of natural origin or produced by recombinant methods. Use of recombinant methods allows multiple copies of the protamine to be produced or modifications may be made in the molecular size and amino acid sequence of the protamine. Corresponding compounds may also be chemically synthesized. When an artificial protamine is synthesized, the procedure used may include, for example, replacing amino acid residues which have functions in the natural protamine that are undesirable for the transporting function (e.g., the condensation of DNA) with other suitable amino acids.
As described above, protamine is a strong positively charged protein that also interacts immediately with negatively charged nucleic acids such as siRNA. The uptake of the complex into the cell is mediated via receptor-mediated endocytosis. The antibody binds to the receptor and is internalized via endocytosis in clathrin coated pits. The vesicles are transported into the cell where the siRNA is released from the antibody-protamine complex and can enter the RNAi pathway (Rossi, 2005). "Histones", as used herein, refer to small DNA-binding proteins present in the chromatin having a high pro-portion of positively charged amino acids (lysine and arginine) which enable them to bind to DNA independently of the nucleotide sequence and fold it into nucleosomes, the arginine-rich histones H3 and H4 being particularly suitable. As for the preparation and modifications thereof, the remarks made above in relation to protamines apply here as well. "Synthetic polypeptides" include peptides such as homologous polypeptides (polylysine, polyarginine) or heterologous polypeptides (that include two or more representatives of positively charged amino acids). "Synthetic cationic polymer" includes polymeric cations, preferably polyethylene comprising cations, more preferably polyethyleneimines. The size of the polymeric cation is preferably selected so that the sum of the positive charges is about 10 to 500 and this is, in some embodiments, matched to the nucleic acid which is to be transported
The term "thiol group" is a -SH (sulfur-hydrogene) functional group and can also be referred to "sulhydryl", "-SH" or "sulhydryl-" in the context of the present invention. A "non-covalent binding" is a type of bond that does not involve the sharing of pairs of electrons, but rather involves more dispersed variations of electromagnetic interactions. There are four commonly mentioned types of non-covalent interactions: hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. For example at a given pH, proteins have charged groups that may participate in binding them to each other or to other types of molecules. For example, negatively-charged carboxyl groups on aspartic add (Asp) and glutamic acid (Glu) residues may be attracted by the positively-charged free amino groups on lysine (Lys) and arginine (Arg) residues. Non-covalent binding of the present invention includes binding of a preferably the negatively charged nucleic acid molecule to the positively charged molecule.
The term "coupling" in the sense of the present invention means the connection of a positively charged molecule with the peptide via a linker. So, the coupling likewise includes coupling of at least one portion of the linker to the peptide, as well as coupling of at least one portion of the linker with the positively charged molecule. This connection can be achieved via different reactive groups or via the same reactive groups. Thus, coupling of the positively charged molecule with the peptide via a linker can take place in one reaction. In one embodiment, first coupling of the positively charged molecule with a portion of a linker takes place in a first step, while the coupling of the linker/positively charged molecule molecule with the peptide takes place in a second step.
In one embodiment of the method of the present invention, the coupling of the peptide comprising a binding domain to the hetero- or homobifunctional linker is achieved via a NH2-group of said peptide.
In another embodiment of the method of the present invention, the coupling of the peptide comprising a binding domain to the hetero- or homobifunctional linker is achieved via a thiol-group of said peptide.
In another embodiment of the method of the present invention, the coupling of the peptide comprising a binding domain to the hetero- or homobifunctional linker is achieved via a carboxyl-group of said peptide.
In one embodiment of the method of the present invention, the coupling of the peptide comprising a binding domain to the heterobifunctional linker is achieved via a NH2-group of said peptide.
In another embodiment of the method of the present invention, the coupling of the peptide comprising a binding domain to the heterobifunctional linker is achieved via a thiol-group of said peptide.
In another embodiment of the method of the present invention, the coupling of the peptide comprising a binding domain to the heterobifunctional linker is achieved via a carboxyl-group of said peptide.
In one embodiment of the method of the present invention, the coupling of the peptide comprising a binding domain to the homobifunctional linker is achieved via a NH2-group of said peptide.
In another embodiment of the method of the present invention, the coupling of the peptide comprising a binding domain to the homobifunctional linker is achieved via a thiol-group of said peptide.
In another embodiment of the method of the present invention, the coupling of the peptide comprising a binding domain to the homobifunctional linker is achieved via a carboxyl-group of said peptide.
In another embodiment of the method of the present invention, the coupling to the hetero- or homobifunctional linker in a) achieved via a NH2 group of said positively charged molecule.
In another embodiment of the method of the present invention, the coupling to the hetero- or homobifunctional linker in a) achieved via a thiol group of said positively charged molecule.
In another embodiment of the method of the present invention, the coupling to the hetero- or homobifunctional linker in a) is achieved via a carboxyl group of said positively charged molecule.
In one embodiment of the method of the present invention, the coupling to the heterobifunctional linker in a) is achieved via a NH2-group of said positively charged molecule.
In another embodiment of the method of the present invention, the coupling to the heterobifunctional linker in a) achieved via a thiol group of said positively charged molecule.
In another embodiment of the method of the present invention, the coupling to the heterobifunctional linker in a) is achieved via a carboxyl group of said positively charged molecule.
In another embodiment of the method of the present invention, the coupling to the homobifunctional linker is achieved via a NH2-group of said positively charged molecule and via a NH2-group of the peptide comprising a binding domain.
In another embodiment of the method of the present invention, the coupling to the heterobifunctional linker is achieved via a NH2-group of said positively charged molecule and via a thiol-group of the peptide comprising a binding domain.
In another embodiment of the method of the present invention, the coupling to the heterobifunctional linker is achieved via a NH2-group of said positively charged molecule and via a carboxyl group of the peptide comprising a binding domain.
In another embodiment of the method of the present invention, the coupling to the hetero- or homobifunctional linker is achieved via a thiol group of said positively charged molecule and via a thiol group of the peptide comprising a binding domain.
In another embodiment of the method of the present invention, the coupling to the heterobifunctional linker is achieved via a thiol group of said positively charged molecule and via a carboxyl group of the peptide comprising a binding domain.
In another embodiment of the method of the present invention, the coupling to the heterobifunctional linker is achieved via a thiol group of said positively charged molecule and via a NH2-group of the peptide comprising a binding domain.
In another embodiment of the method of the present invention, the coupling to the homobifunctional linker is achieved via a carboxyl group of said positively charged molecule and via a carboxyl group of the peptide comprising a binding domain.
In another embodiment of the method of the present invention, the coupling to the heterobifunctional linker is achieved via a carboxyl group of said positively charged molecule and via a thiol group of the peptide comprising a binding domain.
In another embodiment of the method of the present invention, the coupling to the heterobifunctional linker is achieved via a carboxyl group of said positively charged molecule and via a NH2-group of the peptide comprising a binding domain.
Functional groups on the peptide or the positively charged molecule that can be targeted for crosslinking including primary amines, sulhydryls, carbonyls, hydroxyls, carbohydrates, carboxylic acids and the like, preferably via amine and/or sulfhydryl and/or carboxyl. In one embodiment, the method of the present invention includes that coupling to the heterobifunctional linker is achieved via a NH2-(primary amine) group of the positively charged molecule. In another embodiment, the method of the present invention includes that coupling to the heterobifunctional linker is achieved via a carboxyl-group of the positively charged molecule. In another embodiment, the method of the present invention includes that coupling to the heterobifunctional linker is achieved via a thiol group of the positively charged molecule.
In another embodiment, the method of the present invention includes that coupling to the homobifunctional linker is achieved via a thiol group of the positively charged molecule. In a further embodiment, the method of the present invention includes that coupling to the homobifunctional linker is achieved via a thiol group of the positively charged molecule and wherein this is a direct reaction.
In another embodiment, the method of the present invention includes that coupling to the homobifunctional linker is achieved via a thiol group of peptide comprising a binding domain. In a further embodiment, the method of the present invention includes that coupling to the homobifunctional linker is achieved via a thiol group of the peptide comprising a binding domain and wherein this is a direct reaction.
In another embodiment, the method of the present invention includes that coupling to the homobifunctional linker is achieved via a carboxyl group of the positively charged molecule. In a further embodiment, the method of the present invention includes that coupling to the homobifunctional linker is achieved via a carboxyl group of the positively charged molecule and wherein this is a direct reaction.
In another embodiment, the method of the present invention includes that coupling to the homobifunctional linker is achieved via a carboxyl group of the peptide comprising a binding domain. In a further embodiment, the method of the present invention includes that coupling to the homobifunctional linker is achieved via a carboxyl group of the peptide comprising a binding domain and wherein this is a direct reaction. A "carboxyl" or "carboxy" group is an organic acid characterized by the presence of at least one carboxyl group. The general formula of a carboxyl is -COOH. A carboxyl group (or carboxy) is a functional group consisting of a carbonyl (RR'C=0) and a hydroxyl (R-O-H), which has the formula -C(=0)0H, usually written as -COOH or -C02H, wherein R is any organic group. This group is present in molecules comprising a carboxylic acid.
In one embodiment, the molecule of the present invention includes that the coupling via the carboxyl group results in an ester bond. A "direct" reaction means that there are no additional steps between the coupling of the positively charged molecule to the hetero-or homobifunctional linker and the coupling of at least one thiol, amino or carboxyl group of the peptide with the hetero- or bifunctional linker. In one embodiment, the method of the present invention includes that there is no purification after the coupling of the positively charged molecule to the hetero- or homobifunctional linker and before coupling of the positively charged molecule /linker molecule to the peptide.
When referred to herein the terms "nucleotide sequence(s)", "polynucleotide(s)", "nucleic acid(s)", "nucleic acid molecule" are used interchangeably and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length. Nucleic acid sequences include DNA, cDNA, genomic DNA, RNA such as e.g. mRNA, siRNA, synthetic forms and mixed polymers, both sense and antisense strands, or may contain non-natural or derivatives nucleotide bases, as will be readily appreciated by those skilled in the art. Further, non-limiting examples of such nucleic acids include but are not limited to any type of RNA interfering (RNAi), whether single stranded or double stranded, that perform gene cessation and/or gene knockdown, including gene knockdown of message (mRNA) by degradation or translational arrest of the mRNA, inhibition of tRNA and rRNA functions or epigenetic effects; short (or small) interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA and non-coding RNA or the like, Short RNAs activity on DNA, and Dicer-substrate siRNAs (DsiRNAs) (DsiRNA are cleaved by the RNase III class endoribonuclease dicer into 21-23 base duplexes having 2-base 3 '-overhangs siRNA), and UsiRNA (UsiRNAs are duplex siRNAs that are modified with non-nucleotide acyclic monomers, termed unlocked nucleobase analogs (UNA), in which the bond between two adjacent carbon atoms of ribose is removed); and Self-delivering RNA (sdRNA) including rxRNA™ (of RXi Therapeutics), and agents inhibiting the pre-mRNA maturation step of poly A tail addition such as the Ul adaptor (IDT Inc.). The Ul adaptor consists of two parts, a target-gene binding domain and a U'l domain that attracts and inhibits the cellular splicing apparatus. By combining both capabilities in the same molecule, the Ul adaptor can inhibit the pre-mRNA maturation step of polyA tail addition. Further, the domains of the oligonucleotide are independent so transcript binding and splicing inhibition can be independently optimized and adapted to a wide array of genes. As another example the nucleic acids can also include microRNA. The microRNA could also induce an upregulation (apart from downregulation) of expression both in cellular and non-cellular messages such as the effect of microRNA 122 on Hepatitis C virus (HCV) replication; microRNA 122 enhances HCV replication. Also the nucleic acid could include aptamers, tripel-helix formation, DNAzymes, antisense and ribozyme.
The nucleic acid utilized by the present invention effects a target cell. E.g. via provision of the nucleic acid molecule the expression of a specific molecule or protein is reduced or increased in a target cell. Preferably, the expression of a specific molecule protein is reduced by utilization of a nucleic acid molecule.
In one embodiment, the method of the present invention includes nucleic acid molecules selected from siRNA or miRNA. Even more preferably the nucleic acid molecule is siRNA. In another embodiment, the method of the present invention includes nucleic acid molecules selected from EZH2 SiRNA, KRAS siRNA, BRAF siRNA, MEK1 siRNA, CDK1 siRNA, CDK4 siRNA, CDK6 siRNA, FLT3 siRNA, MLL siRNA, CSF1R siRNA, hAES riRNA, preferably the KRAS siRNA is KRAS esiRNA.
The molecule of the invention also comprises siRNA molecules that are designed to target and suppress or block the expression of a gene or protein associated with cancer or is involved in the development and/or progression of cancer.
In one embodiment, the method of the present invention includes determining the quantity of free thiol groups of the peptide and/or the positively charged molecule by an adequate assay.
For determining free thiol groups different tests are known to a person skilled in the art. For example, Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB) is a chemical that can be used to quantify the number or concentration of thiol groups in a sample/solution (Ellman (1959) "Tissue sulfhydryl groups" Arch Biochem Biophys, 82(1):70-7). A solution of this compound produces a measurable yellow-colored product when it reacts with sulfhydryls. More precisely, DTNB reacts with a free sulfhydryl group to yield a mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB; see Figure 2). The target of DTNB in this reaction is the conjugate base (R—S-) of a free sulfhydryl group. Therefore, the rate of this reaction is dependent on several factors: 1) the reaction pH, 2) the pKa' of the sulfhydryl and 3) steric and electrostatic effects. TNB is the "colored" species produced in this reaction and has a high molar extinction coefficient in the visible range. The molar extinction coefficient of TNB is reflected by a value of 14,150 M-lcm-1 at 412 nm (Riddles etal. (1983) Reassessment of Ellman's reagent. Meth. EnzymoL;91,49-60; Riddles et. al. (1979) Ellman's reagent: 5,5'-dithiobis(2-nitrobenzoic acid) - a reexamination. Anal. Biochem. 94,75-81). The extinction of TNB is not affected by changes in pH between 7.6 and 8.6 (Riddles et. al. (1979) cit. loc.). However, the extinction of TNB can be different in other solvents. Sulfhydryl groups may be estimated in a sample by comparison to a standard curve composed of known concentrations of a sulfhydryl-containing compound such as cysteine. Alternatively, sulfhydryl groups may be quantitated by reference to the extinction coefficient of TNB.
Also other tests are comercially available such as the Thiol Quantification Assay Kit (Fluorometric) from abeam or Sensylyte ® Thiol Quantification Kit (colorimetric) from ANASPEC. A "molecule" of the present invention includes a molecule comprising of a stable system composed of two or more different portions. A stable system describes a system wherein the portions have a tendency to remain localized in one or more regions of space. Preferably, the molecule of the present invention comprises 4 portions, namely a nucleic acid molecule, a linker, a positively charged molecule and a peptide portion. Thus a general formula of the molecule of the present invention includes: N'+P-L-Pbd wherein N' is a nucleic acid molecule, which is non-covalently bound to the positively charged molecule; *P is a positively charged molecule; -L- is a homo- or heterobifunctional linker; P80 is a peptide comprising a binding domain capable of binding to a cell surface molecule, which is preferably internalized, wherein at least one thiol, amino or caboxylic acid group of the peptide is coupled to a portion of the homo- or heterobifunctional linker.
The nucleic acid molecule (or nucleic acid molecule portions) itself may comprise 1,2, 3,4, 5,6, 7, 8, 9.10.11.12.13.14.15.16.17.18.19.20 or more nucleic acid molecules.
