WO2021119669A1 - Méthodes et compositions pour le traitement d'une leucémie aiguë myéloïde - Google Patents

Méthodes et compositions pour le traitement d'une leucémie aiguë myéloïde Download PDF

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WO2021119669A1
WO2021119669A1 PCT/US2020/070905 US2020070905W WO2021119669A1 WO 2021119669 A1 WO2021119669 A1 WO 2021119669A1 US 2020070905 W US2020070905 W US 2020070905W WO 2021119669 A1 WO2021119669 A1 WO 2021119669A1
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inhibitor
icam
patient
mcsc
stem cells
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PCT/US2020/070905
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English (en)
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Yi XU (David)
Huynh CAO
Jeffrey XIAO
David Baylink
Mark Reeves
Chien-Shing Chen
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Loma Linda University Pathology Medical Group, Inc.
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Publication of WO2021119669A1 publication Critical patent/WO2021119669A1/fr

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    • 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
    • C07K16/2884—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD44
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the disclosure relates to methods and compositions for treatment of acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • AML is a hematopoietic cancer that involves a complex interplay between different cells of the bone marrow, cytokines, growth factors, and neural modulation, all of which contribute to the survival and growth of leukemia stem cells (LSCs).
  • LSCs leukemia stem cells
  • the release of cytokines has been reported to be up-regulated in the AML microenvironment (niche), including vascular endothelial growth factor (VEGF) and interleukins (ILs) that enhance vascularization and support LSCs.
  • VEGF vascular endothelial growth factor
  • ILs interleukins
  • plastic-adherent MSC are actually a heterogeneous mixture of cells with varying cell-surface antigen profiles including CD44 (Cluster of Differentiation 44), CD90, CD105, and CD73.
  • CD44 Cluster of Differentiation 44
  • CD90 CD90
  • CD105 CD105
  • CD73 CD73
  • Recent studies of patient bone marrow revealed that AML MSC displayed inferior capabilities in both proliferation and differentiation.
  • the disturbed osteo-hematopoietic interaction has been reported to transform normal hematopoietic stem cells into LSCs.
  • Increased angiogenesis and disorganized vascularization has been documented in AML bone marrow, leading to disease progression and relapse.
  • One such method includes the step of administering a therapeutically effective amount of an inhibitor of CD44 (Cluster of Differentiation 44) to the patient and inhibiting proliferation of the mesenchymal cancer stem cells.
  • the inhibitor of CD44 can be an agent that interferes with expression of the CD44 gene.
  • the inhibitor of CD44 can be an agent that interferes with modification or activity of the CD44 protein or its variants.
  • the inhibitor of CD44 can be an agent that interferes with interactions of CD44 and glycosaminoglycan hyaluronan.
  • the inhibitor of CD44 can be an anti-CD44 antibody.
  • the CD44 inhibitor can be provided as part of a treatment regimen involving one or more of an angiogenic inhibitor, an ICAM-1 (Intercellular Adhesion Molecule 1) inhibitor, or a compound to inhibit release of cytokine by mesenchymal cancer stem cells in a bone marrow of the patient.
  • an angiogenic inhibitor an ICAM-1 (Intercellular Adhesion Molecule 1) inhibitor
  • ICAM-1 Intercellular Adhesion Molecule 1
  • a compound to inhibit release of cytokine by mesenchymal cancer stem cells in a bone marrow of the patient a compound to inhibit release of cytokine by mesenchymal cancer stem cells in a bone marrow of the patient.
  • Another method of treatment of cancers includes the step of administering a therapeutically effective amount of a compound to inhibit release of cytokine by the mesenchymal cancer stem cells. Inhibition of the release of cytokine by the mesenchymal cancer stem cells inhibits their proliferation.
  • the compound to inhibit release of cytokine by the mesenchymal cancer stem cells can be one or more of an anti-interleukin 6 agent, an inhibitor of interleukin 6 receptor (IL-6R) signaling, a corticosteroid, a nonsteroidal anti-inflammatory drug, an anti- interleukin-2 receptor (IL-2R) agent, an anti-Tumor Necrosis Factor a monoclonal antibody (TNFa mAh), and an interleukin-1 receptor (IL-lR)-based inhibitor.
