WO2003076465A2 - Downregulation of ca 125 tumor antigen and uses thereof - Google Patents
Downregulation of ca 125 tumor antigen and uses thereof Download PDFInfo
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- WO2003076465A2 WO2003076465A2 PCT/CA2003/000341 CA0300341W WO03076465A2 WO 2003076465 A2 WO2003076465 A2 WO 2003076465A2 CA 0300341 W CA0300341 W CA 0300341W WO 03076465 A2 WO03076465 A2 WO 03076465A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
Definitions
- This invention relates to CA 125 tumor antigen. More specifically, it relates to modulators of CA 125 tumor antigen and to their uses in the treatment and prevention of diseases wherein CA 125 tumor antigen is overexpressed.
- Ovarian cancer is one of the leading causes of death in women over 40. Although most patients respond to initial treatment, the majority relapses partially due to the appearance of chemo-resistant tumor cells. In order to improve therapy, it is essential to understand the underlying mechanisms responsible for the occurrence of ovarian cancer.
- CA125 antigen is the most important clinical marker of ovarian cancer
- CA125 tumor antigen is the most important clinical marker of ovarian cancer as it is used to monitor response to chemotherapy. Rising or falling blood levels of CA125 correlate with progression or regression of the disease.
- CA125 antigen was first detected in the early 80's using the MAb OC125 which was raised against the human ovarian carcinoma cell line OV433 isolated from a patient with serous papillary cystadenocarcinoma (1).
- the specific reactivity of the OC125 Mab to a variety of human ovarian carcinoma cell lines and paraffin-embedded ovarian carcinoma tissues has led to the development of a radioimmunoassay to detect the CA125 antigen in serum from ovarian cancer patients (2).
- CA125 is a high molecular weight glycoprotein having properties of a mucin-type molecule (15). In these studies however, a definite consensus regarding the molecular nature of CA 125 could not be elaborated and no information about its function was provided.
- CA125 A partial cDNA encoding CA125 was recently identified as MUC16.
- the deduced amino acid sequence proposed an extracellular domain composed of 9 tandem repeats rich in serine, threonine and proline followed by a unique region, a potential transmembrane domain and a short cytoplasmic tail.
- CA125 is expressed in more than 80% of epithelial ovarian cancer but is not detectable in normal ovary tissues. However its role in the disease is unknown.
- An object of the present invention is to provide a therapeutic target that satisfies the above mentioned need.
- the present inventors developed a novel strategy to study the role of proteins that could not be previously studied because the gene was not known or not available. Using this strategy, they derived unique modulators of CA125 tumor antigen.
- the present inventors propose a functional link between CA125 tumor antigen and the pathogenesis of ovarian cancer as well as other diseases where CA 125 tumor antigen is overexpressed, a non exclusive list of which comprises endometriosis, cervical cancer, fallopian tube cancer, cancer of the uterus, prostate cancer and lung cancer.
- the inventors' results have lead to the identification of CA 125 tumor antigen and CA 125 tumor antigen function as novel therapeutic targets for the treatment and prevention of these diseases in mammals.
- an object of the present invention provides for a modulator capable of negatively modulating a CA 125 tumor antigen in a mammalian cell.
- the modulator negatively modulates cell surface expression of CA 125 tumor antigen.
- the modulator sequesters CA 125 tumor antigen or a fragment thereof within an organelle of a mammalian cell, such as the endoplasmic reticulum, the trans-golgi, the golgi, the mitochondrion, the cytoplasm or any other cellular compartment.
- the modulator is a single-chain antibody that specifically binds to CA 125 tumor antigen or a fragment thereof.
- the single-chain antibody is preferably derived from the OC 125 monoclonal antibody or VK-8 monoclonal antibody.
- the single-chain antibody preferably comprises a fragment coded by at least one sequence of the group consisting of SEQ. ID NOS 1 to 6.
- a recombinant nucleic acid comprising at least one sequence selected from the group consisting of SEQ ID NOS 1 to 6.
- a vector comprising a recombinant nucleic acid as defined by the present invention.
- a host cell comprising at least one element selected from the group consisting of a modulator as defined by the present invention, a recombinant nucleic acid as defined by the present invention and a vector as defined by the present invention.
- a pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one element selected from the group consisting of a modulator as defined by the present invention, a recombinant nucleic acid as defined by the present invention, a vector as defined by the present invention, and a host cell as defined by the present invention.
- a seventh object of the present invention there is provides a use of at least one element selected from the group consisting of a modulator as defined by the present invention, a recombinant nucleic acid as defined by the present invention, a vector as defined by the present invention, a host cell as defined by the present invention and a pharmaceutical composition as defined by the present invention for preventing and treating a CA 125-tumor-antigen-associated disease in a mammal.
- a method of prevention or treatment of a CA 125-tumor-antigen-associated disease in a mammal comprising the step of administrating to that mammal at least one element selected from the group consisting of a modulator as defined by the present invention, a recombinant nucleic acid as defined by the present invention, a vector as defined by the present invention, and a host cell as defined by the present invention.
- a ninth object of the present invention there is provided a method for negatively modulating a CA 125 tumor antigen in a mammalian cell comprising the step of introducing into that cell at least one element selected from the group consisting of a modulator as defined by the present invention, a recombinant nucleic acid as defined by the present invention, and a vector as defined by the present invention.
- Figure 1 shows the proposed structure of the CA 125 tumor antigen.