The positively charged molecule (or positively charged molecule portion) itself may comprise 1, 2, 3, 4.5.6.7.8.9.10.11.12.13.14.15.16.17.18.19.20 or more positively charged molecules.
The iyTP-L-PBD conjugates (or "complexes") also include that the peptide is an antibody or functional fragment thereof that targets a cell, preferably a cancer cell to selectively deliver associated nucleic acid molecule(s), preferably siRNA(s) to that cell. An antibody or functional antibody fragment is a molecule that includes one or more portions of an immunoglobulin or immunoglobulin-related molecule that specifically binds to, or is immunologically reactive with an antigen, preferably a cancer-related antigen or another cancer biomarker.
In one embodiment, the general formula of the molecule of the present invention can include: N-+p-l-pB0 wherein N' is a siRNA, preferably anti-KRAS siRNA or anti-EZH2 siRNA, which is non-covalently bound to protamine; +P is protamine; -L- is a homo or heterobifunctional linker; PB0 is an antibody, preferably an E6FR antibody, more preferably Cetuximab, wherein at least one thiol, amino or carboxyl group of the antibody is coupled with the linker.
The present invention also relates to N^P-P80 conjugates, wherein the term "conjugate" means that the antibody and the protamine/siRNA complex are connected together via a hetero- or homobifunctional linker, thereby forming an antibody-protamine-siRNA complex.
The present invention further relates to a molecule which is obtainable by a method of the present invention.
In one embodiment of the present invention, the molecule obtainable by a method of the present invention comprises per lmol peptide lmol positively charged molecule and 2mol nucleic acid molecule.
In another embodiment of the present invention, the molecule obtainable by a method of the present invention comprises per lmol peptide lmol positively charged molecule and 6mol nucleic acid molecule.
In yet another embodiment of the present invention, the molecule obtainable by a method of the present invention comprises per lmol peptide 3mol positively charged molecule and 2mol nucleic acid molecule.
In a further embodiment of the present invention the molecule obtainable by a method of the present invention comprises per lmol peptide 3mol positively charged molecule and 6mol nucleic acid molecule.
In another embodiment of the present invention, the molecule obtainable by a method of the present invention comprises per lmol peptide 3mol positively charged molecule and lOmol nucleic acid molecule.
In another embodiment of the present invention, the molecule obtainable by a method of the present invention comprises per lmol peptide 7mol positively charged molecule and 6mol nucleic acid molecule.
In another embodiment of the present invention, the molecule obtainable by a method of the present invention comprises per lmol peptide 7mol positively charged molecule and lOmol nucleic acid molecule.
The present invention further relates to a molecule comprising: i) a peptide comprising a binding domain capable of binding to a cell surface molecule, which is internalized; ii) a positively charged molecule; iii) a heterobifunctional linker, with one portion coupled to the peptide and another portion coupled to the positively charged molecule, wherein at least one thiol, amino or carboxyl group of the peptide is coupled to a portion of the heterobifunctional linker; iv) a nucleic acid molecule; wherein the nucleic acid molecule is non-covalently bound to the positively charged molecule.
Additionally, the present invention relates to a molecule comprising: i) a peptide comprising a binding domain capable of binding to a cell surface molecule, which is internalized; ii) a positively charged molecule; iii) a homobifunctional linker, with one portion coupled to the peptide and another portion coupled to the positively charged molecule, wherein at least one thiol, amino or carboxyl group of the peptide is coupled to a portion of the homobifunctional linker; iv) a nucleic acid molecule; wherein the nucleic acid molecule is non-covalently bound to the positively charged molecule.
In one embodiment, the molecules of the present invention include coupling of the thiol group in iii) resulting in a thioether bond. A "portion of a heterobifunctional linker" means that a heterobifunctional linker has different reactive portions (reactive groups), which can react with the molecules/proteins to be coupled. A heterobifunctional linker comprises at least 2 portions, which can react with groups on the targeted molecule/protein. Preferably, the heterobifunctional linker has 2 portions, which react with distinct reactive groups. The heterobifunctional linker can also comprise at least 3,4,5, 6, 7, 8, 9,10,11,12, 13, 14, 15, 16, 17, 18, 19,20 different portions. These portions target at least 2, 3, 4, 5, different groups on the target molecule(s)/protein(s). The portions can for example target primary amines, sulhydryls, carbonyls, hydroxyls, carbohydrates, carboxyls and the like on a molecule/protein. Preferably, at least one portion of the heterobifunctional linker targets a primary amine on one targeted molecule/protein and at least one second portion of the heterobifunctional linker targets a sulhydryl group on a second targeted molecule/protein. Most preferably, one portion of the heterobifunctional linker targets a primary amine on the positively charged molecule and one second portion of the heterobifunctional linker targets a sulhydryl group on the peptide comprising a binding domain capable of binding to a cell surface molecule. Reactive portions or groups, which target amines as a functional group on the target molecule/protein, preferably the nucleic acid molecule include carbodiimide, hydroxymethyl phosphine, imidoester, vinyl sulfone, NHS-ester, PFP-ester and the like. Reactive portions or group, which target sulfhydryls as a functional group on the target molecule/protein, preferably the peptide, include maleimide, pyridyl disulfide, vinyl sulfone and the like. In general such functional groups that may be used for linking sulfhydryl and amine or other groups are known to the person skilled in the art. A "portion of a homobifunctional linker" as used herein refers to a homobifunctional linker having different reactive portions (reactive groups), which can react with the molecules/proteins to be coupled. A homobifunctional linker comprises at least 2 portions, which can react with groups on the targeted molecule/protein. Preferably, the homobifunctional linker has 2 portions, which react with the same reactive groups or which react with the same target functional group. The homobifunctional linker can also comprise at least 3,4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16,17,18, 19,20 different portions. These portions target at least 2, 3, 4, 5, groups on the target molecule(s)/protein(s), which are preferably sulhydryl groups. The portions can for example target primary amines, sulhydryls, carbonyls, hydroxyls, carbohydrates, carboxyls and the like on a molecule/protein. Preferably at least one portion of the homobifunctional linker targets a sulfhydryl on one targeted molecule/protein, preferably the positively charged molecule, and at least one second portion of the homobifunctional linker targets a sulhydryl group on a second targeted molecule/protein, preferably the peptide comprising a binding domain capable of binding to a cell surface molecule. Reactive portions or group, which target sulfhydryls as a functional group on the target molecule/protein include maleimide, pyridyl disulfide, vinyl sulfone and the like. In general such functional groups that may be used for linking sulfhydryl groups are known to the person skilled in the art.
In another embodiment, the molecule of the present invention includes a coupling between the portion of the heterobifunctional linker and the positively charged molecule, which is achieved via a NH2-group of said positively charged molecule. A "NH2-group" means organic compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are derivatives of ammonia, wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group. The NH2 group is a primary amine -Primary amines arise when one of three hydrogen atoms in ammonia is replaced by an alkyl or aromatic. Important primary alkyl amines include methylamine, ethanolamine (2-aminoethanol), and the buffering agent tris, while primary aromatic amines include aniline. Thus a primary amine can also be expressed by R-NH2, wherein R is an organic group.
In one embodiment the molecule of the present invention includes that the coupling via the NH2-group results in an amine bond. A "amine bond" in this context means, that the NH2 group on the target molecule/protein, which was reacted with the linker, preferably a heterobifunctional linker, is converted into a secondary, tertiary or quaternary amine- now coupling/connecting the linker with the target molecule/protein. This means that the amine bond can be expressed as:
Figure LU92353A1D00421
wherein R1, R2, R3, R4 are each independently an organic group.
In one embodiment the molecule of the present invention includes that the coupling via the NH2-group results in an amine bond, which is a secondary amine.
In another embodiment, the molecule of the present invention includes that the coupling between the portion of the hetero- or homobifunctional linker and the positively charged molecule is achieved via a thiol-group of said positively charged molecule. Which linker or reactive groups can achieve such a coupling is described above. In another embodiment, the molecule of the present invention includes that the coupling between the portion of the hetero- or homobifunctional linker and the peptide comprising the binding domain is achieved via a thiol-group of said positively charged molecule.
In a further embodiment, the molecule of the present invention includes that the coupling of the thiol group results in a thioether bond. A "thioether bond" means a functional group with the connectivity C-S-C (carbon-sulfur-carbon). It can also be expressed by the formula R1-S-R2, wherein R1 is an organic group and R2 is an organic group. A thioether is similar to ether, except that it contains a sulfur atom in place of the oxygen. The thiol group is on the target molecule/protein, which was reacted with the linker, preferably a heterobifunctional or homobifunctional linker, is converted into a thioether- now coupling/connecting the linker with the target molecule/protein. Which linker or reactive groups can achieve such a coupling is described above.
The present invention further relates to a pharmaceutical composition comprising the molecules of the present invention and/or the molecule obtainable by the method of the present invention.
The term "pharmaceutical composition" relates to a composition for administration to a patient, preferably a human patient. Pharmaceutical compositions or formulations are usually in such a form as to allow the biological activity of the active ingredient to be effective and may therefore be administered to a subject for therapeutic use as described herein. Usually, a pharmaceutical composition comprises suitable (i.e. pharmaceutically acceptable) formulations of carriers, stabilizers and/or excipients. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the aforementioned molecules, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In one embodiment, the pharmaceutical composition is a composition for parenteral, trans-dermal, intra-luminal, intra-arterial, intrathecal and/or intranasal administration or for direct injection into tissue. It is in particular envisaged that said composition is administered to a patient via infusion or injection. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intra-peritoneal, subcutaneous, intra-muscular, topical or intra-dermal administration. The composition of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, liposomes, etc. Compositions comprising such carriers can be formulated by well-known conventional methods.
In accordance with the present embodiments, the term "therapeutically effective amount" refers to an amount of the molecules of the present invention and/or the molecule obtainable by the method of the present invention that is effective for the treatment of diseases associated with cancer. Preferred dosages and preferred methods of administration are such that after administration the molecules of the present invention and/or the molecule obtainable by the method of the present invention is present in the blood in effective dosages. The administration schedule can be adjusted by observing the disease conditions and analysing serum levels of the molecule decreasing the expression of target molecules in laboratory tests followed by either extending the administration interval e.g. from twice per week or once per week to once per two weeks, once per three weeks, once per four weeks, and the like, or, alternatively, reducing the administration interval correspondingly. In the case of cancer, the therapeutically effective amount of the molecules or compositions disclosed herein may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow and/or stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow and/or stop) tumor metastasis; inhibit tumor growth; and/or relieve one or more of the symptoms associated with the cancer.
In another embodiment, the pharmaceutical composition is suitable to be administered in combination with an additional drug, i.e. as part of a co-therapy. In said co-therapy, an active agent may be optionally included in the same pharmaceutical composition as the molecule of the invention, or may be included in a separate pharmaceutical composition. In this latter case, said separate pharmaceutical composition is suitable for administration prior to, simultaneously as or following administration of said pharmaceutical composition comprising the molecule of the invention. The additional drug or pharmaceutical composition may be a non-proteinaceous compound or a proteinaceous compound. In the case that the additional drug is a proteinaceous compound, it is advantageous that the proteinaceous compound be capable of providing an activation signal for immune effector cells. Preferably, said proteinaceous compound or non-proteinaceous compound may be administered simultaneously or non-simultaneously with the molecule (or preparation) of the invention as defined hereinabove, a vector as defined as defined hereinabove, or a host as defined as defined hereinabove.
The pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and by clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohofic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishes (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. In addition, the pharmaceutical composition in accordance with the present invention might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin. It is envisaged that the pharmaceutical composition in accordance with the invention might comprise, in addition to the above described molecules further biologically active agents, depending on the intended use of the pharmaceutical composition. Such agents might be drugs acting on the gastro-intestinal system, drugs acting as cytostatica, drugs preventing hyperurikemia, drugs inhibiting immunoreactions (e.g. corticosteroids), drugs modulating the inflammatory response, drugs acting on the circulatory system and/or agents such as cytokines known in the art.
To analyse the effect of the molecules of the present invention and/or the molecule obtainable by the method of the present invention for example in cancer therapy, outcome measures can be selected e.g. from pharmacokinetics, immunogenicity, and the potential to decrease the size of a cancer by e.g. MRI imaging as well as patient reported outcomes.
Another major challenge in the development of drugs such as the pharmaceutical composition in accordance with the invention is the predictable modulation of pharmacokinetic properties. To this end, a pharmacokinetic profile of the drug candidate, i.e. a profile of the pharmacokinetic parameters that affect the ability of a particular drug to treat a given condition, is established. Pharmacokinetic parameters of the drug influencing the ability of a drug for treating a certain disease entity include, but are not limited to: half-life, volume of distribution, hepatic first-pass metabolism and the degree of blood serum binding. The efficacy of a given drug agent can be influenced by each of the parameters mentioned above. "Half-life" means the time where 50% of an administered drug are eliminated through biological processes, e.g. metabolism, excretion, etc. By "hepatic first-pass metabolism" is meant the propensity of a drug to be metabolized upon first contact with the liver, i.e. during its first pass through the liver. "Volume of distribution" means the degree of retention of a drug throughout the various compartments of the body, like e.g. intracellular and extracellular spaces, tissues and organs, etc. and the distribution of the drug within these compartments. "Degree of blood serum binding" means the propensity of a drug to interact with and bind to blood serum proteins, such as albumin, leading to a reduction or loss of biological activity of the drug.
Pharmacokinetic parameters also include bioavailability, lag time (Tlag), Tmax, absorption rates and/or Cmax for a given amount of drug administered. "Bioavailability" means the amount of a drug in the blood compartment. "Lag time" means the time delay between the administration of the drug and its detection and measurability in blood or plasma. “Tmax" is the time after which maximal blood concentration of the drug is reached, the absorption is defined as the movement of a drug from the site of administration into the systemic circulation, and "Cmax" is the blood concentration maximally obtained with a given drug. The time to reach a blood or tissue concentration of the drug which is required for its biological effect is influenced by all parameters.
The term "toxicity" as used herein refers to the toxic effects of a drug manifested in adverse events or severe adverse events. These side events might refer to a lack of tolerability of the drug in general and/or a lack of local tolerance after administration. Toxicity could also include teratogenic or carcinogenic effects caused by the drug.