  • an anti-interleukin 6 agent an inhibitor of interleukin 6 receptor (IL-6R) signaling
  • a corticosteroid a nonsteroidal anti-inflammatory drug
  • an anti- interleukin-2 receptor (IL-2R) agent an anti-Tumor Necrosis Factor a monoclonal antibody (TNFa mAh)
  • IL-lR interleukin-1 receptor
  • the compound to inhibit release of cytokine by mesenchymal cancer stem cells in a bone marrow of the patient can be provided as part of a treatment regimen involving one or more of a CD44 inhibitor, an ICAM-1 inhibitor, or an angiogenic inhibitor.
  • Another method of treatment of cancers includes the step of administering a therapeutically effective amount of an angiogenic inhibitor to the patient.
  • this method also includes administering a therapeutically effective amount of an inhibitor of CD44 to the patient.
  • the inhibitor of CD44 can be an agent that interferes with expression of the CD44 gene.
  • the inhibitor of CD44 can be an agent that interferes with modification or activity of the CD44 protein or its variants.
  • the inhibitor of CD44 can be an agent that interferes with interactions of CD44 and glycosaminoglycan hyaluronan.
  • the inhibitor of CD44 can be an anti-CD44 antibody.
  • this method also includes administering a therapeutically effective amount of a compound to inhibit release of cytokine by mesenchymal cancer stem cells in a bone marrow of the patient.
  • the compound to inhibit release of cytokine by the mesenchymal cancer stem cells can be one or more of an anti-interleukin 6 agent, an inhibitor of interleukin 6 receptor signaling, a corticosteroid, a nonsteroidal anti inflammatory drug, an anti-interleukin-2 receptor agent, an anti-Tumor Necrosis Factor a monoclonal antibody, and an interleukin- 1 receptor-based inhibitor.
  • the angiogenic inhibitor can be provided as part of a treatment regimen involving one or more of a CD44 inhibitor, an ICAM- 1 inhibitor, or a compound to inhibit release of cytokine by mesenchymal cancer stem cells in a bone marrow of the patient.
  • Another method of treating acute myeloid leukemia in a patient includes the step of inhibiting proliferation of the mesenchymal cancer stem cells by administering a therapeutically effective amount of a CD44 inhibitor and an ICAM-1 inhibitor to the patient.
  • the ICAM-1 inhibitor can be an agent that interferes with expression of the ICAM-1 gene.
  • the ICAM-1 inhibitor can be an agent that interferes with function of the ICAM-1 protein or its variants.
  • the ICAM-1 inhibitor is an anti-ICAM-1 antibody.
  • One such method includes the step of evaluating level of ICAM-1 in a bodily fluid sample of the patient; and in response to increased level of ICAM-1 in the bodily fluid sample, administer a therapeutically effective amount of a CD44 inhibitor and an ICAM-1 inhibitor to the patient.
  • One such method includes the steps of (i) incubating the bone marrow sample in a lysing buffer to remove red blood cells; (ii) washing residual cells with phosphate buffered saline and collecting them as a cell pellet by centrifugation at room temperature; (iii) reconstituting the cell pellet in a culture medium suitable for derivation and expansion of mesenchymal progenitor cells; (iv) plating the mesenchymal progenitor cells on a surface in presence of a growth medium to allow mesenchymal stem cells to attach to the surface; (v) removing the growth medium periodically to collect non-adherent floating cells; and (vi) isolating mesenchymal cancer stem cells by continuous culture of the non-adherent floating cells.
  • the step of isolating mesenchymal cancer stem cells includes transferring
  • FIGS. 1A - 1Z provide images and graphical plots demonstrating the isolation, expansion, and characterization of CD90 CD13 CD44 + Mesenchymal Cancer Stem Cells (MCSC) from an AML patient bone marrow ex vivo.
  • MCSC Mesenchymal Cancer Stem Cells
  • FIGS. 1A-1D are four phase bright images of MSC and MCSC at different passages (P). Red arrows indicate pseudopodia in the middle of gap space in MCSC cultures.
  • FIGS. IE and IF are images of FACS analysis for the incorporation of BrdU and expression of CD44 in P7 MSC and P7 MCSC samples that were collected 24 hours after feeding BrdCT.
  • FIG. 1G is a graphical representation of the percentage of BrdU + CD44 + cells in MSC and MCSC.
  • FIGS. 1H and II are images of FACS analysis for the expressions of CD44 and CD 13 by P3 MSC cells.
  • FIGS. 1 J and IK are images of FACS analysis for the expressions of CD44 and CD13 by P3 MCSC cells.
