WO2010043582A1 - Méthode pour le traitement du cancer - Google Patents

Méthode pour le traitement du cancer Download PDF

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WO2010043582A1
WO2010043582A1 PCT/EP2009/063250 EP2009063250W WO2010043582A1 WO 2010043582 A1 WO2010043582 A1 WO 2010043582A1 EP 2009063250 W EP2009063250 W EP 2009063250W WO 2010043582 A1 WO2010043582 A1 WO 2010043582A1
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lna
seq
cancer
antibody
inhibitor
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PCT/EP2009/063250
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Margit Wissenbach
Jens Bo Rode Hansen
Anja Høg
Keith Mccullagh
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Santaris Pharma A/S
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    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C12N2310/11—Antisense
    • 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
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • 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
    • C12N2320/31—Combination therapy

Definitions

  • Leukocyte Biology 72 reports on the modulation of malignant B cell activation and apoptosis by Bcl-2 antisense oligonucleotides and immunostimulatory CpG oligonucleotides, and their results suggested that modulation of B cell apoptosis by G3139 depended on its immunostimulatory properties rather than on antisense-mediated reduction of Bcl-2 expression.
  • WO 2005/061710 discloses 16 nucleobase phosphorothioate LNA gapmers which comprises a target binding domain that is specifically hybridizable to a region ranging from base position No. 1459 (5') to No. 1476 (3') of the human Bcl-2 mRNA (a region corresponding to the first six codons).
  • the invention provides for a method for the treatment of cancer, said method comprising the steps of i) administering an LNA oligomer targeting Bcl-2 and ii) administering an inhibitor of CD-20 to a patient who is in need for the treatment of cancer.
  • Figure 1 Treatment effects on survival time of SCID mice bearing disseminated Raji human lymphoma.
  • the mice received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj or SEQ ID NO 3 (control) at 10 mg/kg/inj for 14 consecutive days from D4 to D17 (Q1 Dx14) alone or in combination with 6 IV injections of Rituximab at 1 mg/kg/inj once a day twice weekly for 3 consecutive weeks at D4, D7, D11 , D14, D18 and D21 (TWx3).
  • Figure 2 Mean hCD45 positive cells in bone marrow of 4 SCID mice out of 12 bearing disseminated Raji human lymphoma at day 18.
  • naturally occurring variant thereof refers to variants of the BCL-2 polypeptide of nucleic acid sequence which exist naturally within the defined taxonomic group, such as mammalian, such as mouse, rat, monkey, and preferably human.
  • the term also may encompass any allelic variant of the BCL-2 encoding genomic DNA which are found at the Chromosome 18: 58.94 - 59.14 Mb (NM 000633) by chromosomal translocation or duplication, and the RNA, such as mRNA derived therefrom.
  • “Naturally occurring variants” may also include variants derived from alternative splicing of the BCL-2 mRNA.
  • the contiguous nucleotide sequence comprises no more than a single mismatch when hybridizing to the target sequence, such as the corresponding region of a nucleic acid which encodes a mammalian BCL-2.
  • the nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 90% homologous to the reverse complement of a corresponding sequence present in SEQ ID NO: 1 , such at least 91 %, at least 92%at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97%, at least 98%, at least 99%, such as 100% homologous (identical).
  • R 1* , R 2 , R 3 , R 5 , R 5* are hydrogen.
  • R 1* , R 2 , R 3 are independently selected from the group consisting of hydrogen, halogen, Ci -6 alkyl, substituted Ci -6 alkyl, C 2-6 alkenyl, substituted C 2-6 alkenyl, C 2- 6 alkynyl or substituted C 2- 6 alkynyl, Ci -6 alkoxyl, substituted Ci -6 alkoxyl, acyl, substituted acyl, C 1-6 aminoalkyl or substituted C 1-6 aminoalkyl. For all chiral centers, asymmetric groups may be found in either R or S orientation.
  • R 1* , R 2 , R 3 are hydrogen.
  • R 1* , R 2 , R 3 , R 5 , R 5* are independently selected from the group consisting of hydrogen, halogen, C 1-6 alkyl, substituted C 1-6 alkyl, C 2-6 alkenyl, substituted C 2-6 alkenyl, C 2-6 alkynyl or substituted C 2-6 alkynyl, C 1-6 alkoxyl, substituted C 1-6 alkoxyl, acyl, substituted acyl, C 1-6 aminoalkyl or substituted C 1-6 aminoalkyl.