The terms "safety", "in vivo safety" or "tolerability" as used herein define the administration of a drug without inducing severe adverse events directly after administration (local tolerance) and during a longer period of application of the drug. "Safety", "in vivo safety" or "tolerability" can be evaluated e.g. at regular intervals during the treatment and follow-up period. Measurements include clinical evaluation, e.g. organ manifestations, and screening of laboratory abnormalities. Clinical evaluation may be carried out and deviating to normal findings recorded/coded according to NCI-CTC and/or MedDRA standards. Organ manifestations may include criteria such as allergy/immunology, blood/bone marrow, cardiac arrhythmia, coagulation and the like, as set forth e.g. in the Common Terminology Criteria for adverse events vB.O (CTCAE). Laboratory parameters which may be tested include for instance haematology, clinical chemistry, coagulation profile and urine analysis and examination of other body fluids such as serum, plasma, lymphoid or spinal fluid, liquor and the like. Safety can thus be assessed e.g. by physical examination, imaging techniques (i.e. ultrasound, x-ray, CT scans, Magnetic Resonance Imaging (MRI), other measures with technical devices (i.e. electrocardiogram), vital signs, by measuring laboratory parameters and recording adverse events. The term "effective and non-toxic dose" as used herein refers to a tolerable dose of the,molecules of the present invention and/or the molecule obtainable by the method of the present invention, preferably the antibody as defined herein, which is high enough to cure or stabilize the disease of interest without or essentially without major toxic effects. Such effective and non-toxic doses may be determined e.g. by dose escalation studies described in the art and should be below the dose inducing severe adverse side events (dose limiting toxicity, DLT).
The pharmaceutical composition of the present invention may have different formulations. The formulation (sometimes also referred to herein as "composition of matter”; "composition", or "solution") may preferably be in various physical states such as liquid, frozen, lyophilized, freeze-dried, spray-dried and reconstituted formulations, with liquid and frozen being preferred. "Liquid formulation" as used herein refers to a composition of matter that is found as a liquid, characterized by free movement of the constituent molecules among themselves but without the tendency to separate at room temperature. Liquid formulations include aqueous and non-aqueous liquid, with aqueous formulations being preferred. An aqueous formulation is a formulation in which the solvent or main solvent is water, preferably water for injection (WFI). The dissolution of the molecules of the present invention and/or the molecule obtainable by the method of the present invention in the formulation may be homogenous or heterogeneous, with homogenous being preferred as described above.
Any suitable non-aqueous liquid may be employed provided that it provides stability to the formulation of the invention. Preferably, the non-aqueous liquid is a hydrophilic liquid. Illustrative examples of suitable non-aqueous liquids include: glycerol; dimethyl sulfoxide (DMSO); polydimethylsiloxane (PMS); ethylene glycols, such as ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol ("PEG") 200, PEG 300, and PEG 400; and propylene glycols, such as dipropylene glycol, tripropylene glycol, polypropylene glycol ("PPG") 425 and PPG 725. "Mixed aqueous/non-aqueous liquid formulation" as used herein refers to a liquid formulation that contains a mixture of water, preferably WFI, and an additional liquid composition.
When used herein a "formulation" or "composition" is a mixture of the molecules of the present invention and/or the molecule obtainable by the method of the present invention (i.e., the active drug/substance) and further chemical substances and/or additives required for a medicinal product which is preferably in a liquid state. A formulation of the invention includes a pharmaceutical formulation.
The preparation of the formulation includes the process in which different chemical substances, including the active drug, are combined to produce a final medicinal product such as a pharmaceutical composition. The active drug of the formulation of the invention is the molecule of the present invention and/or the molecule obtainable by the method of the present invention.
In certain embodiments, the molecules of the present invention and/or the molecule obtainable by the method of the present invention can be formulated essentially pure and/or essentially homogeneous (i.e., substantially free from contaminating substances, e.g. proteins, etc. which can be product-related and/or process-related impurities). The term "essentially pure" means a composition comprising at least about 80%, preferably about 90% by weight of the compound, preferably at least about 95% by weight of the compound, more preferably at least about 97% by weight of the compound or most preferably at least about 98% by weight of the compound, preferably of the compound in a monomeric state. The term "essentially homogeneous" means a composition comprising at least about 99% by weight of the compound, preferably of the compound in a monomeric state, excluding the mass of various stabilizers and water in solution.
When used herein, the term "about" is understood to mean that there can be variation in the respective value or range (such as pH, concentration, percentage, molarity, number of amino acids, time etc.) that can be up to 5%, up to 10%, up to 15% or up to and including 20% of the given value.
For example, if a formulation comprises about 5 mg/ml of a compound, this is understood to mean that a formulation can have between 4 and 6 mg/ml, preferably between 4.25 and 5.75 mg/ml, more preferably between 4.5 and 5.5 mg/ml and even more preferably between 4.75 and 5.25 mg/ml, with the most preferred being 5 mg/ml. As used herein, an interval which is defined as "(from) X to Y" equates with an interval which is defined as "between X and Y". Both intervals specifically include the upper limit and also the lower limit. This means that for example an interval of "5 mg/ml to 10 mg/ml" or "between 5 mg/ml and 10 mg/ml" includes a concentration of 5,6, 7,8, 9, and 10 mg/ml as well as any given intermediate value. A "stable" formulation is one in which the molecules of the present invention and/or the molecule obtainable by the method of the present invention therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage and/or does not show substantial signs of aggregation, precipitation, fragmentation, degradation and/or dénaturation compared to a control sample, preferably upon visual examination of colour and/or clarity, or as measured by UV light scattering or by size exclusion chromatography. Various further analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993), for example. "During storage," as used herein, means a formulation that once prepared, is not immediately used; rather, following its preparation, it is packaged for storage, either in a liquid form, in a frozen state, or in a dried form for later reconstitution into a liquid form or other form. A "subject" in accordance with the present invention is a vertebrate, preferably a mammal, more preferably a human subject. A "vertebrate" includes vertebrate fish, birds, amphibians, reptiles and mammals. A "mammal" includes dogs, cats, horses, rats, mice, apes, rabbits, cows, pigs, sheep, and preferably humans. To a human can also be referred to by the term patient.
The present invention further relates to the molecule obtainable by the method of the present invention or the molecule of the present invention and/or the pharmaceutical composition of the present invention for use in a method for treating cancer in a subject.
The term "cancer" and "cancerous" as used by the present invention means a condition in vertebrates, preferably mammals, more preferably humans that is typically characterized by unregulated cell growth.
Cancers are classified by the type of cell that the tumor cells resemble and are therefore presumed to be the origin of the tumor. These types include: carcinoma, sarcoma, lymphoma and leukemia, germ cell tumors, blastoma. A "carcinoma" when referred to herein can include cancers derived from epithelial cells. When referred herein to a carcinoma prostate cancers, lung cancers, pancreas cancers, breast cancers, ovaries cancers and colon cancers can be included. Examples can include adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma. There are also a large number of rare subtypes of anaplastic, undifferentiated carcinoma. Examples include Askin's tumor, sarcoma botryoides, chondrosarcoma, Ewing's, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcomas such as cystosarcoma, angiosarcoma, epithelioid sarcoma, liposarcoma, and the like. A "lymphoma and leukemia" when referred to herein can include two classes of cancer arising from hematopoietic (blood-forming) cells that leave the marrow and tend to mature in the lymph nodes and blood, respectively. When referred herein to leukemia, bone marrow-derived cells that normally mature in the bloodstream can be included. When referring herein to a lymphoma, bone marrow-derived cells that normally mature in the lymphatic system can be included.
Examples of a lymphoma can include follicular lymphoma, diffuse large B ceil lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia/lymphoma, MALT lymphoma, Burkitt's lymphoma, mycosis fungoides peripheral T-cell lymphoma, not-otherwise-specified nodular sclerosis form of Hodgkin lymphoma, Hodgkin's lymphoma.
Examples of leukemia can include acute lymphoblastic leukemia (ALL) for example precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt's leukemia, and acute biphenotypic leukemia; chronic lymphocytic leukemia (CLL) like e.g. B-cell prolymphocytic leukemia; acute myelogenous leukemia (AML) for example acute promyelocytic leukemia, acute myeloblastic leukemia, and acute megakaryoblastic leukemia; chronic myelogenous leukemia (CML) for example chronic monocytic leukemia; hairy cell leukemia (HCL) or T-cell prolymphocytic leukemia (T-PLL) Large granular lymphocytic leukemia and adult T-cell leukemia. A "germ cell tumor" when referred to herein can include cancers derived from pluripotent cells, most often presenting in the testicle or the ovary (seminoma and dysgerminoma, respectively). Examples can include germinoma (including dysgerminoma and seminoma), dysgerminom, seminoma, embryonal carcinoma, endodermal sinus tumor, choriocarcinoma, teratoma including mature teratoma, dermoid cyst, immature teratoma, teratoma with malignant transformation, polyembryoma, gonadoblastoma. Such tumors can be localized in the head, neck, mediastinum, pelvis, ovary, testis. A "blastoma" when referred to herein can include cancers derived from immature "precursor" cells or embryonic tissue. Examples include hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma, glioblastoma multiforme. "Metastasis or métastasés" when referred to herein refers to the process by which cancer spreads from the location at which the cancer initiated as a tumor to one or more distant locations in the body by migration of one or more cancerous cells. These terms can also include micro-metastasis wherein the formation of tumors at distal locations corresponds to small aggregates of cancer ceils that are visible microscopically. These terms can also refer to the secondary cancerous growth resulting from the spread of the primary tumor from the original location.
Thus in one embodiment, the molecule obtainable by a process of the present invention or the molecules of the present invention and/or the molecule obtainable by the method of the present invention is used in a method for treating cancer, wherein the cancer is a carcinoma, sarcoma, lymphoma or leukemia, germ cell tumors, blastoma or metastasis.
In a further embodiment, the molecule obtainable by a process of the present invention or the molecule of the present invention or the pharmaceutical composition of the present invention is used in a method for treating cancer, wherein the cancer is a therapy resistant cancer mediated by mutant KRAS.
In another embodiment, the molecule obtainable by a process of the present invention or the molecule of the present invention or the pharmaceutical composition of the present invention is used in a method for treating cancer, wherein the cancer is a prostate cancer, breast cancer, bladder cancer, gastric cancer, lung cancer, hepatocellular cancer, lymphoma or myeloid neoplasm.
According to the present invention, the term "patient" or "subject" refers to vertebrates, preferably mammals, and more preferably, humans. For the purpose of the present application, a "patient undergoing treatment with the molecule of the present invention" is defined as a patient who is expected to receive, receiving or having received the molecules of the present invention and/or the molecule obtainable by the method of the present invention. In some embodiments, the patient is diagnosed with cancer.
The term "treatment" as used herein, means to alleviate, reduce, stabilize, or inhibit progression of a cancer which are associated with the presence and/or progression of a disease or pathological condition.
The present invention further relates to the molecule obtainable by a process of the present invention or the molecule of the present invention or the pharmaceutical composition of the present invention which is used in a method for inhibiting and/or controlling tumor growth in a subject. A "tumor" or "neoplasm" is an abnormal mass of tissue as a result of abnormal growth or division of cells. The growth of neoplastic cells exceeds, and is not coordinated with that of the normal tissues around it. However, a tumor in the sense of the present invention does also include leukemia, and carcinoma in situ. A tumor can be benign, pre-malignant, or malignant. In a preferred embodiment the tumors are pre-malignant or malignant. Most preferably, the tumor is malignant.
The present invention further relates to the molecule obtainable by a process of the present invention or the molecule of the present invention or the pharmaceutical composition of the present invention for delivering a nucleic acid molecule to the site of a tumor in a subject.
In one embodiment of the present invention, the molecule obtainable by a process of the present invention or the molecule of the present invention or the pharmaceutical composition of the present invention for use of the present invention includes a siRNA selected from the group consisting of EZH2 SiRNA, KRAS siRNA, BRAF siRNA, MEK1 siRNA, CDK1 siRNA, CDK4 siRNA, CDK6 siRNA, FLT3 siRNA, MLL siRNA, CSF1R siRNA. So an siRNA of the present invention can target EZH2, KRAS, BRAF, MEK1, CDK1, CDK4, CDK6, FLT3, MLL or CSF1R. Preferably the siRNA reduces the expression of EZH2, KRAS, BRAF, MEK1, CDK1, CDK4, COK6, FLT3, MLL, CSF1R of a cell. Preferably, the expression of these targets is decreased. In another embodiment, the expression of these targets is increased.
In one embodiment, of the present invention, the molecule obtainable by a process of the present invention or the molecule of the present invention or the pharmaceutical composition of the present invention for the use of the present invention includes a siRNA targeting KRAS and/or EZH2. In a further embodiment, the siRNA reduces KRAS expression of a ceil. In another embodiment, the siRNA molecule reduces EZH2 expression of a cell.
In a further embodiment, the siRNA increases KRAS expression of a cell. In another embodiment, the siRNA molecule increases EZH2 expression of a cell. A "siRNA targeting" means the target which is recognized by a specific siRNA. siRNAs can be constructed in different ways. For example a siRNA can be targeting mRNA.
In general the design of a siRNA is known to the skilled artesian. See for example Reynolds et al., (Reynolds et al., (2004) "Rational siRNA design for RNA interference" Nature Biotechnology 22,326 -330) or Judge et al., (Judge et al., 2006) "Design of Noninflammatory Synthetic siRNA Mediating Potent Gene Silencing in Vivo" Molecular Therapy (2006) 13, 494-505) or Sioud and Leirdal (Sioud and Leirdal (2004) "Potential design rules and enzymatic synthesis of siRNAs" Methods Mol Biol. 2004;252:457-69.
The term "expression" or "gene expression" means the transcription of a specific gene or specific genes or specific genetic construct. The term "expression" or "gene expression" in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product. The mRNA is then translated into peptide/polypeptide chains, which are ultimately folded into the final peptide/polypeptides/proteins. Protein expression is commonly used by proteomics researchers to denote the measurement of the presence and abundance of one or more proteins in a particular cell or tissue. The expression of a protein of a cell can be measured by various means. For example, with immunohistochemistry or western blot analysis. Here the obtained results should be evaluated in comparison, to a healthy cell, or control standard. A lower expressing cell shows a staining, which is decreased e.g. in intensity, when compared to a control cell. A higher expressing cell shows a staining, which is increased e.g. in intensity, when compared to a control cell in the same setting. Also the expression of the mRNA can be measured e.g. by RT-PCR. Here, a lower expressing cell shows e.g. a higher number of amplification cycles to overt a detectable signal when compared to a control cell in the same setting. The person skilled in the art knows different techniques, how to determine the expression of a protein, mRNA of a cell.
In one embodiment, the molecule obtainable by a process of the present invention or the molecule of the present invention or the pharmaceutical composition of the present invention is used according to the present invention, wherein the nucleic acid is a siRNA targeting KRAS and/or EZH2 or wherein the siRNA reduces KRAS or EZH2 expression of a cell and wherein said cell is present in a subject.
For example, the cell can be present in the blood, liver, stomach, mouth, skin, lung, lymphatic system, spleen, bladder, pancreas, bone marrow, brain, kidneys, intestines, gallbladder, brain, larynx or pharynx of the subject.
In one embodiment, the molecule obtainable by a process of the present invention or the molecule of the present invention or the pharmaceutical composition of the present invention is used according to the present invention, wherein the subject is mammal, preferably a human being.
In another embodiment the present invention relates to a preparation obtainable by a method of the present invention. A "preparation" in accordance with the present invention is obtainable by a method of the present invention.
In one embodiment, the preparation can therefore comprise the molecule of the present invention or the molecule obtainable by the method of the present invention. The preparation can also comprise further molecules and/or proteins and/or substances e.g. a left over from a buffer used in the method of the present invention due to e.g. less efficient purification of the molecules of the present invention. However, the preparation is also envisaged to comprise compositions comprising the molecule of the present invention or the molecule obtainable by the method of the present invention and at least one further ingredient such as a protein, molecule, etc.