  • FIGS. 1L - lO are graphical representations of the percentage of MSC and MCSC cells positive for each biomarker — CD44 + , CD90 + , CD13 + , and CD90 CD13 CD44 + , respectively.
  • FIG. IP is aphase bright image of floating cells fromP5 MSC culture.
  • FIG. IQ is an image of FACS analysis for the expression of CD90+ and CD13+ by P5 MSC following gating on CD44+ cells.
  • FIG. 1R is a phase bright image of floating cells from P5 MCSC culture.
  • FIG. IS is an image of FACS analysis for the expression of CD90+ and CD13+ by P5 MSC following gating on CD44+ cells.
  • FIG. IT is a graphical representation of the percentage of CD90 CD13 CD44 + MSC floating cell populations from MSC and MCSC.
  • FIG. 1U is a graphical representation of the cumulative counts of clusters per 100,000 cells generated from MSC and MCSC cultures at different time points measured as passages.
  • FIGS. IV and 1W are phase bright images of floating tube-like structures from P8 MCSC culture. The black thick arrow in FIGS. IV and 1W indicates the magnified image of the same round tube indicate the same round tube at 2X and 10X magnification.
  • FIG. IX is a phase bright image of GFP+MOLM-14 cells cultured with MSC after 24 hours’ plating.
  • FIG. 1Y is a phase bright image of GFP+MOLM-14 cells co-cultured with MCSC after 24 hours’ plating.
  • FIG. 1Z is an image of FACS analysis for the expression of endothelial cell biomarkers like VE- Cadherin/CD144 by P7 MCSC following gating on CD44+ cells.
  • FIGS. 2A - 2C provide the proteome analyses indicating the significant increase in angiogenic protein release from MCSC cultures.
  • FIG. 2A and 2B are images of partial blot films developed for proteome analyses of supernatants from P7 MSC and P7 MCSC cultures.
  • FIG. 2C is a proteome comparison (fold change) of angiogenic proteins between supernatants from P7 MSC and P7 MCSC cultures. Fold Changes represents MCSC versus MSC.
  • FIG. 3 is a diagrammatic representation of the processes between healthy regeneration and AML relapse.
  • FIGS. 4A-4H illustrate the lack of significant difference in differentiation capabilities of P4 MSC and P4 MCSC during ex vivo cultures.
  • FIG. 4A is a FACS plot of CD34 CD13 + MSC, which differentiated into bone (Alizalin staining) (FIG. 4B), fat (phase bright) (FIG. 4C), and cartilage (Alcian blue staining) (FIG. 4D).
  • FIG. 4E is a FACS plot of CD34 CD13 + MCSC, which differentiated into bone (Alizalin staining) (FIG. 4F), fat (phase bright) (FIG. 4G), and cartilage (Alcian blue staining) (FIG. 4H).
  • FIGS. 5A-5D illustrate the inhibition of the cluster formation and proliferation of P5 MCSC by the anti-CD44 monoclonal antibodies.
  • FIG. 5A is a phase bright image of floating clusters from P5 MCSC without treatment.
  • FIG. 5B is a phase bright images of floating cells from P5 MCSC with treatment of anti-CD44.
  • FIG. 5C is a graphical representation of the cumulative cluster count data from P5 MCSC treated with anti-CD44 antibody or without treatment.
  • FIG. 5D is a graphical representation of the cumulative cluster count data from P7 MCSC treated with anti- CD44 antibody or without treatment.
  • FIG. 6A is a graphical representation of the differences in cell proliferation of MSCs and MCSCs.
  • FIG. 6B is an image of FACS analysis of the MCSCs for expression of Caspase 3 and CD44+.
  • FIG. 7 is a graphical representation of the proteome comparison of ICAM-1 between supernatants from P5 MCSCs and P7 MCSC cultures.
  • AML is a hematopoietic cancer that has a heterogeneous cell population and is an aggressive malignancy with poor prognosis.
  • AML is extremely difficult to treat with the current chemotherapeutic regimens.
  • Increased angiogenesis appears to play an important role in leukemia progression. Therefore, identification of the cellular and molecular contributors to angiogenesis and cytokine release in AML bone marrow facilitates the development of less toxic and more cost- effective treatments to prevent AML progression and relapse.
  • MSC Mesenchymal Cancer Stem Cells
  • the MCSC differ from MSC in isolation, expansion, differentiation, immunophenotype, and cytokine release profile.
  • MCSC do not express CD90 or CD13, but they express CD44 (MCSC are CD90 CD13 CD44 + ) and grow quickly in floating clusters.