  • R 1* , R 2 , R 3 , R 5 , R 5* are independently selected from the group consisting of hydrogen, halogen, Ci -6 alkyl, substituted Ci -6 alkyl, C 2-6 alkenyl, substituted C 2-6 alkenyl, C 2-6 alkynyl or substituted C 2-6 alkynyl, Ci -6 alkoxyl, substituted Ci -6 alkoxyl, acyl, substituted acyl, Ci -6 aminoalkyl or substituted Ci -6 aminoalkyl.
  • R 1* , R 2 , R 3 , R 5 , R 5* are hydrogen.
  • LNA is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particular beta-D-oxy-LNA.
  • an oligomeric compound may function via non RNase mediated degradation of target mRNA, such as by steric hindrance of translation, or other methods, however, the preferred oligomers of the invention are capable of recruiting an endoribonuclease (RNase), such as RNase H.
  • RNase endoribonuclease
  • the oligomer of the invention may comprise or consist a nucleotide sequence which comprises both DNA nucleotides and LNA nucleotide, and may be in the form of a gapmer.
  • region A consists of 1 , 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as between 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units; and/or region C consists of 1 , 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as between 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units.
  • LNA units such as between 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units.
  • B consists or comprises of 5, 6, 7, 8, 9, 10, 11 or 12 consecutive nucleotides which are capable of recruiting RNAse, or between 6-10, or between 7-9, such as 8 consecutive nucleotides which are capable of recruiting RNAse.
  • region B consists or comprises at least one DNA nucleotide unit, such as 1-12 DNA units, preferably between 4-12 DNA units, more preferably between 6-10 DNA units, such as between 7-10 DNA units, most preferably 8, 9 or 10 DNA units.
  • the 5' monomer at the end of an oligomer does not comprise a 5' linkage group, although it may or may not comprise a 5' terminal group.
  • linkage group or "internucleotide linkage” are intended to mean a group capable of covalently coupling together two nucleotides, two nucleotide analogues, and a nucleotide and a nucleotide analogue, etc. Specific and preferred examples include phosphate groups and phosphorothioate groups.
  • all remaining linkage groups are either phosphodiester or phosphorothioate, or a mixture thereof.
  • all the internucleotide linkage groups are phosphorothioate.
  • linkages are phosphorothioate linkages
  • alternative linkages such as those disclosed herein may be used, for example phosphate (phosphodiester) linkages may be used, particularly for linkages between nucleotide analogues, such as LNA, units.
  • phosphate (phosphodiester) linkages may be used, particularly for linkages between nucleotide analogues, such as LNA, units.
  • C residues are annotated as 5'methyl modified cytosine
  • one or more of the Cs present in the oligomer may be unmodified C residues.
  • the oligomers of the invention may, for example, be selected from the group consisting of the LNA oligomers targeting the human Bcl-2 shown in table 1.
  • the following LNA oligomers targeting Bcl-2, shown in Table 1 are disclosed in WO2005/061710, and US provisional application US 61/012185, which are both hereby incorporated by reference.
  • the oligomeric compounds are referred to by means of the specified sequence number, e.g. "SEQ ID NO: 2".
  • the compound "SEQ ID NO: 4" is also called Oblimersen sodium and is used herein as a reference compound.
  • the oligomer is selected from the group consisting of 2, and 5 - 34. In some embodiments the oligomer is SEQ ID NO 2. In some embodiments the oligomer is SEQ ID NO 12. In some embodiments is selected from an oligo of SEQ ID 35 - 67. In some embodiments the oligomer is selected from the group consisting of SEQ ID NO 36, 37, 38, 42, 43, 50 and 61. In some embodiments the oligomer is SEQ ID NO 36. In some embodiments the oligomer is SEQ ID NO 37. In some embodiments the oligomer is SEQ ID NO 38. In some embodiments the oligomer is SEQ ID NO 42. In some embodiments the oligomer is SEQ ID NO 43. In some embodiments the oligomer is SEQ ID NO 50. In some embodiments the oligomer is SEQ ID NO 61.
  • the oligomers of the invention may also be conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • active drug substances for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • the conjugated moiety is a sterol, such as cholesterol.
  • the conjugated moiety comprises or consists of a positively charged polymer, such as a positively charged peptides of, for example between 1 -50, such as 2 - 20 such as 3 - 10 amino acid residues in length, and/or polyalkylene oxide such as polyethylglycol(PEG) or polypropylene glycol - see WO 2008/034123, hereby incorporated by reference.
  • a positively charged polymer such as a positively charged peptides of, for example between 1 -50, such as 2 - 20 such as 3 - 10 amino acid residues in length
  • polyalkylene oxide such as polyethylglycol(PEG) or polypropylene glycol - see WO 2008/034123, hereby incorporated by reference.