Therefore, in one embodiment the preparation of the present invention comprises the molecule of the present invention or the molecule obtainable by the method of the present invention.
The present invention also relates to a kit comprising one or more coupling buffer/reagents and protocol suitable for performing the method of the present invention.
In one embodiment, the kit comprises one or more coupling buffer/reagents and protocol suitable for performing the method of the present invention.
The present invention relates to a kit comprising buffer/reagents and protocol suitable for performing the method of the present invention and optionally means to purify or enrich for e.g. molecules of the present invention or molecules obtained by the method of the present invention and/or means to wash said molecules and/or means to store said molecules. Said molecules and the additional means are thereby preferably packaged together in one sealed package or kit.
The present invention also relates to a kit that comprises the molecule of the present invention and/or the molecule obtainable by a method of the present invention.
The present invention relates to a kit comprising the molecule of the present invention and/or the molecule obtainable by a method of the present invention and/or optionally means to purify or enrich said molecules and/or means to wash said molecules and/or means to store said molecules.
Said molecules and the additional means are thereby preferably packaged together in one sealed package or kit.
Parts of the kit (or the "kit of parts") of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.
The kit of the present invention may comprise one or more container(s), optionally with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic, and are preferably sterilized. The container holds a composition having an active ingredient or comprising a buffer which is effective for the method of the present invention. Further container may hold suitable buffers (for example reaction buffers) which allow the specific reactions to take place. It is also envisaged that containers are included which hold diverse buffers, for example reaction buffers and/or buffers for the purification of the molecules of the present invention and/or the molecule obtainable by the method of the present invention etc. The active agent in the composition is preferably the molecule obtainable by the method of the present invention or the molecule of the present invention or the pharmaceutical composition of the present invention.
The kit may also comprise written instructions for performing the method of the present invention in accordance with the methods and uses of the present invention. Said kit may further comprise a label or imprint indicating that the contents can be used for the augmentation of the molecule in accordance with the present invention and/or for said molecules of the present invention.
It is also envisaged that the kit of the present invention, further comprises for example buffers, vials, control(s), stabilizer(s), written instructions which aid the skilled person in the preparation or use of the DNA polymerase of the present invention.
In addition the present invention also relates to the use of the molecule of the present invention or the molecule obtainable by the method of the present invention or the pharmaceutical composition of the present invention, in the treatment of cancer in a subject.
The present invention also relates to a method of treating cancer in a subject, comprising administering a therapeutically effective amount of the molecule of the present invention or the molecule obtainable by the method of the present invention or the pharmaceutical composition of the present invention to said subject.
The term "administration" means administering of a therapeutically or diagnostically effective dose of the aforementioned molecules of the present invention to a subject. Different routes of administration are possible and are described above.
The present invention also relates to the use of the molecule of the present invention or the molecule obtainable by the method of the present invention or the pharmaceutical composition of the present invention, for the preparation of a medicament.
For example, for a medicament effective in the treatment of cancer.
The present invention is further characterized by the following list of items:
Item 1. Use of a molecule of the present invention or the pharmaceutical composition of the present invention in the treatment of cancer in a subject.
Item 2. A method of treating cancer in a subject, comprising administering a therapeutically effective amount of a molecule of the present invention ora pharmaceutical composition of the present invention to said subject.
Item 3. Use of a molecule of the present invention or the pharmaceutical composition of the present invention for the preparation of a medicament.
SEQUENCES
Below, sequences used in the present application are listed. Tablel: Sequences
Figure LU92353A1D00561
Figure LU92353A1D00571
Figure LU92353A1D00581
Examples
The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the claims.
Example 1A: The following materials and methods were utilized for the examples IB-7.
Peptides
The anti-NRP-1 peptides listed below were used as a carrier for siRNA delivery into cultured cell. In total three different anti-NRP-1 peptides with different sequences were tested. The sequence of each peptide with its according reference is listed in table 2. The 9R-linker (RRR RRR RRR) has the function to bind siRNA. The cysteine (C) residue on the N-terminus (H2N) can be coupled by the chemical reagents like EDC Sulfo-NHS and Sulfo-SMCC.
Table 2: Sequence of the anti-Neuropilin-1 peptides
Figure LU92353A1D00591
Table 3: Sequence of the control peptides
Figure LU92353A1D00592
Table 4: Sequence of the anti-EGFR peptide
Figure LU92353A1D00593
Antibodies
Several antibodies were used for different procedures like flow cytometry, western blot analysis or as carriers for siRNA delivery into the cell.
Antibodies directly labeled with a fluorescent conjugate for flow cytometry analysis are listed in table 5.
Table 5: Antibodies for flow cytometry analysis
Figure LU92353A1D00594
Cell lines
According to their malignancies the cell lines and the appropriate media are listed in the following tables.
Table 6: Acute myeloid leukemia cell lines
Figure LU92353A1D00601
Media
In general two different media were used for cell culture cultivation, RPMI1640 and DMEM obtained from SIGMA (Steinheim, Germany). For colony formation assays methylcellulose media and IMDM (Iscove's Modified Dulbecco's Media) was used. IMOM was obtained from Life Technologies (Grand island, NY, USA). The mixture of the methyiceliuiose media is listed in the table below (table 10).
Table 10: Mixture of methylcellulose media
Figure LU92353A1D00611
Cell culture
Cells were cultures at 37*C in a humidified incubator of 5% C02 (HeraCeil 150). Cells were split under sterile conditions at the Laminar flow/ clean bench (Hera Safe) ever 2-3 days. In general all cells are maintained in a 75 cm2 cell culture flask with 20 mL of the appropriate medium. AH suspension cell lines (HL-60, U-937, Kasumi, Mv4-ll) and the adherent LoVo cell line were cultured in RPMI-1640 (Roswell Park Memorial Institute) plus 10 % FCS, 1 % glutamine and 1 % penicillin-streptomycin (pen-strep). All adherent cell lines (HCT-116, MDA-MB468, HTB-56, A-549) were cultured in DMEM (Dulbecco's modified eagle's medium) with 10 % FCS, 1 % glutamine and 1 % pen-strep.
For cell culture cultivation and further procedures of adherent cell lines the media is discarded, the cells are washed twice in PBS (phosphate buffered saline) and are incubated with 2 mL trypsin (75 cm2 flask) for several minutes (depending on the cell line) at 37eC in the incubator. The protease activity of trypsin is stopped by adding media to the cells. The celts were transferred to a new cell culture flask and were provided with fresh media. The rest of the cells were used for further experiments.
Suspension cell lines were centrifuged for 5 min. at 400 x g, the supernatant is discarded, the cell pellet is washed twice in PBS. The cells were provided with fresh media and were transferred into a new cell culture flask. The rest was used for further experiments.
Vital cell counts were determined with a Neubauer-counting chamber and trypan blue, which stains only dead cells. [Cell/mL = cell counts of all 4 quadrants/4 * dilution factor * 104]
Delivery of antibody or peptide - siRNA complex into the cells
The cells are incubated with the antibody or peptide-siRNA complex in the cell culture media for several hours, depending on the gene of interest and on the assay which is performed. The complex was dispensed directly to the cells. The cell culture flask was slightly moved in order to disperse the complex and to gain a high efficiency of siRNA delivery into the cell.
Flow cytometry analysis - Cell staining with fluorescent labeled antibodies
To determine an antigen on the cell surface (in this case Neuropilin and EGFR) the cells were stained with fluorescent labeled antibodies and flow cytometry analysis is carried out with a flow cytometer (FACS-Calibur). For flow cytometric analysis 3*105 cells were harvest and stained either with a primary antibody and a secondary fluorescent labeled antibody (in the case of Neuropilin and CD33) or directly with a specific fluorescent labeled antibody (in the case of EGFR) for 30 minutes on ice in the dark. Afterwards the cells were washed twice in PBS and then resuspended in 300 μΙ PBS. The cells were kept in the dark on ice until flow cytometric analysis was carried out.
Flow cytometry Assay
In order to visualize a reduction of an antigen which was internalized by receptor mediated endocytosis a shift to the left site can be illustrated in the histogram plot. A left shift indicates a lower concentration of a certain antigen while a shift to the right indicates a higher concentration and therefore a higher expression of the tested antigen.
In order to see a shift, cells were incubated with and without an antibody with the ability of internalizing antibody-antigen complexes. For example Cetuximab (Erbitux) binds to the EGF-receptor and internalizes the antibody-antigen complex. This leads to less EGFR expression on the cell surface and would be indicated by a left shift compared to cells incubated without Cetuximab. Cells are incubated with the receptor internalizing antibody for several time points at different temperatures, 37eC in the incubator, 4eC on ice and 20°C on room temperature (RT) in order to see a left-shift.
Apoptosis Assay
To distinguish living cells from dead, early or late apoptotic cells, cells were stained with Annexin V-FITC and propidium iodide (PI) using the Annexin-V FITC apoptosis detection kit. The kit contains Annexin-V labeled FITC and PI. Annexin V binds with a high affinity to phosphatidyldserine (PS) whereas PI intercalates in the DNA of dead and late apoptotic cells. Annexin V is a Ca2+-dependent phospholipids-binding protein. PS is a negative charged phospholipid and is usually located in the inner layer of the cell membrane. In the early phase of apoptosis the cell loses the asymmetry of their membrane phospholipids, PS is then exposed at the surface of the cell and can be recognized by
Annexin V. Because of this mechanism dead, early and late apoptotic cells can be distinguished by Annexin V and PI (Annexin V-FITC Apoptosis Detection Kit, Beckman Coulter).
Annexin V-FITC and propidium iodide staining of the cells
The apoptosis assay was performed according to the company's instructions (Annexin V-FITC Apoptosis Detection Kit, Beckman Coulter). The cells were washed in ice cold PBS and were centrifuged 5 min. at 500 x g at 4CC. The supernatant was discarded, the pellet was resuspended in 100 μί lx ice cold binding buffer (5 x 105 - 5 x 106 cells per mL binding buffer). 1 μΙ of Annexin V-FITC and 5 μΙ of PI were added to the cell suspension and were mixed. The cells were incubated in the dark at 4°C for approximately 15 min. 400 μΙ of lx binding buffer was added and mixed. Until flow cytometric analysis was performed the cells were kept at 4‘C in the dark.
Flow cytometric analysis of apoptotic cells
The absorption maximum of Annexin V-FITC is 492 nm whereas the emission maximum is 520 nm, the absorption of PI is 370 nm and 550 nm, the emission range lies between 560 nm and 680 nm.
Vital cells are stained negatively for Pi as well as for Annexin V, early apoptotic cells are positive stained for Annexin V, but negative for PI. Late apoptotic or necrotic cells are positive stained for Annexin V and PI, dead cells are only stained positively for PI. According to these requirements the assay can be evaluated.
Coupling mechanism of siRNA to peptides and antibodies
For peptide or antibody mediated siRNA delivery, the siRNA has to be linked to its carrier. It is taken advantage of the fact that the siRNA is negatively charged. Therefore we used either protamine or a positive arginine (R) linker which was already manufactory linked to the peptide. Cetuximab was linked with either Sulfo-SMCC or EDC-Sulfo NHS to protamine. The anti-Neuropilin peptide (H2N -RRR RRR RRR 66S GGS RPARPAR - COOH; SEQ ID NO: 3) is already positively charged due to the 9R linker, negatively charged siRNA can directly bind to the positive arginines. Protamine has a molecular weight of approximately 5 kDa. The sequence contains lots of positively charged arginines (R) (Sequence of protamine: H2N - P RRRR SSS RPV RRRRR PRVS RRRRR GG RRRR - COOH; SEQ ID NO: 13). Coupling siRNA to protamine or to the positive 9R linker was done overnight at 4”C
Coupling mechanism of siRNA to an antibody via Suifo-SMCC and protamine
Protamine-sulfate and Sulfo-SMCC were linked in a molar ratio of 1:10. 2 mg of Sulfo-SMCC were dissolved in 200 μΙ H20. The molar mass of Sulfo-SMCC is 436 g/mol. 10 mg protamine-sulfate was dissolved in 1 mL PBS. 30 μΙ of protamine-sulfate in PBS and 70 μΙ Sulfo-SMCC in H20 were combined. If the solution changed form clear to cloudy, 800 μΙ PBS were added to dilute the reaction. The reaction is incubated for 2h at 4°C. Subsequently the reaction was diluted by a total end volume of 1000 μΙ this results in a molar concentration of 62 μΜ. The complex was linked to Erbitux in a molar ratio of 2:1. Cetuximab (Erbitux) has a molecular weight of approximately 166 kDa, with a the stock solution of 5 mg/mL the molar concentration equals 30 μΜ. 1000 μ! of the reaction product are resuspended in 1000 μΙ stock solution of Erbitux (5 mg/mL). The reaction was incubated over night at 4°C. The next day, the Sulfo-SMCC-antibody-protamine complex was desalted with a Zeba Desalt Spin Column (Thermo Scientific) according to the manufactures instructions. Subsequently, the whole complex was linked in a ratio of 1:5 or 1:6 to a siRNA. The complex/conjugate is displayed in Figure 9A.
Cell lysis and protein extraction
At least 5x10s cells for each assay were harvest and counted. Cells were centrifuges at 400 x g for 5 min. (Eppendorf, 5414D). Cells were washed once in PBS. The supernatant was discarded and the pellet was provided with freshly made lysis buffer. The lysis buffer can be used up to 14 days. The cells (5x10s) were resuspended in 50 μΙ lysis buffer, vortexed and incubated on ice for at least 30 min. up to lh 30 min. The cells were centrifuged at 4eC, at 13.000 rpm for 10 min. (Eppendorf, 5417R). The supernatant with its protein was transferred into a new 1,5 mL tube, the pellet containing cell debris was discarded.
Example IB: Peptide mediated siRNA delivery by anti-Neuropilin-1 peptides
Flow cytometry analyses were carried out to analyze the expression of Neuropilin-1 (NRP-1) in leukemic cells as a precondition for anti-NRP-1 peptide dependent delivery of siRNA into tumor cells. After peptide binding, the corresponding receptor internalizes, leading to a decrease of cell surface NRP-1 levels. To proof this, FACS-shift assays were performed. The effect of three different anti-NRP-1 peptides was tested in apoptosis assays by flow cytometry. Western blots, luciferase and colony-formation assays were performed to analyze the impact of anti-NRP-1 dependent delivery of siRNA.
Neuropilin-1 expression in leukemic cells
The expression of NRP-1 was detected by flow cytometry in HL-60, Kasumi and Mv4-ll cells. Human breast cancer cells (MDA) were used as a negative control. The cells were first stained with an unlabeled anti-NRP-1 antibody and in turn with a secondary Aiexa-488 labeled GaR-antibody. According to flow cytometry analysis leukemic cells were all tested positive for NRP-1 expression in contrast to MDA cells. The highest NRP-1 expression of all tested cell lines was detected in Mv4-ll cells. HL-60 and Kasumi cells expressed the same amount of NRP-1, whereas MDA cells showed the lowest expression of all tested cell lines. The results are depicted in a histogram plot in Figure 1A. In every experiment the same cell number of 3*105 cells, an equal amount of the anti-NRP-1 antibody and the secondary Alexa-488 labeled GaR-antibody were used to compare NRP-1 cell surface levels of each cell line (Fig. 1A).
In addition to flow cytometry, western blot analysis of U937, Mv4-ll and HL-60 cells were performed in order to analyze the expression of NRP-1. A protein band representing NRP-1 was predicted at approximately 130-kDa. All of the tested cell lines showed a high-intensity band at 130-kDa indicating expression of NRP-1 (data not shown).
Thus, neuropilin-1 might be a promising candidate for targeted drug delivery in anti-cancer therapy. NRP-1 is highly expressed in a variety of cancer types for example in brain tumors, colon cancer, leukemia, breast cancers, lung cancer and in NSCLC, but in contrast, also to some extent on endothelial cells. Overexpression of NRP-1 is associated with increased tumor aggressivity, enhanced tumor angiogenesis and poor prognosis (Ellis, 2006; Hong et al., 2007; Lu et al., 2008). Due to the fact that NRP-1 is expressed on a variety of cancer cells as well as on endothelial cells, the antigen does not fulfill the criteria that the target should exclusively be expressed on the cell of interest. This has to be considered when anti-NRP-1 peptides are used in anti-cancer therapies. According to Barr et al. it is assumed that anti-NRP-1 peptides might be more effective using them in combination with inducers of apoptosis, angiogenesis inhibitors and conventional chemo- or radiotherapy (Barr et al., 2005). NRP-1 can be targeted in different ways according to anti-cancer therapy. NRP-1 can be directly targeted by antibodies resulting in a blockade of signaling according to an inhibition of ligand binding. NRP-1 expression can also be blocked by the mechanisms of RNAi to prevent synthesis of NRP-1 in the cells (Ellis, 2006; Mac Gabhann and Popel, 2006). It was demonstrated that targeting NRP-1 by siRNA significantly reduces tumor growth, metastasis formation and angiogenesis in a variety of human cancers, especially in AML and in lung cancer (Grandclement and Borg, 2011). Additionally, NRP-1 can also be used as a target to deliver siRNA into tumor cells according to anti-NRP-1 peptides linked to siRNA. According to this mechanism, anti-NRP-1 peptides bind to the NRP-1 receptor on the cell surface, induce internalization of the peptide-receptor complex and release the siRNA in the cell.