- Figures 2 A and B show the construction of an ScFv library.
- Figures 3 A and B show the selection of soluble ScFvs through a "colony lift assay".
- Figure 4 shows selection of soluble ScFvs (periplasmic extracts)
- FIG. 5 shows expression of ScFvs (ELISA).
- Figure 6 shows expression of ScFvs in the pCantab-5E prokaryotic expression system with and without induction.
- Figure 7 shows selection of ScFvs binding to CA 125 (ELISA).
- Figure 8 shows selection of ScFvs binding to CA 125 (ELISA).
- Figure 9 shows cloning of ScFvs binding to CA 125 in eukaryotic expression system.
- Figures 10 A, B, C and D show Western blots showing expression of ScFvs directed to the golgi in OVCAR-3 and directed to the ER in OVCAR-3.
- Figure 11 shows expression of ScFv OC125 golgi 3.11 compared with expression ⁇ of CA 125.
- Figure 12 show expression of ScFv VK-8 KDEL 1.9 compared with expression of CA 125.
- Figure 13 shows expression of control linker compared with expression of CA 125.
- Figure 14 shows expression of ScFv in golgi and expression of ScFv in ER compared with expression of proteins native to golgi and ER, respectively.
- Figure 15 illustrates construction and in vitro validation of anti-CA125 scFvs.
- NIH:OVCAR-3 cells were transiently transfected with pSTCF.Golgi-OC125-3.11 or pSTCF.KDEL-VK-8-1.9 constructs and 48hrs later the cells were fixed in ice- cold methanol. Localization of scFvs was detected with the anti-c-myc A14 polyclonal antibody and compared with ER and Golgi residents using anti- calreticulin PA3-900 and anti-ADP ribosylation factor MA3-060 monoclonal antibodies, respectively. Oregon green anti-rabbit and texas red anti-Mouse secondary antibodies were used.
- NIH:OVCAR-3 cells were transiently transfected with pSTCF.Golgi-OC125-3.11 or pSTCF.KDEL-VK-8-1.9 constructs and 48hrs later the cells were fixed in ice- cold methanol.
- Expression of scFvs and CA125 was detected using the anti-c- myc A14 polyclonal antibody and anti-CA125 M11 monoclonal antibody. Oregon green anti-rabbit and Texas red anti-mouse secondary antibodies were used.
- Figure 17 shows Cell surface down modulation of CA125 in stable NIH:OVCAR- 3 clones expressing the ER-VK-8-1.9 anti-CA125scFv and relevant control.
- Stable transfectants expressing the ER-targeted VK-8-1.9 and VK-8-4.5 scFvs and parental cell line NIH:OVCAR-3 were fixed in ice-cold methanol generated and expression of CA125 at the cell surface and scFv was assesed by immunofluorescence using anti-c-myc A14 polyclonal antibody and anti-CA125 M11 monoclonal antibody, respectively. Oregon green anti-rabbit and Texas red anti-mouse antibodies were used as secondary antibodies.
- CA125 expression in the stable trasnfectants was analysed by FACS using anti- CA125 M11 monoclonal antibody and a Phyco-Erythrin-anti-mouse antibody and compared with parental cell line NIHOVCAR-3; Black, OVCAR-3 levels of CA125 expression at cell surface; grey, CA125 levels in stable transfectants.
- Figure 18 shows decreased CA125 cell surface expression influences the proliferation rate, cell-cell interaction and cell migration.
- ER-VK-8-4.5#12 and parental cells OVCAR-3 were inoculated subcutaneously in nude mice Tumors were allowed to grow for 6 weeks after which tumorw were excised and tumor weight was measured and plotted for each transfectant.
- Figure 18.1 shows results of clonogenic assay.
- NEDO cDNA clone FLJ 14303 encodes a part of CA125.
- Cos-7 cells negative for CA125 by Western blot and ELISA were transfected with an expression vector encoding the cDNA from the NEDO clone FLJ 14303. Reactivity of anti-CA125
- OC125 and VK-8 antibodies with the expression product of this cDNA was analysed by western blot and compared to CA125 expression in OVCAR-3 cells as well as in mock-transfected Cos-7 cells.
- CA125 cytoplasmic tail was cloned in the pGBDU and pGAD for the yeast two- hybrid system.
- the S.Cerevisiae strain PJ69-4a was transformed with the pGBDU empty vector (EV) or with the vector Containing CA125 cytoplasmic tail
- Figure 20 shows cisplatin sensibility of stable NIH:OVCAR-3 clones expressing the ER-VK-8-1.9 anti-CA125scFv and relevant controls.
- Figure 20.1 shows the relative distribution of cells in each phase of the cell cycle for transfectants ERVK-8-1.9#9 and ERVK-8-4.5#12.
- Figures 20.1A1 to A4 show the different relative profiles of cell cycle progression: graphs 001 to 007 (ERVK-8-4.5#12) and 008 to 014(ERVK-8-1.9#9) at 0, 8, 16, 24, 32, 40 and 48 hrs.
- Figure 20.1 B is a table form of cell cycle progression and shows the relative percentages of cells in each phase of the cell cycle as determined by FACS analysis.
- Figure 21 shows IC50 of cisplatin for the stable NIH:OVCAR-3 clones expressing the ER-VK-8-1.9 anti-CA125scFv and relevant controls. Inhibitory concentrations of cisplatin resulting in 50% survival of cells were calculated from curves of graph in figure 20 (red line in figure 20). .