  • the positively charged polymer, such as a polyalkylene oxide may be attached to the oligomer of the invention via a linker such as the releasable inker described in WO 2008/034123.
  • conjugate moieties may be used in the conjugates of the invention: 5'- OLIGOMER -3'
  • a functional moiety will comprise a chemical group that is capable of covalently bonding to the oligomer via, e.g., a 3'-hydroxyl group or the exocyclic NH 2 group of the adenine base, a spacer that is preferably hydrophilic and a terminal group that is capable of binding to a conjugated moiety (e.g., an amino, sulfhydryl or hydroxyl group).
  • this terminal group is not protected, e.g., is an NH 2 group.
  • the terminal group is protected, for example, by any suitable protecting group such as those described in "Protective Groups in Organic Synthesis" by Theodora W.
  • Suitable hydroxyl protecting groups include esters such as acetate ester, aralkyl groups such as benzyl, diphenylmethyl, or triphenylmethyl, and tetrahydropyranyl.
  • suitable amino protecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl, triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groups such as trichloroacetyl or trifluoroacetyl.
  • the functional moiety is self- cleaving.
  • the functional moiety is biodegradable. See e.g., U.S. Patent No. 7,087,229, which is incorporated by reference herein in its entirety.
  • activated oligomers of the invention are synthesized with monomers that have not been functionalized, and the oligomer is functionalized upon completion of synthesis.
  • the oligomers are functionalized with a hindered ester containing an aminoalkyl linker, wherein the alkyl portion has the formula (CH 2 ) W , wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group is attached to the oligomer via an ester group (-0-C(O)- (CH 2 ) W NH).
  • the oligomers are functionalized with a hindered ester containing a (CH 2 ) w -sulfhydryl (SH) linker, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group attached to the oligomer via an ester group (-O-C(O)-(CH 2 ) W SH)
  • sulfhydryl-activated oligonucleotides are conjugated with polymer moieties such as polyethylene glycol or peptides (via formation of a disulfide bond).
  • Activated oligomers containing hindered esters as described above can be synthesized by any method known in the art, and in particular by methods disclosed in PCT Publication No. WO 2008/034122 and the examples therein, which is incorporated herein by reference in its entirety.
  • such activated oligomers have a functionalizing reagent coupled to a 5'-hydroxyl group of the oligomer. In other embodiments, the activated oligomers have a functionalizing reagent coupled to a 3'- hydroxyl group. In still other embodiments, the activated oligomers of the invention have a functionalizing reagent coupled to a hydroxyl group on the backbone of the oligomer. In yet further embodiments, the oligomer of the invention is functionalized with more than one of the functionalizing reagents as described in U.S. Patent Nos. 4,962,029 and 4,914,210, incorporated herein by reference in their entirety. Methods of synthesizing such functionalizing reagents and incorporating them into monomers or oligomers are disclosed in
  • 5'-Amino-Modifier C6 is also available from ABI (Applied Biosystems Inc., Foster City, Calif.) as Aminolink-2, and 3'-Amino-Modifier is also available from Clontech Laboratories Inc.
  • the CD20 inhibitor may be a CD-20 binding molecule, such as an antibody or fragment thereof.
  • CD20 binding molecules refer to any molecule that specifically binds to a portion of CD20 under cellular and/or physiological conditions for an amount of time sufficient to inhibit the activity of CD20 expressing cells and/or otherwise modulate a physiological effect associated with CD20; to allow detection by ELISA, western blot, or other similarly suitable binding technique described herein and/or known in the art and/or to otherwise be detectably bound thereto after a relevant period of time (for instance at least about 15 minutes, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 12 hours, such as about 1-24 hours, about 1-36 hours, about 1-48 hours, about 1-72 hours, about one week, or longer).
  • the CD20 antibody is Ocrelizumab (Genetch/Roche/Biogen prototype), In some embodiments of the invention the CD20 antibody is Pro131921 (Genentech),
  • WO07059188A reports that 'several anti-CD20 antibodies, including rituximab, have been shown to induce apoptosis in vitro when crosslinked by a secondary antibody or by other means.
  • These anti-CD20 antibodies specifically bind to the CD20 antigen of (ostensibly) both normal and malignant B cells; the antibody bound to the CD20 surface antigen may lead to the destruction and depletion of neoplastic B cells
  • chemical agents or radioactive labels having the potential to destroy the tumor can be conjugated to the anti-CD20 antibody such that the agent is specifically "delivered" to the neoplastic B cells.