  • MCSC can differentiate into bone, fat and cartilage, and generate vessel -like structures in vitro.
  • MCSC Proteome assays revealed that MCSC release significantly increased levels of angiogenic cytokines and growth factors when compared to MSC. Blocking CD44 inhibited the proliferation and cluster formation of MCSC. Based on the disclosure herein, AML appears to include two types of disease: hematopoietic malignancy and stromal cancer. MCSC appears to be responsible for angiogenesis and cytokine release that contributes to the survival, proliferation, and metastatic routes of AML blasts. Therefore, embodiments include inhibitory agents that target the MCSC and their cytokine release to stop disease progression and increase survival of AML patients.
  • CD44 is a non-kinase transmembrane glycoprotein that functions as a cell surface adhesion receptor. It is highly expressed in many cancers and regulates metastasis via recruitment of CD44 to the cell surface. In humans, CD44 is encoded by 19 exons with 10 of these exons constant in all isoforms. The standard form of CD44 is encoded by the ten constant exons. The variant isoforms of CD44 is generated by alternative splicing and possess the ten constant exons and any combination of the remaining nine variant exons.
  • AML therapies have been developed to target LSCs because of their significant role in initiating uncontrolled clonal proliferation and manipulating the naive bone marrow niche to allow AML progression and relapse.
  • the novel MCSC isolated here displayed pro-angiogenic properties, including vessel structure formation and the release of both cytokines and growth factors.
  • Embodiments for treatment of AML include compositions to inhibit the proliferation and cluster formation of MCSC.
  • Therapeutic strategies for treatment of AML include compositions to inhibit the cytokine release from MCSC to prevent AML progression and relapse. In certain aspects, these compositions are delivered to the bone marrow of a patient with AML to target MCSC and their cytokine release and to prevent AML progression and relapse.
  • Embodiments include methods of treatment of acute myeloid leukemia in a patient.
  • One such method includes the step of inhibiting proliferation of the mesenchymal cancer stem cells by administering a therapeutically effective amount of an inhibitor of CD44 to the patient.
  • the CD44 inhibitor can be provided as part of a treatment regimen involving one or more of an angiogenic inhibitor, an ICAM-1 inhibitor, or a compound to inhibit release of cytokine by mesenchymal cancer stem cells in a bone marrow of the patient.
  • an inhibitor of CD44 is any chemical or biological agent that (i) inhibits or reduces or interferes with the expression of the CD44 gene or the modification of the CD44 protein and its variants; (ii) suppresses or reduces or interferes with the activity of CD44; or (iii) inhibits or interferes with the interactions of CD44 and glycosaminoglycan hyaluronan (HA).
  • suitable inhibitors of CD44 include antibodies, peptides, aptamers, pharmacological inhibitors, and hyaluronic acid oligomers.
  • the inhibitor of CD44 is an anti-CD44 antibody.
  • the inhibitor of CD44 is anti-CD44 humanized antibodies (Pan or Subsets). In another embodiment, the inhibitor of CD44 is a CD44 binding peptide. In an embodiment, the inhibitor of CD44 is a CD44 ectodomain (competitor). In an embodiment, the inhibitor of CD44 is CD44-specific RNA compound, such as small interfering RNA (siRNA) or short hairpin (shRNA) or microRNAs (miRNAs) that affect CD44 expression. In another embodiment, the inhibitor of CD44 is anti-CD44-based nanoparticles. In another embodiment, the inhibitor of CD44 is HA-based amino acid or nucleotide entity.
  • siRNA small interfering RNA
  • shRNA short hairpin
  • miRNAs microRNAs
  • An inhibitor of CD44 for use in the methods of treatment of cancers will inhibit proliferation of mesenchymal cancer stem cells by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, or at least 200%) as compared to proliferation of mesenchymal cancer stem cells in the absence of the inhibitor of CD44.
  • Embodiments include methods of treating AML patients by targeting the proangiogenic CD44 + MCSCs.
  • One such method includes treating an AML patient with an angiogenic inhibitor to suppress malignant angiogenesis to prevent metastatic routes and inappropriate growth nutrients.
  • angiogenic inhibitors include any chemical or biological agent that are inhibitors of one or more of VEGF, vascular endothelial growth factor receptors (VEGFR), fibroblast growth factor-2 (FGF-2), fibroblast growth factor receptors (FGFR), platelet-derived growth factors (PDGF), and platelet-derived growth factor receptors (PDGFR). These proteins are especially important for both angiogenesis and stem cell proliferation, which should be inhibited in high risk AML patients.