- Figure 22 shows expression of E-cadherin and ⁇ v ⁇ 5 integrin in NIH:OVCAR-3 cells.
- NIH:OVCAR-3 were fixed in ice-cold methanol generated and expression of E-cadherin and ⁇ v ⁇ integrin at the cell surface was assesed by immunofluorescence using E-cadherin clone 36 and anti- ⁇ v ⁇ 5 integrin clone P1 F6 antibody and Texas red labelled secondary anti-mouse antibody.
- Figure 23 shows expression of E-cadherin and scFv in NIH:OVCAR-3 stable transfectant expressing the ER-VK-8-1.9 anti-CA125 scFv without induction with doxycycline.
- Cells were grown on glass slides for 48hrs and fixed in ice-cold methanol generated and expression of E-cadherin and scFv was assesed by immunofluorescence using E-cadherin clone 36 and anti-c-myc A14 antibody and Texas red or Oregon green-conjugated secondary anti-mouse and anti-rabbit antibodies.
- Figure 24 shows expression of E-cadherin and scFv in NIH:OVCAR-3 stable transfectant expressing the ER-VK-8-1.9 anti-CA125 scFv when induced with doxycycline.
- Cells were grown in presence of doxycycline for 48 hrs and then fixed in ice-cold methanol generated and expression of E-cadherin and scFv was assessed by immunofluorescence using E-cadherin clone 36 and anti-c-myc A14 antibody and Texas red or Oregon green-conjugated secondary anti-mouse and anti-rabbit antibodies.
- Figure 25 shows expression of E-cadherin and scFv in NIH:OVCAR-3 stable transfectant expressing the ER-VK-4.5 control scFv without induction with doxycycline.
- Cells were grown on glass slides for 48hrs and fixed in ice-cold methanol generated and expression of E-cadherin and scFv was assesed by immunofluorescence using E-cadherin clone 36 and anti-c-myc A14 antibody and Texas red or Oregon green-conjugated secondary anti-mouse and anti-rabbit antibodies.
- Figure 26 shows expression of E-cadherin and scFv in NIH:OVCAR-3 stable transfectant expressing the ER-VK-4.5 control scFv when induced with doxycycline.
- Cells were grown on glass slides in the presence of doxycycline for 48hrs and fixed in ice-cold methanol generated and expression of E-cadherin and scFv was assesed by immunofluorescence using E-cadherin clone 36 and anti-c-myc A14 antibody and Texas red or Oregon green-conjugated secondary anti-mo ⁇ se and anti-rabbit antibodies.
- Figure 27 shows expression of ⁇ v ⁇ 5 integrin and scFv in NIH:OVCAR-3 stable transfectant expressing the ER-VK-8-1.9 anti-CA125 scFv when induced or not with doxycycline.
- Cells were grown in the absence or presence of doxycycline for 48hrs and subsequently fixed in ice-cold methanol generated and expression of ⁇ v ⁇ 5 integrin and scFv was assesed by immunofluorescence using anti- ⁇ v ⁇ 5 integrin clone P1 F6, anti-c-myc 9E10 antibody and Texas re or Oregron green labelled secondary antibodies.
- Figure 28 shows expression of ⁇ v ⁇ integrin and scFv in NIH:OVCAR-3 stable transfectant expressing the ER-VK-8-4.5 anti-CA125 scFv when induced or not with doxycycline.
- Cells were grown in the absence or presence of doxycycline for 48hrs and subsequently fixed in ice-cold methanol generated and expression of ⁇ v ⁇ 5 integrin and scFv was assesed by immunofluorescence using anti- ⁇ v ⁇ 5 integrin clone P1 F6, anti-c-myc 9E10 antibody and Texas re or Oregron green labelled secondary antibodies.
- Figure 29 shows alignment of deduced amino acid sequence for anti-CA125 scFvs VK-8-1.9 and OC125-3.11 as well as control scFv VK-8-4.5
- Nucleotidic sequences encoding the anti-CA125 scFvs and their control were determined from pCANTAB5E/scFv constructs using the scFv specific primers S1 and S6 from the pCANTAB ⁇ sequencing primer set (Amersham Pharmacia Biotech, Piscataway, NJ). Sequences were determined using the LI-COR automatic sequencing system (Bio S&T Inc., Lachine, QUE).
- Amino acid sequence was deduced from the nucleotidic sequences and aligned using the Alibee multiple alignment software available at www.qenebee.msu.su/services/malign reduced.html.
- the area in boxes represent consensus sequences of frameworks 1-4 of heavy and light chain, asterisks correspond to differences between VK-8-1.9 and OC125-3.11 anti-CA125 scFvs whereas arrows identify differences between VK-8-1.9 and VK-8-4.5 scFvs.
- Figure 30 shows SEQ ID NO: 1.
- the nucleotide sequence of the linker is underlined.
- Figure 31 shows SEQ ID NO: 2.
- the nucleotide sequence of the linker is underlined.
- Figure 32 shows SEQ ID NO: 3. The 5' to 3' coding sequence of the V H portion of the single-chain antibody VK-8-1.9.
- Figure 33 shows SEQ ID NO: 4.
- Figure 34 shows SEQ ID NO: 5.
- Figure 35 shows SEQ ID NO: 6.