  • a primary goal is to destroy the tumor; the specific approach can be determined by the particular anti- CD20 antibody that is utilized, and thus, the available approaches to targeting the CD20 antigen can vary considerably. More recently, rituximab has been shown to have antiproliferative effects in tritiated thymidine-incorporation assays and to induce apoptosis directly, while other anti-CD 19 and anti-CD20 antibodies do not.'
  • the antibody may be a Radioimmunotherapeutic, such as lbritumomab (Zevalin ® )or Tositumomab (Corixa) BEXXAR ® .
  • the CD-20 antibody is Ofatumumab (HUMAX-CD20, Genmab, DK).
  • the CD-20 antibody is Rituximab (US 5,736,137).
  • the CD-20 antibody is Ocrelizumab, also referred to as R1594 - a humanised anti-CD20 monoclonal antibody developed by Roche.
  • the invention provides for a pharmaceutical composition
  • a pharmaceutical composition comprising an inhibitor of CD-20, such as Rituximab, and an LNA oligomer targeting BCL-2, such as SEQ ID NO 2 and a pharmaceutically acceptable diluent, carrier, or salt.
  • PCT/DK2006/000512 provides suitable pharmaceutically acceptable diluent, carrier and adjuvants - which are hereby incorporated by reference.
  • suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in PCT/DK2006/000512 - which are also hereby incorporated by reference.
  • the LNA oligomer may be used in standard therapeutic antibody compositions, such as those used for Rituximab or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 1 1 B8, 7D8, 2C6, Veltuzumab, AME-133v
  • the LNA oligomer, such as SEQ ID NO 2 and the CD-20 inhibitor, such as Rituximab or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 1 1 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan, Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159 and GA-101 , are in independent compartments within the kit.
  • the LNA oligomer is inhibiting (reducing) expression of Bcl-2
  • the CD-20 inhibitor is inhibiting the function of CD-20 in the patient, preferably within (or in the case of surface bound CD-20 on the surface of) the cancer cells within the patient.
  • the concurrent activity of the CD-20 inhibitor such as Rituximab or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 11 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan, Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159 and GA-101 , and the LNA oligomer, such as SEQ ID NO 2, therefore refers to the two active ingredients are in operation at the same time, existing in the patient at pharmacologically active concentrations at the same time.
  • the LNA oligomer targeting LNA oligomer targeting Bcl-2 and the CD-20 inhibitor are administered in the same dosage formulation. In some embodiments, the LNA oligomer targeting Bcl-2 and the inhibitor of CD-20 are administered separately. In some embodiments, the LNA oligomer targeting Bcl-2 and the CD-20 inhibitor are used concurrently. In some embodiments, the LNA oligomer targeting Bcl-2 and the inhibitor of CD-20 are administered together.
  • the patient is a subject who is in need for the treatment of cancer.
  • the patient may be a mammal, such as a rat, a mouse, a monkey etc., or, preferably a human being, who is suffering from cancer.
  • the patient may have a history of unsuccessful treatment with the CD-20 inhibitor - and may, in some embodiments be a relapsed or refractory patient.
  • the patient is suffering from non-Hodgkin's lymphoma.
  • Hodgkin's Lymphoma including but not limited to Follicular NHL, lymphoplasmacytic NHL, Waldenstrom's macroglobulinemia.
  • the cancer is relapsed or relapsed partially sensitive to treatment with a CD-20 inhibitor. In some embodiments, the cancer is refractory and not sensitive to treatment with a CD-20 inhibitor.
  • the cancer cell(s) is substantially CD20- or CD20-negative, i.e. less than 1 % of a population of the cancer cells stain positive for CD20 protein.
  • the cancer cell is substantially insensitive to said CD20 inhibitor, for example when applied to the cell at a concentration of 0.1 , 1 , 10 or 100ug/ml. in the absence of the LNA oligomer targeting Bcl-2, but is sensitive to the CD20 inhibitor in the presence of the LNA oligomer targeting Bcl-2, such as at the concentrations referred to herein.
  • the LNA oligomer targeting Bcl-2 may be administered at regular intervals (Dose intervals, Dl) of between 3 days and two weeks, such as 4, 5, 6, 7, 8, 9, 0, 1 1 , 12, 13 days, such as about 1 week, such as 6, 7 or 8 days.
  • Dl Dose intervals
  • at least two doses are provides with a Dl period between the two dosages, such as 3, 4, 5, 6, 7, 8, 9 or 10 dosages, each with a dose interval (Dl) between each dose of LNA oligomer.