In a second step the internalization activity of the NRP-1 receptor induced by an anti-NRP-1 peptide was tested in FACS-shift assays.
Example 2: The effect of the CGFYWLRSC anti-NRP-1 peptide on tumor cells
The internalization activity of the anti-NRP-1 peptide with the following sequence C6F YWL RSC GGS RRR RRR RRR (SEQ ID NO: 5} was tested by flow cytometry in leukemic cells. FACS-shift assays with different conditions were performed to obtain the best requirements for the internalization of the peptide-receptor complex. The incubation time of the cells with the corresponding peptide varied between 30 min., 2h and overnight. The temperature was changed between 4°C, 37°C and room temperature (RT). Different peptide concentrations ranging from 0.01 μΜ to 200 μΜ were tested as well.
Subsequently, the ceils were stained with an anti-NRP-1 antibody and a secondary Alexa-488 labelled GaR-antibody in order to detect cell surface levels of NRP-1 by flow cytometry. Untreated cells served as a negative control. Internalization of the receptor results in a decrease of cell surface levels of NRP-1 and is indicated by a left-shift in the histogram plot. None of the tested conditions showed a left-shift indicating internalization of the receptor due to an anti-NRP-1 peptide. Instead of the expected left-shift, a right-shift increasing with higher concentration of the anti-NRP-1 peptide was observed (data not shown).
Surprisingly, none of the tested anti-NRP-1 peptides showed reduced cell surface levels of NRP-1 in FACS-shift assays (Example 2). This could be due to the fact that the anti-NRP-1 peptide might either function as a cell penetrating peptide and does not specifically bind to the NRP-1 receptor and penetrates receptor-independent into the cells.
Example 3: Propidium iodide staining of Mv4-ll cells with 100 μΜ of the anti-NRP-1 peptide A right shift usually indicates either an increase of expression or autofluorescence of the cells, which in general represents apoptotic cells. To determine if the anti-NRP-1 peptide has an apoptotic effect on the cells, the viability of the cells was tested by flow cytometry. HL-60 cells were incubated with different anti-NRP-1 peptide concentration in a range of 0.01 μΜ to 100 μΜ for 45 min. on ice. Afterwards the cells were stained with propidium iodide (PI) to distinguish apoptotic cells from living cells by flow cytometry. Peptides with an unspecific sequence not able to bind to the NRP-1 receptor were used as a negative control. The results are shown in a bar graph in Figure IB.
Only 5% of the HL-60 cells became apoptotic due to an anti-NRP-1 peptide concentration of 0.01 μΜ and 0.1 μΜ. It was observed that the amount of apoptotic cells increased with increasing peptide concentration. 29½ of the HL-60 cells were stained PI positive due to a peptide concentration of 1 μΜ. Almost 60½ apoptotic cells were detected by a peptide concentration of 20 μΜ. 100 μΜ of the anti-NRP-1 peptide resulted in nearly complete cell death, 90½ of the cells became apoptotic. A concentration of 100 μΜ of the control peptides resulted in only 13½ PI positive cells.
Increasing concentrations of the anti-NRP-1 peptide resulted in dose-dependent increase of apoptosis. To test if the apoptotic effect of the anti-NRP-1 peptide is receptor dependent, the NRP-1 receptor of Mv4-ll cells was first blocked by an anti-NRP-1 antibody and then incubated with the anti-NRP-1 peptide. Cells were exposed to peptide concentrations of either 100 nM or 100 μΜ. The results of the PI staining of Mv4-ll cells with a peptide concentration of 100 μΜ are illustrated in a histogram plot in Figure 2. A concentration of 100 nM of the anti-NRP-1 peptide did not result in an apoptotic effect, whereas 100 μΜ of the peptide led to nearly complete cell death of the Mv4-ll cells. Blocking the receptor by a specific anti-NRP-1 antibody before incubating the cells with the anti-NRP-1 peptide had no impact on the viability of the cells. This is indicated by a positive PI signal in the histogram plot in Figure 2.
Another apoptosis assay was performed to analyze if the apoptotic effect of the anti-NRP-1 peptide is receptor dependent. Therefore human breast cancer cells (MDA), which do not express NRP-1 (Figure 1A) were incubated with different concentration of the corresponding anti-NRP-1 peptide. Additionally it was tested if the positively charged 9R-linker on the C-terminus of the anti-NRP-1 peptide (CGFYWLRSC GGS 9R) contributes to the apoptotic effect. Mv4-ll, U937 and MDA cells were incubated with the anti-NRP-1 peptide in comparison to an anti-EGFR peptide containing a Bit-sequence (YHWYGYTPQNVIGG GGS 9R; SEQ ID NO: 1) as well. Different concentration of either 100 nM or 100 μΜ of each peptide were used. The results of a PI staining of the cells are shown in a bar graph in Figure 3.
None of the two peptides with a concentration of 100 nM induced apoptosis in Mv4-ll, U937, HL-60 and MDA cells, whereas 100 μΜ of the anti-NRP-1 resulted in almost complete cell death in leukemic cells as well as in MDA cells. 100 μΜ of the anti-EGFR control peptide resulted in 37% apoptotic Mv4-11 cells, 42% apoptotic U937 cells and almost 50% of the HL-60 cells became apoptotic, whereas only 14% of the MDA cells were stained PI positive
Example 4: The impact of the RPARPAR anti-NRP-1 peptide on leukemic cells
The following sequence RPARPAR (present in SEQ ID NO: 3 and 4) of the two other anti-NRP-1 peptides is deduced from the binding site of the NRP-1 ligand VEGF165 that binds to NRP-1 and causes internalization of the NRP-receptor complex, reducing cell surface levels of NRP-1. The internalization properties of the two anti-NRP-1 peptides were tested by FACS-shift assays in HL-60 and Kasumi cells. One of the peptide contained a 9R-residue (RRR RRR RRR GGS GGS RPARPAR; SEQ ID NO: 3) as a siRNA binding motive, whereas the other peptide contained a Cysteine-residue instead of the 9R linker on the N-terminus for directional coupling of other ligands. HL-60 and Kasumi cells were incubated with either one of the peptides. To optimize the internalization process of the receptor complex, different experiments were carried out. According to a published Kd value of 1.7 ± 0.4 μΜ (Teesalu et al., 2009), an anti-NRP-1 peptide concentration of 1.3 μΜ was used. Cell surface levels of NRP-1 in untreated HL-60 and Kasumi cells and the levels of NRP-1 due to the anti-NRP-1 peptides are illustrated in a histogram plot in Figure 4.
None of the two anti-NRP-1 peptides reduced NRP-1 cell surface levels, indicated by a left shift in the histogram plot.
Example 5: Preparation and Characterization of the anti-EGFR-single chain antibodys (scFv 225).
Now, single chain antibodies were utilized for siRNA transport. The scFVs were generated by the group of Prof. Dr. Fey, Erlangen and detect EGFR (epidermal growth factor receptor). They are genetic dérivâtes of the Cetuximab. The sequence of the construct from which the scFVs were obtained is depicted in SEQ. ID NO: 11 and 12 (pASK-6\-\225-VLVH-scFv\-\free\cystein also shown in Figure 16).
After binding to its receptor, the receptor is endocytosed into the cell. We purified EGFR-specific single chain Fv (scFv)-antibodies expressed by E. coli from E. coii inclusion bodies (Figure 5A-C). Then these were folded back from their denaturized condition and reactive scFv were generated, which detected EGFR and could be internalized upon binding (Figure 5D). This scFv could be coupled to a positively charged arginine peptide via an oxidative disulfide-coupling. This peptide serves as an electrostatic binding partner for the negatively charged siRNA as subject of transportation. Construct 225 and 425 were tested.
It turned out that the backfolding and stabilization of the purified protein is a critical point. Upon back-folding from the denaturized condition about 95% of the purified protein were lost upon stabilization, only 5% stayed back folded in solution. From these 5% in the next days a portion precipitated. The resulting protein is active and internalizes as can be seen by the FACS-left-shift Figure 14. However, the preparation was accompanied by huge losses of the protein.
Without being bond to any theory the system of coupling was considered the biggest obstacle. For the oxidative coupling of scFV-SH > SH-polyArginine terminal cysteines are necessary. Yet, these terminal cysteine, which are known for this process called expressed protein ligation overt a disturbing effect on the backfolding and protein stability.
Example 6: The expression and internalization of the EGF-receptor in tumor cells
Thus, FACS-shift assay were performed in order to detect a decrease of "full-length" EGFR antibody due to internalization of the antibodv-antisen complex. The effect of Cetuximab on K-Ras wildtvDe and mutant cell lines was tested In a viability assay by flow cytometry. Antibody dependent delivery of siRNA was tested in a variety of western blots to detect a gene silencing effect according to antibody mediated delivery of a specific siRNA.
Decreased cell surface levels of EGFR according to Cetuximab
First of all the expression of EGFR was tested on a variety of cell lines. Human breast cancer cells (MDA), colon carcinoma cell lines (LoVos, HCT-116) and bronchial carcinoma cell lines (A549) were directly stained with an anti-EGFR fluorescent labeled antibody to detect the amount of cell surface EGFR by flow cytometry. All of these tested cell lines expressed EGFR on their cell surface. In a second step, the cells were incubated for 30 min. on ice with 50 nM Cetuximab, an anti-EGFR antibody. Subsequently, the cells were directly stained with an anti-EGFR fluorescent labeled antibody to compare the levels of cell surface EGFR of treated and untreated cells. EGFR cell surface levels of A549, HCT-116 and MDA cells with and without Cetuximab are illustrated in Figures 6 and 7.
To interact with siRNA, Cetuximab needs to be linked to protamine via different chemical reagents. To test if the coupling reaction of protamine onto Cetuximab does reduce the affinity of the mAb, flow cytometry analyses were carried out. Therefore MDA cells were incubated with Cetuximab and Cetuximab linked to protamine to analyze receptor internalization in MDA cells. The coupling reaction of protamine onto Cetuximab via Sulfo-SMCC or EDC Sulfo-NHS had no impact on the internalization (Figure 7). Exemplary, the coupling of protamine via Sulfo-SMCC onto Cetuximab is depicted in Figure 9B.
Example 7: The influence of RAS mutations on Cetuximab resistance
In order to find the most suitable cell line for Cetuximab dependent delivery of siRNA, EGFR expressing cell lines were tested in apoptosis assays. The influence of RAS mutations on Cetuximab resistance in K-RAS wild type and mutant cell lines was analyzed in an Annexin-V staining. MDA cells display K-RAS wild type, whereas HCT-116 and Lovos are K-RAS mutated. MDA and HCT-116 cells were incubated with 50 nM of Cetuximab for 72h at 37°C. The antibody concentration and incubation conditions are comparable to western blot analysis. The results are illustrated in Figure 8. Each bar in the diagram indicates the amount of either early apoptotic, late apoptotic or living cells in %. 60% of the MDA cells treated with 50 nM of Cetuximab became apoptotic in comparison to untreated cells. HCT-116 cells showed no significant difference in the amount of apoptotic cells according to an incubation of the cells with and without Cetuximab. Cetuximab had no apoptotic effect on HCT-116 cells.
Due to the fact that some patients with EGFR overexpressing tumors are resistant to EGFR inhibition it has to be mentioned that EGFR expression itself is not a good indicator of response to EGFR targeted therapy (Bianco et al., 2006). The influence of RAS mutations and also EGFR mutations on Cetuximab resistance has to be taken into consideration if Cetuximab is used as a carrier for siRNA delivery into tumor cells. It is estimated that 30-40% of colon carcinoma patients carry a KRAS mutation. It was shown in several clinical trials that patients with a KRAS mutations are resistant towards Cetuximab and do not respond to the mAb, whereas patients with a KRAS wildtype are sensitive to Cetuximab (Dunn et al., 2011). The same effect was demonstrated in cell lines, HCT-116 cells with a KRAS mutation are resistant to Cetuximab, whereas MDA cells with a KRAS wildtype are much more sensitive and undergo apoptosis due to Cetuximab binding (Figures 18 and 19). According to these results Cetuximab was used only in KRAS mutated cell lines as a carrier for siRNA; otherwise most of the cells may become apoptotic. On the other hand, both mechanisms, the antibody mediated cytotoxicity and the antibody mediated siRNA-delivery may contribute to the same target, the eradication of tumor cells in a clinical situation.
Example 8: The following methods apply for the examples 9-15
Coupling of a-EGFR antibody to protaminsulfote
Protaminsulfate (1.67 mM) was amino-terminally coupled to the bifunctional crosslinker Sulfo-SMCC (Pierce No. 22622) in a 1:12 molar ratio in amino-free PBS buffer, left to react for lh at RT, then coupled to cysteine residues of a-EGFR monoclonal antibody (31 μΜ stock; Cetuximab, Erbitux™, Merck-Serono) in a 5:1 molar ratio. Non-reacted educts and protamine doublets were separated from the high molecular weight a-EGFR mAB-protamin product by gel filtration chromatography in Zeba spin desalting columns (Pierce No. 89891). The resulting a-EGFR mAB-protamin product displayed a broad band of >170 kDa in SDS-PAGE (not shown). The a-EGFR mAB-protamin adduct was stored at 4°C and was stable for several weeks.
Coupling of siRNA to a-EGFR mAB-protamin siRNA duplexes against KRAS (KRAS-Mission esiRNA EHU114431, Sigma-Aidrich; SEQ ID NO: 9) were bound to a-EGFR mAB-protamin in a 10-fold molar excess at 4°C for 3 h. This complex was prepared freshly before use.
Estimation of siRNA load capacity and serum stability of the complex
Constant concentrations (2.5 μΜ) of control siRNA duplexes were pre-incubated with increasing amounts of a-EGFR mAB-protamin up to a 40 fold fold molar excess for 1 h at 4°C, subjected to agarose gel electrophoresis and stained by ethidium bromide. a-EGFR mAB-protamin complexed siRNA proved to be immobile in 2% agarose whereas the unbound 25 bp siRNA duplex band travelled at expected size.
For siRNA stability estimation, control siRNA coupled to α-EGFR mAB-protamin was exposed to filtered Hctll6 cell culture supernatant including FCS for indicated timespans, subjected to 0.4% agarose gel electrophoresis and stained by ethidium bromide. The α-EGFR mAB-protamin/siRNA adduct was detectable as a weakly mobile complex hardly leaving the gel pouch.
Cell culture MDA-MB-231, Hctll6, A549 and LoVo cells were maintained in DMEM supplemented with 10% FCS, 1% streptomycin and penicillin, 1% glutamine, 1% sodium pyruvate, and 1% non-essential amino acid. MDA cells were KRAS-wild type and proved to be Cetuximab-sensitive (Simi et al., 2008) whereas the other cell lines carry KRAS mutations in codon 12 or 13, respectively, resulting in Cetuximab-resistance (Dunn et al. 2011; Okudela et al., 2004).
Fluorescent microscopy MDA, LoVo and Hctll6 cells were seeded at 2 xlO4 cells/cmz, cultivated on 8 well-chamber slides (Sigma C7057) over night and treated with α-EGFR mAB-protamin or Cetuximab alone incubated with Alexa Fluor 488-labeled scrambled control siRNA (Qiagen 1027284), at 1:10 molar ratio for 3 h at 37°C 5% C02. Subsequently, cells were washed with PBS, fixed with ice-cold methanol, stained with DAPI, mounted with Dako fluorescent mounting medium and photographed on a Zeiss Axioskop.
Flow cytometry FITC-coupled anti-EGFR-antibody (mouse monoclonal antibody no. 528) was purchased from Santa Cruz. 3x10s Hctll6 cells were harvested by trypsinisation, washed with PBS and stained with primary labeled antibodies for 1 h (h or hr=hour) at RT in the dark. For EGF receptor internalization studies with α-EGFR mAB-protamin (Cetuximab-protamin), cells were first treated with the Cetuximab-based antibody constructs for 1 h at RT and then stained for EGFR using the FITC-coupled anti-EGFR antibody.
Western blots 5x10s cells were seeded and cultivated overnight, treated with o-EGFR mAB-protamin coupled to the indicated siRNAs (anti-KRAS or negative control siRNA 1027281, Qiagen) at 1:10 molar ratio for 72 h, harvested, lysed, cleared by centrifugation and subjected to Western blot analysis using standard protocols with the following antibodies: Anti-KRAS (ab55391, ABCAM), anti-EZH2 (clone AC22, No. 3147 Cell Signaling Technologies), and anti ß-Actin mAB (Clone AC-15, Sigma Aldrich). Densitométrie analysis of gel-electrophoretic bands was carried out using the NIH image J package (http://rsb.info.nih.gov/ij/).
Clonogenic growth in soft agar
Briefly, 5000 cells in 40 μΙ per sample were incubated with α-EGFR mAB-protamin coupled to the indicated siRNAs at 50 nM end concentration for one hour at RT, resuspended in 150 μΙ of 0.5% soft agar in DMEM plus 10% FCS, 1% streptomycin and penicillin, 1% glutamine, 1% sodium pyruvate, and 1% non-essential amino add and cultivated for colony formation in 96-well format. Cells were treated twice a week with α-EGFR mAB-protamin coupled to siRNAs as indicated at a final concentration of 50 nM, based on mAB concentration. After two to three weeks, the assays were fixed with 4% PFA, counterstained with 0.1% crystal violet, photographed and counted for colony numbers. All treatments were performed in groups of six wells each.
Mouse xenograft tumor model
Female CD1 nude mice (Charles River) were transplanted subcutaneously with 2xl06 Hctll6 cells and kept till tumors reached sizes of approximately 200 mm3. Mice were divided into groups of 6 and treated with Cetuximab-protamin coupled to KRAS specific siRNA EHU114431 (SEQ 10 NO: 9), negative control siRNA (SEQ ID NO: 10) or uncoupled Cetuximab-protamin at 4 mg/kg twice a week intra-peritoneal. For a single dose, 750 pmol of α-EGFR mAB-protamin was coupled to 3.5 nmol of the respective siRNA. Tumor growth was followed with standard caliper measurements in a blinded fashion twice a week. Tumor volumes were calculated by the formula length χ width2 x 0.52 . At the end of the experiment, animals were euthanized by cervical dislocation in deep C02 anesthesia, primary tumors were surgically removed, and tumor weight was determined.
Statistical Analysis