  • An example of a high risk AML patient includes a patient with a DNA methyltransferase (DNMT) mutation.
  • Another example of high risk AML patient includes a patient with a DNMT mutation along with a medical history of diabetes or hypothyroidism or both.
  • Another such method includes treating an AML patient with anti-CD44 monoclonal antibodies and the angiogenic inhibitor.
  • Another such method includes treating an AML patient with anti-CD44 gene therapies to target MCSCs along with angiogenic inhibitors targeting CD44+ Leukemia stem cells (LSCs).
  • Another such method includes treating an AML patient with an anti cytokine release medication and an angiogenic inhibitor.
  • Embodiments disclosed here include new protocols of managing AML patients according their risk based on genetic mutations and past medical history. For example, an AML patient with both DNMT mutation and diabetes can be treated with a CD44 inhibitor and an angiogenic inhibitor due to a high risk for pro-angiogenesis tendency.
  • Another method of treatment of acute myeloid leukemia includes inhibiting proliferation of the mesenchymal cancer stem cells by administering a therapeutically effective amount of a compound to inhibit release of cytokine by the mesenchymal cancer stem cells.
  • inhibitors of cytokine release include one or more of an anti-interleukin 6 agent, an inhibitor of interleukin 6 receptor signaling, a corticosteroid, a nonsteroidal anti inflammatory drug, an anti-IL-2R agent, an anti-TNFa mAbs (such as infliximab), soluble TNFa receptor (such as etanercept) and IL-lR-based inhibitors (such as anakinra).
  • a compound to inhibit release of cytokine for use in the methods of treatment of cancers will inhibit proliferation of mesenchymal cancer stem cells by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, or at least 200%) as compared to proliferation of mesenchymal cancer stem cells in the absence of the inhibitor of cytokine release.
  • the compound to inhibit release of cytokine by mesenchymal cancer stem cells in a bone marrow of the patient can be a chemical or biological agent and can be provided as part of a treatment regimen involving one or more of a CD44 inhibitor, an ICAM-1 inhibitor, or an angiogenic inhibitor.
  • Embodiments include methods of preventing a relapse of cancer in a patient by inhibiting proliferation of mesenchymal cancer stem cells.
  • One such method includes the step of administering a therapeutically effective amount of an inhibitor of CD44 to the patient to inhibit proliferation of the mesenchymal cancer stem cells.
  • Another method includes administering to the patient a therapeutically effective amount of a compound to inhibit release of cytokine to inhibit proliferation of the mesenchymal cancer stem cells.
  • Another method of treating acute myeloid leukemia in a patient includes the step of inhibiting proliferation of the mesenchymal cancer stem cells by administering a therapeutically effective amount of a CD44 inhibitor and an ICAM-1 inhibitor to the patient.
  • the ICAM-1 inhibitor can be any chemical or biological agent that interferes with expression of the ICAM-1 gene.
  • the ICAM-1 inhibitor can be any chemical or biological agent that interferes with modification or activity of the ICAM-1 protein or its variants.
  • the ICAM-1 inhibitor is an anti- ICAM-1 antibody.
  • An ICAM-1 inhibitor for use in the methods of treatment of cancers will inhibit proliferation of mesenchymal cancer stem cells by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, or at least 200%) as compared to proliferation of mesenchymal cancer stem cells in the absence of the ICAM-1 inhibitor.
  • One such method includes the step of evaluating level of ICAM-1 in a bodily fluid sample of the patient; and in response to increased level of ICAM-1 in the bodily fluid sample, administer a therapeutically effective amount of a CD44 inhibitor and an ICAM-1 inhibitor to the patient.
  • the ICAM-1 inhibitor can be an agent that interferes with expression of the ICAM-1 gene.
  • the ICAM- 1 inhibitor can be an agent that interferes with modification or activity of the ICAM-1 protein or its variants.
  • the ICAM-1 inhibitor is an anti-ICAM-1 antibody.
  • the anti-ICAM-1 antibody is enlimomab.
  • the ICAM-1 inhibitor is an antisense oligonucleotide that down-regulates ICAM-1 mRNA, such as alicaforsen.
  • a “therapeutically effective amount” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy).
  • a therapeutically effective dose can be administered in one or more administrations.