- the present invention is directed to modulators of CA 125 tumor antigen, recombinant nucleic acids, vectors, host cells, pharmaceutical compositions, and methhods of use of the foregeoing for negatively modulating CA 125 tumor antigen in mammalian cell in order to prevent and treat a CA 125 tumor antigen associated disease in a mammal.
- the present invention is directed to modulators capable of negatively modulating of CA 125 tumor antigen in a mammalian cell. More specifically, the modulators of the invention negatively modulate the function and or expression of CA 125 tumor antigen. Negative modulation is to be understood herein therefore as a significant decrease and preferably the abolition of the function and or expression of CA 125 tumor antigen.
- the present inventors show herein that CA 125 tumor antigen is responsible for the promotion of CA 125 associated diseases in mammals. More particularly, the inventors have discovered that CA 125 tumor antigen function and CA 125 tumor antigen expression both constitute novel therapeutic targets for combatting these diseases. The modulators of the present invention are therefore aimed at these novel therapeutic targets which are CA 125 tumor antigen expression and CA 125 tumor antigen function.
- CA125 tumor antigen is a protein associated with the majority of human epithelial ovarian cancers, the most common form of the disease. It is also known to be overexpressed in other diseases such as endometriosis, cervical cancer, cancer of the uterus, fallopian tube cancer, cancer of the endometrium, prostate cancer, lung cancer, etc. The foregoing diseases are therefore non exclusive examples of what is meant by the expression "CA 125 tumor antigen associated diseases".
- the term "mammal” refers to any mammal that is susceptible to a CA 125 tumor antigen associated disease as defined herein. Among the mammals which are known to be potentially affected, are humans.
- the present invention concerns a modulator capable of negatively modulating CA 125 tumor antigen function and or expression in a mammalian cell.
- a modulator may, for instance, negatively modulate the cell surface expression of CA 125 tumor antigen.
- One way of achieving this downregulation of CA 125 tumor antigen cell surface expression is through the sequestration of newly synthesized CA 125 tumor antigens or peptidic fragments thereof within cellular organelle(s) such as the endoplasmic reticulum, the trans-golgi, the golgi, the mitochondrion, the cytoplasm.
- the sequestration may also alternatively be achieved within any other cellular compartment.
- a prefered modulator contemplated by the present invention is a single-chain antibody that specifically binds to CA 125 tumor antigen or a peptidic fragment thereof.
- a single-chain antibody is preferably derived from the OC 125 monoclonal antibody or the VK-8 monoclonal antibody as are the single-chain antibodies generated by the inventors and designated hereinbelow OC 125-3.11 and VK-8-1.9.
- These particular single-chain antibodies contain fragments coded by SEQ ID Nos 2 and 1 respectively ( Figures 31 and 30).
- the invention also concerns any single-chain antibody comprising a fragment coded by any one or more of the sequences of SEQ.
- the present inventors developed the two above mentioned anti-CA125 single-chain antibodies (scFvs), OC 125-3.11 and VK-8-1.9, and show that they act as CA125- specific negative modulators of CA 125 tumor antigen function and expression as further described hereinbelow.
- scFvs single-chain antibodies
- the anti-CA125 scFvs entrap CA125 within the secretion pathway and therefore prevent its proper cell surface localization in the mammalian cells which results in an increased cell proliferation and sensitivity to chemotherapeutic drugs such as cisplatin, reduced cell adhesion and migration and prevents tumor growth in nude mice.
- the inventors show a functional link between CA125 tumor antigen and cell proliferation, sensitivity to drugs such as cisplatin, cell adhesion and cell migration and tumorigenicity.
- the negative modulators of the present invention are to be understood as comprising any negative modulator of CA 125 tumor antigen function and or expression.
- negative modulators may act at different levels: such as the 'transciptional, post-transcriptional, translational or post-translational levels.
- Such negative modulators may be any type of ligand that specifically bind any CA 125 tumor antigen precursor (such as a CA 125 mRNA), or any CA 125 tumor antigen, or fragments of the foregoing.
- Dominant negative molecules are another example of a negative modulator which allows to achieve the negative modulation of CA 125 tumor antigen function and or expression.
- a negative modulator of the present invention may therefore be any molecule which specifically inhibits, blocks, neutralizes, knocks-out, or downregulates CA 125 tumor antigen function and or expression in a mammalian cell. Such a negative modulator may also act upon CA 125 tumor antigen function or expression from the outside of the cell, for instance, through the extracellular portion of CA 125 tumor antigen.
- the present invention is also directed to any recombinant nucleic acid comprising at least one nucleic acid sequence selected from the group consisting of SEQ ID NOS 1 to 6 (figures 30 to 35 respectively).
- nucleic acid sequence As used interchangeably in the present application and which are meant to designate a precise chain of nucleotides, modified or not.allowing the defintion of a fragment or region of a polynucleotide, comprising or not non natural nucleotides, and that may correspond to double-stranded DNA or a single-stranded DNA. This also includes DNA molecules, RNA molecules, cDNA, artificial sequences and all fragments thereof.
- the present invention does not concern nucleotide sequences in their natural chromosome environment, e.g. in the natural state. Rather it concerns purified or isolated sequences, e.g. sequences that have been directly or indirectly obtained, through a process such as cloning, amplification and or chemical synthesis, their environment having therefore been at least partially modified. It is also understood that a polynucleotide which is introduced in an organism by transformation, genetic engineering, or any other recombinant method is "isolated” or "recombinant” even though it is present inside the said organism.