All data are presented as means ± standard deviation, if not indicated otherwise. The mean values of two groups were compared by Student's t test.
Example 9: Development of a receptor specific siRNA carrier system
Cell type specific delivery of siRNA is a major problem for siRNA based therapies. The coupling of specific siRNAs to therapeutic antibodies against cell surface receptors could be an effective tool for siRNA delivery. In addition, this approach could also enhance the antibody's efficacy and/or might overcome drug resistance. We tested several methods to couple antibodies with siRNA. The most effective method was chemical coupling using protamine as a siRNA complexing agent. Protamine is a positively charged molecule that has previously been used for hemostatic therapy and has been extensively tested in humans (Hansen et al., 1979).
Protamine was coupled to the monoclonal antibody against the EGF receptor (α-EGFR mAB) using a sulfo-SMCC linker (Figure 9A) in a 5:1 molar ratio, enabling the binding of multiple protamine molecules per molecule of mAB. The siRNA can bind to protamine by electrostatic interactions (Choi et al., 2009). The specific siRNA binding capacity of protamine was analyzed by electrophoresis to determine bound vs. free siRNA (Kumar et al., 2008). The siRNA was incubated either with a-EGFR mAB or the a-EGFR mAB-protamine complex. Subsequently, non-bound siRNA was visualized by agarose gel electrophoresis (Fig. 9C, D) and quantities were determined by densitometry. No binding was evident for α-EGFR mAB alone (Fig. 9D), whereas free siRNA was depleted from the pool by incubation with α-EGFR mAB-protamin (Fig. 9C). At least eight molecules of siRNA were able to bind to one molecule α-EGFR mAB-protamine indicating a significant siRNA load for the antibody-protamine complex, while a higher molar excess of siRNA leads to an overflow of unbound siRNA.
To test the stability of the α-EGFR mAB-protamin/siRNA-complex under realistic conditions, we incubated the complex with filtered HCT116 cell culture supernatant including FCS for a timespan from 1 to 22 h. Next, the α-EGFR mAB-protamin-bound siRNA was visualized on a 0.4% agarose gel. The α-EGFR mAB-protamin/siRNA adduct displayed a complex with low net charge and consequently a weak mobility hardly leaving the gel pouch. After ethidium bromide stain, no degradation of the high molecular weight siRNA/protein complex was observed which indicated an increased stability (Fig. 9B).
Internalization of the antibody complex upon binding to the receptor is required for the intracellular siRNA activity. It is known that EGF receptors internalize into the cell after binding of either the EGF ligand or of monoclonal antibodies that bind to the same domain such as the α-EGFR mAB Cetuximab (Sunada et al., 1986). Consequently, surface expression of EGFR disappears upon its internalization which can be detected by flow cytometry. Therefore, first the α-EGFR antibody Cetuximab was incubated either alone or as a complex with protamine and siRNA with HCT116 cells that express EGFR on their surface (Fig. 10A). Then EGFR surface expression was analyzed using a FITC labeled a-EGFR-antibody that bound to a different extracellular epitope of EGFR than Cetuximab and detected the EGF receptor on the cell surface by flow cytometry (Fig. 10A, second panel). EGFR expression on the surface of Hctll6 cells was no longer detectable when the cells were pre-incubated with Cetuximab alone (Fig. 10A, third panel). Also after pre-incubation with the α-EGFR mAB-protamin /siRNA complex, EGFR expression disappeared from the cell surface (Fig. 10A, fourth panel). These analyses indicated that the Cetuximab antibody/siRNA complex was as effectively internalized as the uncoupled antibody and the chemical modification in combination with the highly anionic siRNA load did not interfere with EGF receptor binding and internalization.
To further verify internalization, we coupled α-EGFR mAB-protamin with scrambled control siRNA that was labeled with Alexa Fluor 488. Several carcinoma cell lines (HCT116, LoVo and MDA cells) were incubated with the conjugate and showed widespread and significant siRNA-antibody internalization as evident by the cytoplasmatic localization of the green fluorescence. After three hours of incubation in culture, internalized Alexa Fluor 488-labeled protein was stored in vesicular structures in the cytoplasmic compartment judged by DAPI nuclear stain and phase-contrast exposures (Fig. 10B, left-hand panel). In contrast, 488-siRNA pre-incubated with uncoupled a-EGFR mAB-protamin was not internalized into the cells (Fig. 10B, right-hand panel). This assay was also used to determine the optimal molar coupling ratios between the mAB, protamine and siRNA. An increase in mol protamine per mol of mAB increases siRNA carrier function to EGFR-expressing MDA cells (Figure 15). In line with this, molar increase of siRNA load per mol of mAB-protamin also enhanced carrier construct uptake: Optimal molecular ratios between mAB, protamine and siRNA lead to cell targeting frequencies of 30% in only three hours of incubation (Figure 15).
Example 10: anti-EGFR mAB-directed RNAi reduces target gene expression in EGFR-expressing carcinoma cell lines
Since the α-EGFR mAB-protamin/siRNA complexes were effectively taken up by cells, we now checked the intracellular functionality of RNA interference. For this, we used a specific siRNA against KRAS, which in its mutated form in codons 12,13 and 61 is a strong predictive biomarker for therapy resistance towards Cetuximab due to gain of function in its GTPase activity (De Roock et al., 2011). The KRAS siRNA bound to α-EGFR mAB-protamin (mAB-P) reduced KRAS protein expression in KRAS mutant HCT116 cells to 60% (Fig. 11A). KRAS protein migrated in a double band at 25 and 28 kDA, respectively. In contrast, a scrambled control siRNA did not affect KRAS expression level (Fig. 11A).
To further verify the gene specific effect, a specific siRNA against EZH2was used, a well-known oncogene in multiple solid tumors (Chase and Cross 2011), which is highly expressed in adenocarcinoma cell lines. Experiments were performed in adenocarcinoma cell lines with mutant KRAS (Hctll6 and A549). Cultured cells were exposed to the α-EGFR mAB-protamin/siRNA complexes or different controls in culture medium. After 72 h, protein lysates were prepared and subjected to Western blot analysis for EZH2 expression. Compared to α-EGFR mAB alone - which was used as a control - neither pure siRNA against EZH2 nor a α-EGFR mAB/scrambled-siRNA complex reduced EZH2 protein expression (Fig. 11B). The reduction of EZH2 expression in Hctll6 cells by application of the α-EGFR mAB alone parallels with a reduced protein concentration shown by actin loading control (Fig. 11B, second row, second lane). Taken together, the combination of oc-EGFR mAB-P/EZH2-siRNA exposure in HCT116 and A549 cells repressed EZH2 expression in an antibody-dependent fashion almost completely.
Example 11: Overcoming Cetuximab resistance in KRAS-mutated adenocarcinoma cell lines in vitro
Next it was tested whether the α-EGFR mAB/siRNA complex was active in vitro. We exposed KRAS-wild type MDA cells and KRAS-mutant LoVo, HCT116 and A549 cells to α-EGFR mAB in a complex with either scrambled or anti-KRAS siRNA and analyzed clonogenic growth in semi-solid medium as a read-out for the cancerogenic potential of the respective cell line. α-EGFR mAB-protamin alone reduced clonogenic growth in KRAS-wild type MDA cells by more than 47±7% (Fig. 12A). The o-EGFR mAB-protamin/KRAS-siRNA complex did not reduce the clonogenic growth beyond the effect observed by either Cetuximab alone or α-EGFR mAB-protamin/scrambled-siRNA complex (Fig. 12A). This finding indicated that targeting of a wild type RAS did not affect therapy response towards anti-EGFR antibody.
As expected Cetuximab alone did not inhibit clonogenic growth in KRAS mutant cells (Fig. 12B-D). Similar results were obtained for α-EGFR mAB-protamin coupled to scrambled siRNA: This complex did not affect growth of LoVo (Fig. 24B), Hctll6 (Fig. 12C) or A549 cells (Fig. 12D) which all express mutant KRAS. However, the α-EGFR mAB-protamin/KRAS-siRNA complex inhibited clonogenic growth to 53±16% (LoVo), 62.5±11% (Hctll6) and 60±8% (A549) respectively. Note that the ability for clonogenic growth varied highly between the four cell lines, giving rise to about 50 colonies per optical field in case of MDA cells and more than 500 in case of A549 cells.
This KRAS-specific reduction of colony numbers indicated that resistance induced by mutant gain-of-function-KRAS can be overcome by siRNA-antibody complexes that repress mutant KRAS activity.
Example 12: Antibody-dependent siRNA targeting in vivo. A total of 2xl06 Cetuximab-resistant HCT116 colon adenocarcinoma cells were subcutaneously implanted in CD1 nude mice. After reaching average tumor size of 200 mm3, mice were injected with 4 mg/kg anti-EGFR mAB-Alexa555 labelled siRNA or with the uncoupled siRNA. Tumors were resected 15 h after injection. In mice injected intra-peritoneally (A) or subcutaneously (B) with mAB-568-siRNA, tumors showed Alexa 555 fluorescent signals in the tumor rim. C and D depict parallel sections of A and B stained hematoxylin/Eosin (H/E). In mice injected with uncoupled siRNA-Alexa555 (0 and E), no fluorescent signals were detectable. F and G again show parallel sections of 0 and E stained H/E (see Figure 17).
Example 13: Efficacy of KRAS-siRNA-anti-EGFR complexes in mouse xenografts
Next, the effect of Cetuximab-siRNA complexes in vivo was analyzed. Briefly, Cetuximab-resistant HCT116 carcinoma cells were injected 2xl06 subcutaneously into the flank of CD1 nude mice. Mice with visible tumor growth were separated into groups and were treated twice weekly with a-EGFR mAB-protamin (mAB-P) alone or with mAB-P/siRNA complexes and tumor growth was determined (Fig. 13A). Cetuximab-resistant tumors continued to grow despite with a-EGFR mAB-P therapy (Fig. 13B, black triangles). Also, the scrambled siRNA coupled to a-EGFR mAB-P did not significantly affect tumor growth (Fig. 13B, black squares). In contrast, a-EGFR mAB-protamin/KRAS-siRNA complexes significantly inhibited tumor growth (Fig. 13B, white rhombs). After three weeks, tumor volumes were less than half the size of the a-EGFR mAB only treated tumors. At this time point, ail mice in the a-EGFR mAB and a-EGFR mAB/scrambled siRNA complex group had to be euthanatized due to excessive tumor growth. In contrast, mice treated with a-EGFR mAB-protamin/KRAS siRNA were followed for five more days, since their tumors were significantly smaller. During this time, only minimal additional tumor growth was observed (Fig. 13C) which indicated tumor control by Cetuximab-KRAS-siRNA complexes. Tumors from mice treated with a-EGFR mAB-protamin and a-EGFR mAB -P/scrambled siRNA prepared after 21 of treatment were larger than tumors from the a-EGFR mAB -P/KRAS siRNA group after 26 days (Figure 13D). This was documented by their significantly lower tumor weights depicted in Figure 13E.
Taken together, tumor growth of anti-EGFR resistant cells can be inhibited in vivo by the targeted siRNA application against the EGFR effector molecule KRAS in vivo.
Example 14
In mice bearing tumors treated twice in four days with mAB-KRAS esiRNA, we observed a reduced expression of the general proliferation antigen Ki67 judged by red AEC (3-amino-9-ethy!carbazole) immunohistochemistry in low power and high power magnifications (top left and right hand panel), whereas this effect was not detectable in tumors from mice treated with PBS control or mAB-control-esiRNA (bottom left and right hand panel). Notably, this effect was seen in the tumor rim, but not in the middle regions. Nuclear counterstain was performed using hematoxylin solution (see Figure 18).
Example 15 CD1 mice bearing tumors were treated for one day-one treatment, four days-two treatments and seven days-three treatments with mAB-KRAS esiRNA or mAB-control esiRNA. Tumors were resected and prepared for quantitative KRAS expression RT-PCR analysis. Tumors treated with mAB-KRAS esiRNA consistently expressed reduced KRAS levels compared to mAB-control esiRNA treated tumors. Simultaneous analysis of human GAPDH expression by real-time RT-PCR was used for standardization (see Figure 19).
References
Allen TM. Ligand-targeted therapeutics In anticancer therapy. Nat Rev Cancer 2002;2(10):750-63.
Brand TM, Wheeler DL. KRAS mutant colorectal tumors: Past and present. Small GTPases 2012;3(l):34-9.
Bulk E, Sargin B, Krug U, et al. S100A2 induces metastasis in non-small cell lung cancer. Clin Cancer Res 2009,-15(1):22-9.
Bulk E, Hascher A, Liersch R, et al. Adjuvant therapy with small hairpin RNA interference prevents non-small cell lung cancer metastasis development in mice. Cancer Res 2008,68(6):1896-904.
Castaigne S, Pautas C, Terre C, et al. Effect of gemtuzumab ozogamicin on survival of adult patients with de-novo acute myeloid leukaemia (ALFA-0701): A randomised, open-label, phase 3 study. Lancet 2012;379(9825):1508-16.De Roock W, De Vriendt V, Normanno N, Ciardiello F, Tejpar S. KRAS, BRAF, PIK3CA, and PTEN mutations: Implications for targeted therapies in metastatic colorectal cancer. Lancet Oncol 2011;12(6):594-603.
Chase A, Cross NC. Aberrations of EZH2 in cancer. Clin Cancer Res 2011;17(9):2613-8
Choi YS, Lee JY, Suh JS, et al. The systemic delivery of siRNAs by a cell penetrating peptide, low molecular weight protamine. Biomaterials 2009;31(6):1429-43.
Christie RJ, Matsumoto Y, Miyata K, et al. Targeted polymeric micelles for siRNA treatment of experimental cancer by intravenous injection. ACS Nano 2012,-6(6):5174-89.
Collier HB (1973). "Letter: A note on the molar absorptivity of reduced Ellman’s reagent, 3-carboxylato-4-nitrothiophenolate.
De Roock W, De Vriendt V, Normanno N, Ciardiello F, Tejpar S. KRAS, BRAF, PIK3CA, and PTEN mutations: Implications for targeted therapies in metastatic colorectal cancer. Lancet Oncol 2011;12(6):594-603.
Dunn EF, lida M, Myers RA, et al. Dasatinib sensitizes KRAS mutant colorectal tumors to cetuximab. Oncogene;30(5):561-74
Ellman GL (1959). "Tissue sulfhydryl groups". Arch. Biochem. Biophys. 82 (1): 70-7
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans. Nature 1998;391(6669):806-11.
Gao J, Liu W, Xia Y, Li W, Sun J, Chen J, Li B, Zhang D, Quian W, Meng Y, Deng L, Wang H, Chen J, Guo Y (2009) The promotion of siRNA delivery to breast cancer overexpressing epidermal growth factor receptor through anti-EGFR antibody conjugation by immunoliposomes. Biomaterials 32:3459-3470.
Hansen B, Linde S, Kolendorf K, Jensen F. Absorption of protamine-insulin in diabetic patients. I. preparation and characterization of protamine-1251-insulîn. Horm Metab Res 1979,-11(2):85-90.
Hauser P V, Pippin J W, Kaiser C, Krofft R D, Brinkkoetter P T, Hudkins K L, Kerjaschki D, Reiser J, Alpers C E, Shankland S J (2010) Novel siRNA delivery system to target podocytes in vivo. Plos one 5(3):e9463.
Higuchi Y, Kawakami S, Hashida M (2010) Strategies for in vivo delivery of siRNAs. Biodrugs 24(3): 195-205.
Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010;60(5):277-300.
Judge A, MacLachlan I. Overcoming the innate immune response to small interfering RNA. Hum Gene Ther 2008;19(2):lll-24.
Kaiser PK, Symons RC, Shah SM, et al. RNAi-based treatment for neovascuiar age-related macular degeneration by sirna-027. Am J Ophthalmol 2010;150(l):33,39.e2.
Karp JE, Vener Tl, Raponi M, et al. Multi-institutional phase 2 clinical and pharmacogenomic trial of tipifarnib plus etoposide for elderly adults with newly diagnosed acute myelogenous leukemia. Blood 2012;119(l):55-63.
Kies MS, Harari PM. Cetuximab (imclone/merck/bristol-myers squibb). Curr Opin Investig Drugs 2002,-3(7):1092-100.
Kumar P, Ban HS, Kim SS, et al. T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell 2008;134(4):577-86.
Lambert JM. Drug-conjugated antibodies for the treatment of cancer. Br J Clin Pharmacol 2012.
Lee, Y.K., Kim, K.S., Kim, J.S., Baek, J.E., Park, S., Jeong, H.Y., Yoon, S.S., Jung, K.C., Song, H.G., and Park, Y.S. (2010). Leukemia-specific siRNA delivery by immunonanoplexes consisting of anti-JL 1 minibody conjugated to oligo-9 Arg-peptides. Mol. Cells 29,457-462.
Meade, B.R., and Dowdy, S.F. (2007). Exogenous siRNA delivery using peptide transduction domains/cell penetrating peptides. Adv. Drug Deliv. Rev. 59,134-140.
Misale S, Yaeger R, Hobor S, et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 2012;486(7404):532-6.
Oh Y-K and Park T.G. (2009) siRNA delivery systems for cancer treatment. Adv. Drug Delivery Rev. 61: 850-862.
Okudela K, Hayashi H, Ito T, et al. K-ras gene mutation enhances motility of immortalized airway cells and lung adenocarcinoma cells via akt activation: Possible contribution to non-invasive expansion of lung adenocarcinoma. Am i Pathol 2004;164(1):91-1Q0.
Peer, D., and lieberman, J. (2011). Special delivery: targeted therapy with small RNAs. Gene Ther. 18, 1127-1133.
Petrocca F, Lieberman J. Promise and challenge of RNA interference-based therapy for cancer. J Clin Oncol 2010,-29(6):747-54.
Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002;418(6901):934.
Rossi, JJ. (2005). Protamine-antibody fragment fusions enable delivery of siRNAs to specific cells in mice. Nat. Biotechnol. 23,682-684.
Riddles PW, Blakeley RL, Zerner B (1983). "Reassessment of Ellman's reagent". Meth. Enzymol, Methods in Enzymology 91:49-60
Riener CK, Kada G, Gruber HJ (2002). "Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4'-dithiodipyridine". Anal Bioanal Chem 373 (4-5): 266-76.
Rothdiener, M., Muller, D., Castro, P.G., Scholu, A., Schwemmlein, M., Fey, G., Heidenreich, 0., and Kontermann, R.E. (2010). Targeted delivery of SiRNA to CD33-positive tumor cells with liposomal carrier systems. J. Control. Release 144,251-258.
Schmidt LH, Spieker T, Koschmieder S, et al. The long noncoding MALAT-1 RNA indicates a poor prognosis in non-small cell lung cancer and induces migration and tumor growth. J Thorac Oncol 2011,-6(12):1984-92.
Scott AM, Allison JP, Wolchok JD. Monoclonal antibodies in cancer therapy. Cancer Immun 2012,12:14.
Sedlak J, Lindsay RH (1968). "Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent". Anal. Biochem. 25 (1): 192-205.
Simt L, Pratesi N, Vignoli M, et al. High-resolution melting analysis for rapid detection of KRAS, BRAF, and PIK3CA gene mutations in colorectal cancer. Am J Clin Pathol 2008;130(2):247-53
Sunada H, Magun BE, Mendelsohn J, MacLeod CL. Monoclonal antibody against epidermal growth factor receptor is internalized without stimulating receptor phosphorylation. Proc Natl Acad Sci U S A 1986;83(ll):3825-9.
Song E, Zhu P, Lee SK, et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat Biotechnol 2005;23(6):709-17.
Verma S, Miles D, Gianni L, et al. Trastuzumab emtansine for HER2-positive advanced breast cancer. N Engl J Med 2012;367(19):1783-91.
Wang, J., Lu, 1., Wientjes, M.G., and Au, J.L.S (2010). Delivery of siRNA therapeutics: Barriers and carriers. APPS12,492-503.