  • the Dl period between each dosage may the same, such as between 3 days and two weeks, such as 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 days, such as about 1 week, such as 6, 7 or 8 days.
  • each dose of the LNA oligomer targeting Bcl-2 may be between about 0.25mg/kg - about 10mg/kg, such as about 0.5mg/kg, about 1 mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg.
  • each does of the LNA oligomer targeting BcI- 2 may be between about 2 mg/kg - about 8mg/kg, or about 4 to about 6 mg/kg or about 4mg/kg to about 5mg/kg.
  • each does of the LNA oligomer targeting Bcl-2 is at least 2mg/kg, such as 2, 3, 4, 5, 6, 7 or 8 mg/kg, such as 6 mg/kg.
  • the dosage regime for the LNA oligomer may be repeated after an initial dosage regime, for example after a rest period where no LNA oligomer targeting Bcl-2 is administered.
  • a rest period may be more than 2 weeks in duration, such as about 3 weeks or about 4 weeks, or about 5 weeks or about 6 weeks.
  • the dosage regimen for the LNA oligomer is one weekly dosage, repeated three, four or five times. This dosage regimen may then be repeated after a rest period of, for example, about 3 - 5 weeks, such as about 4 weeks.
  • the LNA oligomer targeting Bcl-2 is administered during a first dosage regimen at regular dosage intervals (Dl) of between 4 and 13 days for between 2 - 10 administrations.
  • the CD-20 inhibitor is administered during the first dosage regimen.
  • a second dosage regimen follows the first dosage regimen after a rest period when no LNA oligomer targeting Bcl-2 is administered of between 3 - 5 weeks, wherein second dosage regimen comprises the administration of the LNA oligomer targeting Bcl-2 at regular dosage intervals (Dl) of between 4 and 13 days for between 2 - 10 administrations.
  • the first and second dosage regimens may have the same Dl and/or the same number of administrations of the LNA oligomer targeting Bcl-2.
  • the CD-20 inhibitor is administered during the second dosage regimen. In some embodiments, the CD-20 inhibitor is administered during the first and the second dosage regimen.
  • the dosage may, for example, be about weekly dosages of about 375 mg/m 2 .
  • the CD-20 inhibitor may be dosed in a similar regime (in terms of the number of dosages and the Dl), and may for example be four weekly dosages.
  • the first dosage of the CD-20 inhibitor is administered after the first dose of the LNA-oligomer, such as about 1 week after the first dosage of the LNA oligomer.
  • Each dose of the CD-20 inhibitor may however be a sub-optimal dose, i.e. be a dose which is below the standard prescribed dosage for the patient.
  • the CD20 inhibitor is administered in a dosage that is at least 20%, below the recommended dosage of that compound when used for the cancer treatment.
  • the CD20 inhibitor is Rituximab or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 11 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan, Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159, GA-101 and the dosage is less than 300mg/m 2 or less than 250mg/m 2 per administration.
  • the CD20 inhibitor is an antibody, such as those referred to here, including Ritoximab or a CD20 antibody is selected from the group consisting of Ofatumumab (2F2), 1 1 B8, 7D8, 2C6, Veltuzumab, AME-133v Jbritumomab Tiuxetan, Tositumomab , TRU-015 , 2H7.vl6 , Ocrelizumab , Pro131921 , R7159 and GA- 101 , and the dosage is between 20mg/m 2 and 250mg/m 2 ; in some embodiment the dosage is at least 20mg/m 2 , such as at least 50mg/m 2 , such as at least 100mg/m 2 ; in some embodiments the dosage is less than 300mg/ m 2 , such as less than 250mg/ m 2 , such as less than 150mg/ m 2 , such as less than 100mg/ m 2 .
  • the starting dose of the LNA oligomer is given i.v. weekly for two 5-week treatment periods with a 4-week treatment-free period in-between.
  • a standard dose of the CD-20 inhibitor such as 375 mg/m 2 rituximab, weekly for 4 weeks beginning on Week 2.
  • this may be represented as follows:
  • LNA oligomer targeting Bcl-2 for the preparation of a medicament, wherein said medicament is for the use in the treatment of cancer in combination with an inhibitor of CD-20, and wherein the LNA oligomer targeting Bcl-2 is an LNA oligomer targeting BCL-2.
  • CD-20 inhibitor is a CD-20 antibody or a CD-20 antibody fragment, such as a FAB.
  • a pharmaceutical composition comprising an inhibitor of CD-20 and an LNA oligomer targeting BCL-2, and a pharmaceutically acceptable diluent, carrier, or salt, wherein the CD-20 inhibitor and the LNA oligomer targeting Bcl-2 is as according to any one of the preceding embodiments.