  • a therapeutically effective dose of an inhibitor of CD44 or an inhibitor of release of cytokine is an amount of the inhibitor that is sufficient to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of the disease state (e.g., cancer) by inhibiting or preventing or delaying the proliferation of mesenchymal cancer stem cells.
  • Embodiments include methods of inhibiting proliferation of mesenchymal cancer stem cells that cause cancers in a patient.
  • the cancers can include one or more of acute myeloid leukemia, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, and head/ neck squamous cell carcinoma.
  • administering refers to the physical introduction of a therapeutic agent to a subject in need thereof.
  • exemplary routes of administration for agents to inhibit proliferation of mesenchymal cancer stem cells include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
  • a therapeutic agent may be administered via a non-parenteral route, or orally.
  • Non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • Therapeutic agents can be constituted in a composition, e.g., a pharmaceutical composition containing an antibody and a pharmaceutically acceptable carrier.
  • a “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • An embodiment includes an immortalized mesenchymal cancer stem cell line that can be used to develop additional therapeutic regimens.
  • One method for developing an immortalized mesenchymal cancer stem cell line includes transforming mesenchymal cancer stem cells with a vector comprising an immortalizing gene flanked by recombinase target sites to create an immortalized cell line and selecting mesenchymal cancer stem cells that express CD44, but do not express CD90 or CD 13.
  • Another method for developing MSC and MCSC cell systems for use in AML research and screening for pharmaceuticals and biologies includes the SV40 cell immortalization system.
  • the starting cells can contain proangiogenic mutations.
  • DNMT DNA methyltransferase
  • This method includes generating a recombinant pLenti-SV40 lentivirus. Then, MCSCs in a 6-well plate with 1 ml/well culture medium are supplied with the SV40 Lentivirus in the presence of 10pg/ml Polybrene. Then about 12 hours later, the viral supernatant is removed from the wells and the appropriate complete growth medium is added to the cells. The cells are then incubated at 37°C. The gene expression is evaluated 72 hours after transduction by different assays, such as flow cytometry, western blot and qPCR analysis.
  • assays such as flow cytometry, western blot and qPCR analysis.
  • kits containing immortalized mesenchymal cancer stem cell line can include kits containing immortalized mesenchymal cancer stem cell line.
  • Kits typically include a label indicating the intended use of the contents of the kit and instructions for use.
  • the term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
  • One such method includes the steps of (i) incubating the bone marrow sample in a lysing buffer containing ammonium chloride and potassium salts to remove red blood cells; (ii) washing residual cells with phosphate buffered saline and collecting them as a cell pellet by centrifugation at room temperature; (iii) reconstituting the cell pellet in a culture medium suitable for derivation and expansion of mesenchymal progenitor cells; (iv) plating the mesenchymal progenitor cells on a surface in presence of a growth medium to allow mesenchymal stem cells to attach to the surface; (v) removing the growth medium periodically to collect non-adherent floating cells; and (vi) isolating mesenchymal cancer stem cells by continuous culture of the non-adherent floating cells.
  • the step of isolating includes the steps of (i) incubating the bone marrow sample in a lysing buffer containing ammonium chloride and potassium salts to
  • a human AML bone marrow sample was obtained from a donor with informed consent and approved by the Institutional Review Board at the Loma Linda University Medical Center.
  • the AML bone marrow donor is a 32-year old female with a past medical history of diabetes and hypothyroidism. She was diagnosed with AML with a blast count over 50%.
  • MCSC were isolated by the following procedure. After the first week, the supernatant of MSC cultures was transferred to another 24-well plate and allowed to grow for 2 weeks until attachment of cells (MCSC) was observed.
  • the STEMdiffTM Mesenchymal Progenitor Kit Catalog # 05240 STEMCELL TECHNOLOGIES was used as culture medium.
  • cells were suspended in a FACS buffer containing PBS and 0.5% bovine serum albumin (BSA). These cells were incubated and stained with the fixable viability dye eFluorTM 780 (from eBioscienceTM, a product line from Thermo Fisher Scientific Inc.) for 20 min at 4°C. The cells were washed twice with the FACS buffer and further stained with desired surface antibodies at concentrations per the manufacturers’ instructions at 4°C for 30 min.
  • the surface antibodies included CD13, CD34, CD44, CD73, CD90, CD 106, CD 105 and CD117 (from BioLegend, Inc. or BD Biosciences, Inc.).