- sequences of the present invention preferably possess a DNA sequence presenting a percentage of identity of 70% or more with one of the sequences of figures 30 to 35.
- the expression "percentage of identity” is meant to indicate a degree of identity between two nucleic acid sequences along the sequences in their entirety. If the particular sequences are of different lengths, the percentage of identity is expressed relatively to the total length of the shortest sequence of the two. In order to calculate the percentage of identity, both sequences are superposed in such a way to maximize the number of identical bases allowing for intervals, the number identical bases is is then divided by the total number of bases of the shortest sequence.
- polypeptide(s) refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
- Polypeptide(s) refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins.
- Polypeptide(s) include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art.
- Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.
- Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, selenoylation, sulfation and transfer-RNA mediated addition of amino acids to proteins
- Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
- the invention is further directed to cloning or expression vector comprising a polynucleotide sequence as defined above, and more particularly directed to a cloning or expression vector which is capable of directing expression of the polypeptide encoded by the polynucleotide sequences of the present invention in a vector-containing cell or host cell.
- vector refers to a polynucleotide construct designed for transduction/transfection of one or more cell types.
- Vectors may be, for example, "cloning vectors” which are designed for isolation, propagation and replication of inserted nucleotides, "expression vectors” which are designed for expression of a nucleotide sequence in a host cell, or a “viral vector” which is designed to result in the production of a recombinant virus or virus-like particle, or “shuttle vectors", which comprise the attributes of more than one type of vector.
- vectors suitable for stable transfection of cells and bacteria are available to the public (e.g. plasmids, adenoviruses, baculoviruses, yeast baculoviruses, plant viruses, adeno-associated viruses, retroviruses, Herpes Simplex Viruses, Alphaviruses, Lentiviruses), as are methods for constructing such cell lines. It will be understood that the present invention encompasses any type of vector comprising any of the polynucleotide molecule of the invention.
- the invention is also directed to a host, such as a genetically modified cell, comprising a modulator of the present invention, and or a vector of the present invention, and or any of the polynucleotide sequences according to the invention and more preferably, a host capable of expressing the polypeptide encoded by this polynucleotide.
- a host such as a genetically modified cell, comprising a modulator of the present invention, and or a vector of the present invention, and or any of the polynucleotide sequences according to the invention and more preferably, a host capable of expressing the polypeptide encoded by this polynucleotide.
- the host cell is any type of cell.
- it is a mammalian cell, such as a cell from an estblished cell line or an isolated primary cell.
- it may be an insect cell, yeast cell (Saccharomyces cerevisiae, Ktuyveromyces lactis, Pichia pastoris), plant cell, microorganism, or a bacterium (such as E. coli).
- the present invention concerns any pharmaceutical composition
- a pharmaceutically acceptable carrier and at least one element selected from the group consisting of a modulator as defined by the present invention, a recombinant nucleic acid as defined by the present invention, a vector as defined by the present invention, and a host cell as defined by the present invention.
- Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
- Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
- parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
- Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
- Administration may begin before the patient is symptomatic. Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intraarterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, by suppositories, or oral administration.
- Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
- the amount of the elements in the composition of the present invention and amounts to be administered is a therapeutically effective amount.
- a therapeutically effective amount is that amount necessary for obtaining beneficial results without causing overly negative secondary effects in the host to which the composition is administered.
- the exact amount of each of the component elements in the composition and amount of the composition to be administered will vary according to factors such as the type of condition being treated, the other ingredients in the composition, the mode of administration, the age and weight of the individual, etc. If necessary, one may refer to the last edition of the Canadian Compendium of Pharmaceuticals & Specialties (CPS).
- the present invention also concerns a use of at least one element selected from the group consisting of a modulator as defined by the present invention, a recombinant nucleic acid as defined by the present invention and a vector as defined by the present invention for negatively modulating a CA 125 tumor antigen in a mammalian cell.
- the present invention concerns a use of at least one element selected from the group consisting of a modulator as defined by the present invention, a recombinant nucleic acid as defined by the present invention, a vector as defined by the present invention, a host cell as defined by the present invention and a pharmaceutical composition as defined by the present invention for preventing and treating a CA 125-tumor-antigen-associated disease in a mammal.
- the present invention concerns a method of prevention or treatment of a CA 125-tumor-antigen-associated disease in a mammal comprising the step of administrating to that mammal at least one element selected from the group consisting of a modulator as defined by the present invention, a recombinant nucleic acid as defined by the present invention, a vector as defined by the present invention, and a host cell as defined by the present invention.
- the term “treating” refers to a process by which the symptoms of a CA 125 tumor antigen associated disease are alleviated or completely eliminated.
- the term “preventing” refers to a process by which a CA 125 tumor antigen associated disease is obstructed or delayed.
- the present invention concerns a method for negatively modulating a CA 125 tumor antigen in a mammalian cell comprising the step of introducing into that cell at least one element selected from the group consisting of a modulator as defined by the present invention, a recombinant nucleic acid as defined by the present invention, and a vector as defined by the present invention.
- EXAMPLE I Construction and in vitro validation of modulators according to a preferred embodiment of the present invention, namely, anti-CA125 scFvs
- the inventors constructed single-chain antibody libraries derived from the OC125 and VK-8 hybridoma cell lines specific for CA125.