  • a kit for use in the treatment of cancer said kit comprising an inhibitor of CD-20 and an LNA oligomer targeting BCL-2, wherein the CD-20 inhibitor and the LNA oligomer targeting Bcl-2 is as according to any one of the preceding embodiments.
  • CD-20 inhibitor and the LNA oligomer targeting Bcl-2 is as according to any one of the preceding embodiments.
  • a method for the concurrent inhibition of Bcl-2 and CD-20 in a cancer cell comprising administering a LNA oligomer targeting Bcl-2 and a CD-20 inhibitor to said cancer cell, wherein said LNA oligomer targeting Bcl-2 and said CD-20 inhibitor are as according to any one of the preceding embodiments.. 35.
  • said cell is a cancer cell, such as a CD20+ cancer cell.
  • Example 1 In vivo anti-tumour activity of SEQ ID NO 2 and SEQ ID NO 3 in SCID mice IV injected with Raji and Namalwa human burkitt's lymphoma cells.
  • SEQ ID NO 2: b c b .- m r ⁇ s o ⁇ I g o c s c s c s a s a s c s g s t s g s c s g m u s om ⁇ s o a-c _s,
  • Rituximab was injected at 1 and 10 mg/kg/inj (antitumour activity study in SCID mice bearing disseminated Raji human lymphoma) and at 6 and 25 mg/kg/inj (antitumour activity study in SCID mice bearing disseminated NAMALWA human lymphoma). 5
  • the test and reference substances were intravenously (IV, bolus) injected into the caudal vein of mice.
  • Test and reference substances were injected at 10 ml/kg/inj accordingly to the most recent body weight of mice.
  • mice from group 2 received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17),
  • mice from group 3 received one daily IV injection of SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17), • The mice from group 4 received 6 IV injections of Rituximab at 1 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D1 1 , D14, D18 and D21 ),
  • mice from group 5 received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17) in combination with 6 IV injections of Rituximab at 1 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D1 1 , D14, D18 and D21 ).
  • the administration of Rituximab was performed 15 minutes before the administration of SEQ ID NO 2 ,
  • mice from group 6 received one daily IV injection of SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17) in combination with 6 IV injections of Rituximab at 1 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D1 1 , D14, D18 and D21 ).
  • the administration of Rituximab was performed 15 minutes before the administration of SEQ ID NO 3 ,
  • mice from group 9 received 6 IV injections of Rituximab at 10 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D11 , D14, D18 and D21 ).
  • mice tissues At D18, 4 mice per group were terminated. Four not xenografted mice out of 6 were used as negative control.
  • bone marrow were collected processed and analysed by flow cytometry.
  • Bone marrow collection To study the human cells engraftment in mice, bone marrow (from both femurs) was collected at the time of termination. The human cells in bone marrow were detected by FACS analysis using an anti-human CD45 antibody.
  • mice from group 1 received one daily IV injection of saline for 14 consecutive days (Q1 Dx14, from D4 to D17),
  • mice from group 3 received one daily IV injection of SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17), • The mice from group 4 received 6 IV injections of Rituximab at 6 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D11 , D14, D18 and D21 ),
  • mice from group 5 received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17) in combination with 6 IV injections of Rituximab at 6 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D1 1 , D14, D18 and D21 ).
  • the administration of Rituximab was performed 15 minutes before the administration of SEQ ID NO 2 ,
  • mice from group 6 received one daily IV injection of SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days (Q1 Dx14, from D4 to D17) in combination with 6 IV injections of Rituximab at 6 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D1 1 , D14, D18 and D21 ).
  • the administration of Rituximab was performed 15 minutes before the administration of SEQ ID NO 3 ,
  • mice from group 7 received 6 IV injections of Rituximab at 25 mg/kg once a day twice weekly for 3 consecutive weeks (TWx3, at D4, D7, D11 , D14, D18 and D21 ).
  • mice The monitoring of mice was performed as described below.
  • mice tissues At D14, 4 mice per group were terminated. Four not xenografted mice out of 7 were used as negative control. To study the human cells engraftment in these mice, bone marrow were collected, processed and analysed by flow cytometry (as described previously). Animal monitoring and termination: lsoflurane Forene (Minerve, Bondoufle, France) was used to anaesthetize the animals before tumour cells injection, IV treatment and termination. The viability and behaviour of mice were recorded every day. The mice's body weight were recorded twice a week. Efficacy Parameters: The efficacy parameters were chosen to be expressed as a percent (T/C %).
  • Table 2 Treatment effects on survival time of SCID mice bearing disseminated Raji human lymphoma.