Claims (48)

1. A method for producing a molecule comprising: i) a peptide comprising a binding domain capable of binding to a cell surface molecule, which is internalized; ii) a nucleic acid molecule; ni) a hetero- or homobifunctional linker; iv) a positively charged molecule wherein the production comprises: a. coupling of the positively charged molecule to the hetero- or homobifunctional linker; b. coupling of at least one thiol, amino or carboxylic acid group of the peptide with the hetero- or homobifunctional linker coupled to the positively charged molecule obtained in a; c. non-covalent binding of the nucleic acid molecule to the positively charged molecule.
2. The method of claim 1, wherein in a) said coupling to the hetero- or homobifunctional linker is achieved via a NH2-group of said positively charged molecule.
3. The method of claim 1, wherein in a) said coupling to the hetero- or homobifunctional linker is achieved via a thiol group of said positively charged molecule.
4. The method of claim 1, wherein in a) said coupling to the hetero- or homobifunctional linker is achieved via a carboxyl group of said positively charged molecule.
5. The method of one of the preceding claims, wherein the coupling in b) is a direct reaction.
6. The method of one of the preceding claims, wherein the hetero- or homobifunctional linker has no cleavable disulfide bond (S-S).
7. The method of one of the preceding claims, wherein there is no purification after the coupling in a).
8. The method of one of the preceding claims, wherein the peptide is a oligopeptide, polypeptide or protein.
9. The method of one of the preceding claims, wherein the peptide is an antibody.
10. The method of claim 9, wherein the antibody is a single-chain antibody or a Fab-fragment of an antibody.
11. The method of claim 9, wherein the antibody is selected from the group consisting of anti-EGFR antibody, anti-CD44 antibody, anti-EpCAM antibody, anti-CD33 antibody, anti-CD117 antibody.
12. The method of claim 11, wherein the anti-EGFR antibody is Cetuximab.
13. The method of one of the preceding claims, wherein the nucleic acid molecule is selected from siRNA or miRNA.
14. The method of claim 13, wherein the nucleic acid molecule is selected from EZH2 siRNA, KRAS siRNA, BRAF siRNA, MEK1 siRNA, CDK1 siRNA, CDK4 siRNA, CDK6 siRNA, FLT3 siRNA, MU. siRNA, CSF1R siRNA, hAES siRNA.
15. The method of claim 14, wherein the KRAS siRNA is KRAS esiRNA.
16. The method of one of the preceding claims, wherein the positively charged molecule is selected from the group consisting of protamine, Sso7d, histones, poly Lysine, poly Arginine preferably (Arg)9, avidin, synthetic polypeptides, synthetic cationic polymers, carbon nanotubes modified to comprise a net positive charge.
17. The method of one of claim 16, wherein the synthetic polypeptides Is polyetheleneimin.
18. The method of claim 16, wherein the positively charged molecule is protamine.
19. The method of one of the preceding claims, wherein the quantity of free thiol groups of the peptide and/or the positively charged molecule is determined by an adequate assay.
20. A molecule obtainable by a method according to any one of claims 1-19.
21. A molecule comprising: i) a peptide comprising a binding domain capable of binding to a cell surface molecule, which is internalized; ii) a positively charged molecule; iti) a heterobifunctional linker, with one portion coupled to the peptide and another portion coupled to the positively charged molecule, wherein at least one thiol, amino or carboxylic acid group of the peptide is coupled to a portion of the heterobifunctional linker; iv) a nucleic acid molecule; wherein the nucleic acid molecule is non-covalently bound to the positively charged molecule.
22. A molecule comprising: i) a peptide comprising a binding domain capable of binding to a cell surface molecule, which is internalized; ii) a positively charged molecule; iii) a homobifunctional linker, with one portion coupled to the peptide and another portion coupled to the positively charged molecule, wherein at least one thiol, amino or carboxylic acid group of the peptide is coupled to a portion of the homobifunctional linker; iv) a nucleic acid molecule; wherein the nucleic acid molecule Is non-covalently bound to the positively charged molecule.
23. The molecule of any of claims 20,2122, wherein the molecule comprises per lmol peptide lmol positively charged molecule and 2mol nucleic acid molecule.
24. The molecule of any of claims 20,2122, wherein the molecule comprises per lmol peptide lmol positively charged molecule and 6mol nucleic acid molecule.
25. The molecule of any of claims 20,2122, wherein the molecule comprises per lmol peptide 3mol positively charged molecule and 2mol nucleic acid molecule.
26. The molecule of any of claims 20,2122, wherein the molecule comprises per lmol peptide 3mol positively charged molecule and/or 6mol nucleic acid molecule.
27. The molecule of any of claims 20,2122, wherein the molecule comprises per lmol peptide 3 mol positively charged molecule and lOmol nucleic acid molecule.
28. The molecule of any of claims 20,2122, wherein the molecule comprises per lmol peptide 7mol positively charged molecule and 6mol nucleic acid molecule.
29. The molecule of any of claims 20, 2122, wherein the molecule comprises per lmol peptide 7mol positively charged molecule and lOmol nucleic acid molecule.
30. The molecule of claim 21 or 22, wherein the coupling of the thiol group in iii) results in a thioether bond.
31. The molecule of claim 21, wherein in iii) said coupling between the portion of the heterobifunctional linker and the positively charged molecule is achieved via a NH2-group of said positively charged molecule.
32. The molecule of claim 31, wherein said coupling via the NH2-group results in an amine bond.
33. The molecule of claim 22, wherein in iii) said coupling between the portion of the homobifunctional linker and the positively charged molecule is achieved via a thiol-group of said positively charged molecule.
34. The molecule of claim 33, wherein the coupling of the thiol group results in a thioether bond.
35. A pharmaceutical composition comprising the molecule of any one of claims 20-34.
36. The molecule of any of claims 20-34 or the pharmaceutical composition of claim 35, for use in a method for treating cancer in a subject.
37. The molecule of any of claims 20-34 or the pharmaceutical composition of claim 35, for use in a method for inhibiting and/or controlling tumor growth in a subject.
38. The molecule or the pharmaceutical composition for use of claim 36, wherein the cancer is a therapy resistant cancer mediated by mutant KRAS.
39. The molecule or the pharmaceutical composition for use of claim 36, wherein the cancer is a carcinoma, sarcoma, lymphoma or leukemia, germ cell tumors, blastoma or metastasis.
40. A molecule of any of claims 20-34 or the pharmaceutical composition of claim 35, for delivering a nucleic acid molecule to the site of a tumor in a subject.
41. The molecule or the pharmaceutical composition for the use of claim 40, wherein said nucleic acid is a siRNA targeting KRAS and/or EZH2
42. The molecule or the pharmaceutical composition for use of claim 41, wherein said siRNA reduces KRAS expression of a cell.
43. The molecule or the pharmaceutical composition for the use of claim 41, wherein said siRNA molecule reduces EZH2 expression of a cell.
44. The molecule or the pharmaceutical composition for the use of claim 41-43, wherein the cell is present in a subject.
45. The molecule or the pharmaceutical composition for use of claim 44, wherein the subject is a mammal, preferably a human being.
46. A preparation obtainable by a method of any of claims 1-19.
47. A preparation comprising a molecule of any of claims 20-34.
48. A kit comprising one or more coupling buffer/reagents and protocol suitable for performing the method of any one of claims 1-19.
LU92353A 2014-01-14 2014-01-14 Antibody-mediated delivery of RNAI LU92353A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
LU92353A LU92353A1 (en) 2014-01-14 2014-01-14 Antibody-mediated delivery of RNAI