- the two scFv libraries were screened for CA125 binding activity by ELISA using commercially purified human CA125. ScFvs that bound to CA125 by ELISA, OC125-3.11 and VK-8-1.9, and one that did not bind, VK-8-4.5 were selected (figure 15 A-B).
- CA125 antigen would be entrapped during synthesis and thus be unable to localize at cell surface and interact with other intracellular and/or extracellular proteins to achieve its function(s).
- the scFvs were targeted to the ER or trans-median Golgi by sequence fusion with an lg ⁇ secretion leader and a KDEL signal or fusion with the N-terminal 81 amino acids of human beta 1 ,4-galactosyltransferase, a protein resident of the trans-medial Golgi (47-49) in addition ot a c-myc tag at the C-terminus.
- EXAMPLE III Consequences of CA125 cell surface down-regulation in human ovarian cancer cell line NIH:OVCAR-3
- the inventors derived stable clones encoding the ER-targeted VK-8-1.9 and Golgi- OC125-3.11 (both positive for CA125 binding) and ER-VK-8-4.5 (negative for CA125 binding) scFvs in human ovarian cancer cell lines OVCAR-3 (high expresser of CA125), OV-90 (moderate expresser) and SKOV3ip1 cells (low expresser).
- OVCAR-3 high expresser of CA125
- OV-90 moderate expresser
- SKOV3ip1 cells low expresser.
- ⁇ v ⁇ v integrin at the cell surface of ER-VK-8-4.5 transfectant is also not affected by the expression of the scFv (figure 28).
- the stable transfectant expressing the ER-VK-8-1.9 anti- CA125 scFv shows a reduced level of ⁇ v ⁇ v integrin at the cell surface demonstrating that CA125 influences levels of ⁇ v ⁇ v integrin expression at the cell surface.
- clonogenic assays were performed to investigate the growth kinetics of each transfectant when seeded at low density on adhesive support. Increasing amounts of each transfectant, VK-8 KDEL/1 :9 # 9 and KDEL/4:5 # 12, and the parental cell line NIH OVCAR-3 were seeded in 6-well plates and grown in the absence or the presence of doxycycline. Fourteen days later, cells were stained with Giemsa. The number of colonies was scored for each transfectant and plotted against the amount of cells seeded.
- Figure 18.1A shows that no difference in the amounts of colonies formed by each transfectant, regardless of CA125 status, confirming that CA125 does not affect the ability of the cells to form colonies when seeded at low density on adhesive support.
- figures 18.1 B to D show that the size of colonies formed by transfectant VK-8-1.9#9 was much bigger and easier to visualize compared to those from transfectant VK-8-4.5#12 and the parental cell line NIH OVCAR-3 demonstrating that transfectant VK-8-1.9#9 grow at an increased rate compared to the controls.
- Stable clone ER-VK-8-1.9#9, the ER-VK-8-4.5#12 and the parental cell lines were plated in triplicate in 96-well plates and exposed or not to increasing concentrations of cisplatin. Fours days later, cell proliferation was measured with a XTT assay. Percentage of survival was plotted against concentration of cisplatin. Curves represent results from 3 independent experiments (figure 20). Results showed that stable clone ER-VK-8-1.9#9 was more sensitive to cispaltin than control cells.
- the inventors also determined the consequence of reducing CA125 expression levels at the cell surface on cell migration.
- Cells were plated in 6-well plates and when confluent a wound was made in the monolayer using a razor blade. To distinguish between cell proliferation and cell migration, cell proliferation was inhibited with 20mM hydroxyurea (53). Cells were incubated in the presence or the absence of FBS. In the absence of FBS none of the cells, neither the parental cells, were able to migrate and fill in the wound (not shown).
- the inventors also determined whether the loss of CA125 expression at the cell surface affects the in vivo behaviour of human ovarian cancer cells in tumor-bearing mice subcutaneously or intraperitoneally. This was achieved by evaluating tumor growth, tumor burden, formation of ascites, presence of tumor cells in ascites, pattern of metastases spread and survival of mice. The tumorigenicity of each stable transfectant was also determined in nude mice. Stable clone ER-VK-8-1.9#9, the ER- VK-8-4.5#12 and the parental cell lines were inoculated subcutaneously in nude mice and tumors were allowed to grow for 6 weeks after which tumors were excised and tumor weight was measured.
- Figure 18D shows that tumor derived from stable clone ER-VK-8-1.9#9 were significantly much smaller (if existant) than those from the ER- VK-8-4.5#12 and the parental cell lines demonstrating that CA125 influence the tumorigenic potential of ovarian cancer cells.
- stable clone ER-VK-8-1.9#9 showed a significant slower growth, reduced volume of ascites, a decrease in the total number of viable tumor cells in suspension (in ascites) and therefore an overall increased in survival of mice (not shown).
- variable heavy and light chains were amplified from the cDNA by PCR using mouse variable region primers.
- the V H and V DNA fragments were linked together by overlap extension PCR using a (Gly Ser) 3 linker to generate 750bp scFv constructs with flanking Sfil and Notl sites.
- the scFv DNA fragments were inserted into Sfil/Notl sites of the prokaryotic expression vector pCANTAB5E from the Mouse ScFv Module (Amersham Pharmacia Biotech, Piscataway, NJ). Screening of recombinant clones expressing a soluble scFv was accomplished by a colony lift assay as described previously (26).