  • the mice received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj or SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days from D4 to D17 (Q1 Dx14) alone or in combination with 6 IV injections of Rituximab at 1 mg/kg/inj once a day twice weekly for 3 consecutive weeks at D4, D7, D11 , D14, D18 and D21 (TWx3).
  • T Median survival time of mice treated with drugs
  • C Median survival time of mice treated with saline
  • hCD45+ hCD45 positive cells
  • Rituximab 1 mg/kg alone or in combination with SEQ ID NO 2 at 5 mg/kg and SEQ ID NO 3 at 10 mg/kg.
  • a slight level of hCD45+ cells were detected in bone marrow of mice treated with SEQ ID NO 2 at 5 mg/kg (1.32 %).
  • a similar level of hCD45+ cells were detected in bone marrow of mice treated with SEQ ID NO 3 at 10 mg/kg and mice treated with saline (19.6 and 12.4 %).
  • mice treated with SEQ ID NO 2 at 5 mg/kg in combination with Rituximab at 1 mg/kg/inj were increased significantly for mice treated with SEQ ID NO 2 at 5 mg/kg in combination with Rituximab at 1 mg/kg/inj when compared to mice treated with SEQ ID NO 2 and Rituximab alone at the same respective doses (96 days vs 26 and 31 days).
  • the corresponding T/C % parameters were 457.1 % vs 123.8 and 147.6%.
  • the median survival time improvement for mice treated with SEQ ID NO 2 at 5 mg/kg in combination with Rituximab at 1 mg/kg was significantly higher when compared to mice treated with Rituximab at 10 mg/kg alone (96 and 63 days vs 33 days, respectively).
  • mice were terminated (2 in group of mice treated with Rituximab at 1 mg/kg/inj, 3 in group of mice treated with SEQ ID NO 2 at 5 mg/kg/inj in combination with Rituximab at 1 mg/kg/inj, and 1 in group of mice treated with Rituximab at 10 mg/kg/inj).
  • No hCD454+ cells were found in the bone marrow. These mice were considered as cured.
  • mice bearing disseminated NAMALWA human lymphoma.
  • the mice received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj or SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days from D4 to D17 (Q1 Dx14) alone or in combination with 6 IV injections of Rituximab at 6 mg/kg/inj once a day twice weekly for 3 consecutive weeks at D4, D7, D11 , D14, D18 and D21 (TWx3).
  • hCD45+ cells were detected in bone marrow of mice treated with SEQ ID NO 2 at 5 mg/kg (0.36%).
  • a hCD45 level increase was observed for mice treated with Rituximab at 6 mg/kg (7.90 %) similar to that observed for mice treated with Rituximab at 25 mg/kg (9.68 %).
  • a similar level of hCD45+ cells were detected in bone marrow of mice treated with SEQ ID NO 3 at 10 mg/kg and mice treated with saline (14.52 and 16.65 %).
  • SEQ ID NO 2 at 5 mg/kg was found more potent than Rituximab.
  • mice treated with SEQ ID NO 2 at 5 mg/kg in combination with Rituximab at 6 mg/kg/inj when compared to mice treated with SEQ ID NO 2 and Rituximab alone at the same respective doses (43 days vs 31 .5 and 22 days).
  • mice treated with SEQ ID NO 2 at 5 mg/kg in combination with Rituximab at 6 mg/kg was increased significantly when compared to SEQ ID NO 2 and Rituximab administered alone at the same respective doses.
  • Table 5 Mean ( ⁇ SD) CD45 positive cells in bone marrow of 4 SCID mice out of 12 bearing disseminated NAMALWA human lymphoma at D14.
  • the mice received one daily IV injection of SEQ ID NO 2 at 5 mg/kg/inj or SEQ ID NO 3 at 10 mg/kg/inj for 14 consecutive days from D4 to D17 (Q1 Dx14) alone or in combination with 6 IV injections of Rituximab at 6 mg/kg/inj once a day twice weekly for 3 consecutive weeks at D4, D7, D1 1 , D14, D18 and D21 (TWx3).
  • SEQ ID NO 2 were well tolerated by female SCID mice bearing disseminated lymphoma at the tested dose. Based upon the evaluation criteria of antitumour efficacy, SEQ ID NO 2 administered alone displayed a moderate antitumour activity against disseminated Raji tumour xenografted in SCID mice. The optimal T/C% value was 123.8 % for mice treated with SEQ ID NO 2 at 5 mg/kg. In contrast, SEQ ID NO 2 showed a marked and significant improvement of the antitumour activity when combined with Rituximab.