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
LU92353 2014-01-14
LU92353A LU92353A1 (en) 2014-01-14 2014-01-14 Antibody-mediated delivery of RNAI

Publications (1)

Publication Number Publication Date
LU92353A1 true LU92353A1 (en) 2015-07-15

Family

ID=50102155

Family Applications (1)

Application Number Title Priority Date Filing Date
LU92353A LU92353A1 (en) 2014-01-14 2014-01-14 Antibody-mediated delivery of RNAI

Country Status (1)

Country Link
LU (1) LU92353A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022117731A1 (en) 2020-12-02 2022-06-09 Westfälische Wilhelms-Universität Münster Electrostatic nanoparticles and use thereof
WO2022243518A1 (en) 2021-05-21 2022-11-24 Westfälische Wilhelms-Universität Münster Antibody-protamine fusions as targeting compounds of a protamine-based nanoparticle

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000046384A1 (en) * 1999-02-08 2000-08-10 University Of Maryland, Baltimore Nucleic acid uptake and release vehicle
WO2002000914A2 (en) * 2000-06-23 2002-01-03 Huston James S Bioengineered vehicles for targeted nucleic acid delivery
US20030143217A1 (en) * 1994-03-15 2003-07-31 Selective Genetics, Inc. Compositions containing nucleic acids and ligands for therapeutic treatment
US20100310670A1 (en) * 2005-09-02 2010-12-09 Takeda Pharmaceutical Company Limited Sustained-release microsphere containing short chain deoxyribonucleic acid or short chain ribonucleic acid and method of producing the same
WO2011087548A2 (en) * 2009-10-27 2011-07-21 William Marsh Rice University Therapeutic compositions and methods for targeted delivery of active agents
WO2013170071A1 (en) * 2012-05-09 2013-11-14 Gradalis, Inc. Bi-functional short-hairpin rna (bi-shrna) specific for single-nucleotide kras mutations

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030143217A1 (en) * 1994-03-15 2003-07-31 Selective Genetics, Inc. Compositions containing nucleic acids and ligands for therapeutic treatment
WO2000046384A1 (en) * 1999-02-08 2000-08-10 University Of Maryland, Baltimore Nucleic acid uptake and release vehicle
WO2002000914A2 (en) * 2000-06-23 2002-01-03 Huston James S Bioengineered vehicles for targeted nucleic acid delivery
US20100310670A1 (en) * 2005-09-02 2010-12-09 Takeda Pharmaceutical Company Limited Sustained-release microsphere containing short chain deoxyribonucleic acid or short chain ribonucleic acid and method of producing the same
WO2011087548A2 (en) * 2009-10-27 2011-07-21 William Marsh Rice University Therapeutic compositions and methods for targeted delivery of active agents
WO2013170071A1 (en) * 2012-05-09 2013-11-14 Gradalis, Inc. Bi-functional short-hairpin rna (bi-shrna) specific for single-nucleotide kras mutations

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHRISTOPHER HOM ET AL: "Mesoporous Silica Nanoparticles Facilitate Delivery of siRNA to Shutdown Signaling Pathways in Mammalian Cells", SMALL, vol. 6, no. 11, 6 June 2010 (2010-06-06), pages 1185 - 1190, XP055107183, ISSN: 1613-6810, DOI: 10.1002/smll.200901966 *
MASASHI TAKAWA ET AL: "Validation of the histone methyltransferase EZH2 as a therapeutic target for various types of human cancer and as a prognostic marker", CANCER SCIENCE, vol. 102, no. 7, 26 May 2011 (2011-05-26), pages 1298 - 1305, XP055136914, ISSN: 1347-9032, DOI: 10.1111/j.1349-7006.2011.01958.x *
SCHIFFELERS RAYMOND M ET AL: "Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, GB, vol. 32, no. 19, 2004, pages e149, XP002397466, ISSN: 0305-1048, DOI: 10.1093/NAR/GNH140 *
SHIMIZU N ET AL: "IMMUNOGENE APPROACH TOWARD CANCER THERAPY USING ERYTHROCYTE GROWTH FACTOR RECEPTOR-MEDIATED GENE DELIVERY", CANCER GENE THERAPY, APPLETON & LANGE, GB, vol. 3, no. 2, March 1996 (1996-03-01), pages 113 - 120, XP001069258, ISSN: 0929-1903 *
STEFANIE DERER ET AL: "Oncogenic KRAS impairs EGFR antibodies' efficiency by C/EBP[beta]-dependent suppression of EGFR expression", NEOPLASIA (NEW YORK, N.Y.), vol. 14, no. 3, March 2012 (2012-03-01), Canada, pages 190, XP055136911, DOI: 10.1593/neo.111636 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022117731A1 (en) 2020-12-02 2022-06-09 Westfälische Wilhelms-Universität Münster Electrostatic nanoparticles and use thereof
WO2022243518A1 (en) 2021-05-21 2022-11-24 Westfälische Wilhelms-Universität Münster Antibody-protamine fusions as targeting compounds of a protamine-based nanoparticle

Similar Documents

Publication Publication Date Title
JP7420866B2 (en) Nucleic acid polypeptide compositions and uses thereof
CN114375296A (en) Nucleic acid polypeptide compositions and uses thereof
US12220604B2 (en) Treatment of metastatic brain tumor by administration of an antibody-drug conjugate
JP7090343B2 (en) Delivery system for targeted delivery of therapeutically active payload
US11666664B2 (en) Self assembling molecules for targeted drug delivery
JP7177131B2 (en) Compositions and methods for inducing apoptosis
KR20220161378A (en) Compositions and methods for the treatment of muscular dystrophy
WO2024077277A1 (en) Drug linkers and antibody conjugates thereof
WO2015191590A2 (en) Combination therapies targeting tumor-associated stroma or tumor cells and microtubules
LU92353A1 (en) Antibody-mediated delivery of RNAI
US20240024500A1 (en) Electrostatic nanoparticles and use thereof
WO2015191583A2 (en) Combination therapies targeting tumor-associated stroma or tumor cells and topoisomerase
US20230065416A1 (en) Clathrin-chimetic antibody receptor constructs for immune cell activation therapy in vivo
US20240316213A1 (en) Antibody-protamine fusions as targeting compounds of a protamine-based nanoparticle
CN116801909A (en) Electrostatic nanoparticles and uses thereof
WO2024204685A1 (en) Pharmaceutical composition for treating and/or preventing cancer
Moura Paiva Development of active tumor targeting systems for delivery of siRNA and drugs into breast and colorectal cancer
Gangar Self-Assembled Nanostructures For The Cellular Delivery Of Small Molecules, Oligonucleotides And Proteins For The Treatment Of Cancer