  • FIGS. 1A - 1Z provide images and graphical plots demonstrating the isolation, expansion, and characterization of CD90 CD13 CD44 + Mesenchymal Cancer Stem Cells (MCSC) from an AML patient bone marrow ex vivo.
  • FIGS. 1A and 1C are phase bright images of MSCs at different passages (P). MSC could be expanded homogenously for over 10 passages (P) and were over 90% CD90 + CD13 + CD44 + . Certain cells (MCSC) in the supernatant of MSC cultures displayed delayed attachment of about 7 days.
  • FIGS. IB and ID are phase bright images of MCSC at different passages (P). Red arrows indicate pseudopodia in the middle of gap space in MCSC cultures. MCSC displayed mixed morphologies including some with a fibroblast shape and others with a tree-like shape as well as many space gaps (FIG. IB).
  • FIGS. IE and IF are images of FACS analysis for the incorporation of BrdU and expression of CD44 in P7 MSC and P7 MCSC samples that were collected 24 hours after feeding BrdU.
  • FIG. 1G is a graphical representation of the percentage of BrdU + CD44 + cells in MSC and MCSC.
  • FIGS. 1H and II are images of FACS analysis for the expressions of CD44 and CD13 by P3 MSC cells.
  • FIGS. 1J and IK are images of FACS analysis for the expressions of CD44 and CD 13 by P3 MCSC cells. Thick black arrows indicate forward scatter (FSC) gating strategy. The red arrow indicates the population of CD90 CD13 CD44 + cells, many of which were found to be bigger in size and express higher CD44 (red circle of MCSC (FIG. 1J) versus red circle of MSC (FIG. 1H)). Within CD44 + MCSC, there were two different subset cell populations including about 90% CD90 + CD13 + CD44 + and 10% CD90 CD13 CD44 + cells (P3, FIGS. 1J and IK). FIGS.
  • FIG. IP is a phase bright image of floating cells from P5 MSC culture.
  • FIG. IQ is an image of FACS analysis for the expression of CD90+ and CD 13+ by P5 MSC following gating on CD44+ cells.
  • FIG. 1R is a phase bright image of floating cells from P5 MCSC culture.
  • FIG. IS is an image of FACS analysis for the expression of CD90+ and CD 13+ by P5 MSC following gating on CD44+ cells. The red arrow indicates CD90 CD13 CD44 + cells.
  • Floating MCSC cells were 10 micrometers (pm) in diameter with a bright round healthy morphology and generated clusters, in contrast to 2-3 pm single MSC cells (FIGS.
  • FIG. IT is a graphical representation of the percentage of CD90 CD13 CD44 + MSC floating cell populations from MSC and MCSC. After P7, there were few attached MCSC and they expanded quickly in floating cluster conditions (FIG. 1U).
  • FIG. 1U is a graphical representation of the cumulative counts of clusters per 100,000 cells generated from MSC and MCSC cultures at different time points measured as passages.
  • FIGS. IV and 1W are phase bright images of floating tube like structures from P8 MCSC culture.
  • the black thick arrow in FIGS. IV and 1W indicates the magnified image of the same round tube indicate the same round tube at 2X and 10X magnification.
  • FIG. 1X is a phase bright image of GFP+MOLM-14 cells cultured with MSC after 24 hours’ plating.
  • FIG. 1Y is a phase bright image of GFP+MOLM-14 cells co-cultured with MCSC after 24 hours’ plating.
  • FIG. 1Z is an image of FACS analysis for the expression of endothelial cell biomarkers like VE- Cadherin/CD144 by P7 MCSC following gating on CD44+ cells.
  • Both MSC and MCSC are CD34 (FIGS. 4A and 4B).
  • MCSC could be expanded like MSC with a similar plastic attachment pattern at early passages (P1-P4) and with similar multi lineage differentiation capabilities (FIGS. 4B- 4D and 4F - 4H).
  • P1-P4 plastic attachment pattern at early passages
  • FIGS. 4B- 4D and 4F - 4H multi lineage differentiation capabilities
  • 1 c 10 5 MSCs or MCSCs were plated in a 24-well plate. After 12 hours of cell adhesion, 1 ml of osteogenic differentiation medium (StemPro Osteogenesis Differentiation Kit, Gibco) was added into each well. The osteoblast differentiation was normally carried out for 3 weeks. At the end of the cell cultures, extracellular calcium deposition was evaluated by Alizarin Red staining.