- Plasmids - The phagemid pCANTAB5E/scFv contains the anti-CA 125 scFvs encoding sequences under the control of the inducible lac promoter.
- the scFvs are expressed as fusion proteins with a peptide tag (Etag) at the C-terminus to allow easy immunodetection.
- the ER-targeting vector pSTCF.KDEL was previously described (27).
- the selected anti-CA 125 scFv DNA fragments were subcloned into the Sfil/NotI sites of pSTCF.KDEL just upstream and in frame with the c-myc tag/KDEL sequence.
- the pLTR retroviral expression vector was described in details elsewhere (28).
- the same anti-CA 125 scFv DNA fragments were subcloned into the pLTR.KDEL retroviral plasmid as Sfil/NotI sites after insertion of a Sfil/NotI containing polylinker at Xhol site of pLTR.KDEL.
- expression of the scFvs is under the control of a tetracycline-inducible CMV promoter.
- Binding analysis by ELISA - Periplasmic extracts were prepared as previously described (29). Elisa plates were coated with 10 ⁇ g of purified CA 125 tumor antigen in 200 ⁇ l carbonate buffer pH 9.6 and incubated overnight at 4°C in a humidified chamber. Wells were washed 3 times with 200 ⁇ l PBS and blocked with 200 ⁇ l 2% BSA/PBS-0.05% Tween20 (for VK-8 derivative scFvs) or 200 ⁇ l 2% NFDM/PBS-0.1% Tween20 (for OC125 derivative scFvs) for 1 hr at room temperature in a humidified chamber.
- the plates were air dried, 100 ⁇ l of periplasmic extracts were added in 100 ⁇ l of blocking buffer and the plates were incubated at room temperature for 1 hr in humidified chamber.
- Parental monoclonal antibodies VK-8 and OC125 served as positive control and an anti-Bcl-2 scFv and periplasmic extract from bacteria without plasmid served as negative controls.
- the anti-Bcl-2 scFv was described previously (29).
- mice anti-Etag antibody (Pharmacia Biotech, Piscataway, NJ) was added 1 :1000 in 200 ⁇ l 2% BSA/PBS-0.05% Tween20 or 200DI 2% NFDM/PBS-0.1 % Tween20 and plates were incubated for 1 h at 37°C in a humidified chamber.
- Sequencing of DNA encoding anti-CA 125 scFvs - ScFv encoded sequences were determined from pCANTAB5E/scFv constructs using the scFv specific primers S1 and S6 from the pCANTAB ⁇ sequencing primer set (Amersham Pharmacia Biotech, Piscataway, NJ). Sequences were determined using the LI-COR automatic sequencing system (Bio S&T Inc., Lachine, QUE).
- COS-7 cells The human ovarian cancer NIH:OVCAR-3 and the green monkey kidney COS-7 cell lines were obtained from the American Type Culture Collection (Rockville, MD). COS-7 cells were maintained in F12/DMEM (Biomedia, Drummondville, QUE) supplemented with 10% FBS (Biomedia, Drummondville, QUE), 2mM L-glutamine (Biomedia, Drummondville, QUE), 100 units/ml penicillin (Cie, city, state) and 100 ⁇ g/ml streptomycin.
- F12/DMEM Biomedia, Drummondville, QUE
- FBS Biomedia, Drummondville, QUE
- 2mM L-glutamine Biomedia, Drummondville, QUE
- penicillin Cie, city, state
- streptomycin 100 ⁇ g/ml streptomycin
- NIHOVCAR-3 cells were grown in RPMI 1640 (Biomedia, Drummondville, QUE) supplemented with 20% FBS (Biomedia, Drummondville, QUE), 2mM L-glutamine (Biomedia, Drummondville, QUE), 100 units/ml penicillin, 100 ⁇ g/ml streptomycin and 10 ⁇ g/ml insulin. Both cell lines were maintained at 37°C in a humidified 5% C0 2 incubator.
- Immunoblot analysis - Periplasmic extracts (equal volume), cell lysates (equal amounts of proteins) or immunoprecipitates were submitted to SDS-PAGE electrophoresis (12%) and transferred onto PVDF membrane.
- the membranes were probed with either an anti-Etag antibody (Amersham Pharmacia Biotech, Piscataway, NJ) for periplasmic extracts or the anti-c-myc 9E10 antibody (Cie, city, state) for cell lysates.
- a HRP-conjugated rabbit anti-mouse antibody Jackson ImmunoResearch, West Grove, PA
- the immunoblots were developed with chemiluminescence using commercially available ECL system according to the manufacturer's instruction.
- Immunoprecipitation - NIH-OVCAR-3 cells were transiently transfected with the various scFv constructs using commercially available FuGene 6 transfection agent using the manufacturer's instruction. Forty-eight hours later, cell were collected and lysed on ice in NP40 buffer (0.5%NP-40, 0.5% sodium deoxycholate, 0.1 % SDS, 100mM NaCI, 1mM EDTA, 20mM Tris-HCI pH 8.0). Protein concentration was measured by the Bradford method using the Bio-Rad protein assay according to the instruction provided by the manufacturer.
- Stable cell lines expressing anti-CA 125 scFvs - The stable cell lines OVCAR3- 1.9#9, OVCAR3-4.5#12 and OVCAR3-GFP were generated by stable transfection of pLTR.KDEL-anti-CA 125 scFv#1.9, 2.7, 4.5 and GFP respectively. Stable clones were selected under 1 ⁇ g/ml blasticidin exposure. Stable transfection of NIH:OVCAR- 3 cells was performed using commercially available FuGene ⁇ transfection reagent following manufacturer's instructions.