  • SEQ ID NO 2 was found more potent in NAMALWA disseminated tumour model when administered alone and was found more potent in Raji disseminated tumour model when administered in combination with Rituximab. This difference may be attributed to the in vitro level of hCD20 antigen, 20 fold less express on NAMALWA cell line when compared to Raji cell line.
  • PK data indicated that when SEQ ID No 2 was administered by intravenous (i.v.) infusion to a human patient, the maximum plasma concentration (C max ) was reached at 2 hours (end of the infusion). It was followed by a distribution phase with a distribution half-life of 2 hours, indicating that there is rapid tissue uptake.
  • the plasma elimination half-life [VA) was estimated to be 200 hours, representing equilibrium between tissue and plasma. Furthermore, the plasma elimination half-life can be used as a surrogate of the tissue half-life. Due to the plasma elimination half-life of 200 hours, the steady state in plasma concentration during repeated doses would be reached in 30-40 days.
  • Vz volume of distribution
  • AUC area under the plasma concentration/time curve
  • Patients suffering from relapsed follicular or lymphoplasmacytic non-Hodgkin's lymphoma are selected, and undergo the administration regimen shown in Figure 5 using SEQ ID NO 2 and Rituximab.
  • patients are monitored to identify any toxicity, especially relating to the higher dosages of SEQ ID NO 2, and to determine the overall response rate (Complete Response [CR] + Partial Response [PR]) of the combination of SEQ ID NO 2 and rituximab in the treatment of relapsed follicular or lymphoplasmacytic non-Hodgkin's lymphoma.
  • Other variable which are determined include: 1.

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Abstract

La présente invention concerne des thérapies combinées pour le traitement du cancer, utilisant des oligomères ALN ciblant Bcl-2 en association avec des inhibiteurs du CD-20, tels que le rituximab, ou un anticorps anti-CD20 choisi parmi le groupe consistant en l’ofatunumab (2F2), 11B8, 7D8, 2C6, le veltuzumab, l’AME-133v, l’ibritumomab, le tiuxetan, le toxitumomab, TRU-015, 2H7.v16, l’ocrelizumab, Pro131921, R7159 et GA-101.
PCT/EP2009/063250 2008-10-17 2009-10-12 Méthode pour le traitement du cancer WO2010043582A1 (fr)

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Cited By (1)

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WO2020014139A1 (fr) * 2018-07-09 2020-01-16 Millennium Pharmaceuticals, Inc. Administration d'inhibiteur d'enzyme d'activation sumo et anticorps anti-cd20

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WO2004056971A2 (fr) * 2002-12-19 2004-07-08 Genta Incorporated Methodes de traitement d'un trouble associe au bcl-2 a l'aide d'oligomeres anti-sens bcl-2
WO2005061710A1 (fr) * 2003-12-23 2005-07-07 Santaris Pharma A/S Composes oligomeres permettant la modulation de bcl-2
WO2009049841A1 (fr) * 2007-10-15 2009-04-23 F. Hoffmann-La Roche Ag Polythérapie à base d'un anticorps anti-cd20 de type ii avec un agent actif anti-bcl-2

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WO2004056971A2 (fr) * 2002-12-19 2004-07-08 Genta Incorporated Methodes de traitement d'un trouble associe au bcl-2 a l'aide d'oligomeres anti-sens bcl-2
WO2005061710A1 (fr) * 2003-12-23 2005-07-07 Santaris Pharma A/S Composes oligomeres permettant la modulation de bcl-2
WO2009049841A1 (fr) * 2007-10-15 2009-04-23 F. Hoffmann-La Roche Ag Polythérapie à base d'un anticorps anti-cd20 de type ii avec un agent actif anti-bcl-2

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020014139A1 (fr) * 2018-07-09 2020-01-16 Millennium Pharmaceuticals, Inc. Administration d'inhibiteur d'enzyme d'activation sumo et anticorps anti-cd20
CN112384219A (zh) * 2018-07-09 2021-02-19 千禧制药公司 Sumo-激活酶抑制剂和抗cd20抗体的施用
JP2021532067A (ja) * 2018-07-09 2021-11-25 ミレニアム ファーマシューティカルズ, インコーポレイテッドMillennium Pharmaceuticals, Inc. Ｓｕｍｏ活性化酵素阻害剤及び抗ｃｄ２０抗体の投与
JP7352582B2 (ja) 2018-07-09 2023-09-28 武田薬品工業株式会社 Ｓｕｍｏ活性化酵素阻害剤及び抗ｃｄ２０抗体の投与

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