- scFvs Localization of the scFvs was determined by comparing their pattern of expression with that of calreticulin and ADP-ribosylation factor for ER and Golgi localization using anti-calreticulin antibody (1 :10000) anti-calreticulin and anti- ADP-ribosylation factor (1 :400).
- CA125 and the anti-CA125 scFvs in the various NIHOVCAR-3 stable clones were analyzed by FACS.
- One million PBS/EDTA-treated cells were fixed in 2% para-formaldehyde for 20 min at room temperature. Cells were next permeabilized with 0.1% saponine for 20 min at room temperature and incubated with primary antibodies in 2%BSA/PBS locking buffer for 45 min at room temperature.
- CA125 and scFv expression was determined using the mouse anti-CA125 M11 antibody (1 :500) and the polyclonal anti-c-myc antibody A14 (1 :500) respectively.
- Cells were next pelleted and incubated with anti-mouse-PE (1 :1000) and anti-rabbit-FITC (1 :500) secondary antibodies in blocking buffer for 45 min at room temperature. Cells were pelleted, resuspended in an appropriate volume and analyzed using a FACS Scan cytometer (Becton Dickenson, Mississauga, Canada).
- Ishiwata I Ishiwata C, Nazawa S
- Ishiwata H CA125 production by gynecologic tumors in vitro and its modulation induced by dibutyl cyclic adenosine monophosphate. Asia Oceania J Obstet Gynecol 1986, 12: 285-290.
- Bonfrer JM Linders TC, Hageman PC, Hilkens JG, Korse CM and Molthoff CFM. Effect of paclitaxel (taxol) on CA125 expression and release by ovarian cancer cell lines. Tumor Biol 1997, 18: 232-240.
- Mucins (MUC1 and MUC3) of gastrointestinal and breast epithelia reveal different and heterogenous tumr-associated aberrations in glycosylation. J Histochem Cytochem 1997, 45: 1547- 57.
- Transgenic MUC1 interacts with epidermal growth factor receptor and correlates with mitogen-activated protein kinase activation in the mouse mammary gland. J Biol Chem 2001 , 276: 13057-13064.
- RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes and Dev 2001 , 15: 188-200.
- Endothelin-1 induces tumor ptoeinase activation and invasiveness of ovarian carcinoma cells. Cancer Res 2001 , 61 : 8340-8346.
Abstract
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AU2003212142A AU2003212142A1 (en) | 2002-03-11 | 2003-03-11 | Downregulation of ca 125 tumor antigen and uses thereof |
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CA002518695A CA2518695A1 (en) | 2002-03-11 | 2003-03-11 | Downregulation of ca 125 tumor antigen and uses thereof |
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US7429382B2 (en) | 2002-10-16 | 2008-09-30 | Corixa Corporation | Antibodies that bind cell-associated CA 125/O772P and methods of use thereof |
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WO1996013594A1 (en) * | 1994-10-28 | 1996-05-09 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Tumor-specific antibody fragments, fusion proteins, and uses thereof |
-
2003
- 2003-02-28 CA CA002420494A patent/CA2420494A1/en not_active Abandoned
- 2003-03-11 WO PCT/CA2003/000341 patent/WO2003076465A2/en not_active Application Discontinuation
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Non-Patent Citations (2)
Title |
---|
HAISMA H J ET AL: "Antibody-antigen complex formation following injection of OC125 monoclonal antibody in patients with ovarian cancer" INTERNATIONAL JOURNAL OF CANCER, NEW YORK, NY, US, vol. 40, no. 6, 15 December 1987 (1987-12-15), pages 758-762, XP002963264 ISSN: 0020-7136 * |
SCHULTES B ET AL: "Induction of CA125-specific B and T cell responses in MAb-B43.13 injected patients depends on complex formation of the MAb with circulating antigen in vivo" FASEB JOURNAL, FED. OF AMERICAN SOC. FOR EXPERIMENTAL BIOLOGY, BETHESDA, MD, US, vol. 14, no. 6, 20 April 2000 (2000-04-20), page A1003 XP002202309 ISSN: 0892-6638 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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US7429382B2 (en) | 2002-10-16 | 2008-09-30 | Corixa Corporation | Antibodies that bind cell-associated CA 125/O772P and methods of use thereof |
EP2298806A1 (en) | 2002-10-16 | 2011-03-23 | Purdue Pharma L.P. | Antibodies that bind cell-associated CA 125/0722P and methods of use thereof |
US8124086B2 (en) | 2002-10-16 | 2012-02-28 | Purdue Pharma L.P. | Antibodies that bind cell-associated CA 125/O772P and methods of use thereof |
US8299230B2 (en) | 2002-10-16 | 2012-10-30 | Purdue Pharma L.P. | Nucleic acid molecules encoding antibodies that bind cell-associated CA 125/O772P |
EP2891666A1 (en) | 2002-10-16 | 2015-07-08 | Purdue Pharma L.P. | Antibodies that bind cell-associated CA 125/O722P and methods of use thereof |
US9676866B2 (en) | 2002-10-16 | 2017-06-13 | Purdue Pharma L.P. | Antibodies that bind cell-associated CA 125/O772P |
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