  • the cultured cells were fixed with 4% formaldehyde at room temperature for 20 min. After washing with distilled water for three times, Alizarin Red solution (40 mM; TMS-008-C, Millipore) was added. After 30 min of incubation in Alizarin Red solution, the wells were first rinsed with distilled water for three times and then with PBS for three more times. The wells were examined, and images were taken using either a light microscope (DP72, Olympus) or a digital camera. In an example of an in vitro adipogenic differentiation assay, 1 c 10 5 MSCs or MCSCs were plated a 24-well plate.
  • adipogenic medium (StemPro Adipogenesis Differentiation Kit, Gibco) was added into each well. The differentiation was carried out for 21 days. On day 21, the cell cultures were fixed in 4% formaldehyde and stained with Oil Red O solution. Images were taken using either a light microscope (DP72, Olympus) or a digital camera. In an example of an in vitro chondrocyte differentiation assay, 1 c 105 MSCs or MCSCs were plated a 24-well plate. After 12 hours of cell adhesion, 1 ml of a chondrogenic medium (StemPro Chondrogenesis Differentiation Kit, Gibco) was added into each well. The differentiation was carried out for 21 days. On day 21, the cell cultures were fixed in 4% formaldehyde and stained with Alcian blue solution. Images were taken using either a light microscope (DP72, Olympus) or a digital camera.
  • FIGS. 4A-4H illustrate the lack of significant difference in differentiation capabilities of P4 MSC and P4 MCSC during ex vivo cultures.
  • FIG. 4A is a FACS plot of CD34-CD13+MSC, which differentiated into bone (Alizarin staining) (FIG. 4B), fat (phase bright) (FIG. 4C), and cartilage (Alcian blue staining) (FIG. 4D).
  • FIG. 4E is a FACS plot of CD34-CD13+MCSC, which differentiated into bone (Alizarin staining) (FIG. 4F), fat (phase bright) (FIG. 4G), and cartilage (Alcian blue staining) (FIG. 4H).
  • FIG. ID the number of viable floating cells in MCSC cultures increased with increased passage (FIG. ID).
  • FIGS.2A - 2C provide the proteome analyses indicating the significant increase in angiogenic protein release from MCSC cultures.
  • FIG. 2A and 2B are images of partial blot films developed for proteome analyses of supernatants from P7 MSC and P7 MCSC cultures. The black arrows indicate the control dots from the manufacturer. The red arrow in FIG. 2A indicates no protein expression. The red arrowhead in FIG. 2B indicates protein expression at the same location. The green arrow in FIG.
  • FIG. 2A indicates weak protein expression.
  • the green arrowhead in FIG. 2B indicates strong protein expression at the same location.
  • Each antibody has two dot spots according to manufacturer’s specification.
  • FIG. 2C is a proteome comparison (fold change) of angiogenic proteins between supernatants from P7 MSC and P7 MCSC cultures. Fold Changes represents MCSC versus MSC.
  • VEGF vascular endothelial growth factor
  • MCSCs were incubated with 10 pg/ml CD44 monoclonal antibodies (Catalogue #: 103005, BioLegend, Inc.) in a FACS buffer containing PBS and 0.5% bovine serum albumin (BSA) for 1 hour at 4°C.
  • BSA bovine serum albumin
  • the pre-treated cells were washed twice with the PBS and collected by centrifugation.
  • CD44 treated MCSCs were then plated at lx 10 5 /ml in 24-well plates for ex vivo culture. Cell clusters were counted after 2 days.
  • FIGS. 5A - 5D illustrate the inhibition of the cluster formation and proliferation of P5 MCSC by the anti-CD44 monoclonal antibodies.
  • FIG. 5A is a phase bright image of floating clusters from P5 MCSC without treatment.
  • FIG. 5B is a phase bright images of floating cells from P5 MCSC with treatment of anti-CD44.
  • FIG. 5C is a graphical representation of the cumulative cluster count data from P5 MCSC treated with anti- CD44 or without treatment.
  • FIG. 6A is a graphical representation of the differences in cell proliferation of MSCs and MCSCs.
  • the P10 MCSCs were found to proliferate much faster than P10 MSCs. ** P ⁇ 0.01.
  • FIG. 6B is an image of FACS analysis of the MCSCs for expression of Caspase 3 and CD44+. These rapidly proliferating MCSCs do not express cleaved Caspase3 (Cell Signaling Technology, Cat#9664S) and continue to express strong CD44.
  • FIG. 7 is a graphical representation of the proteome comparison of ICAM-1 between supernatants from P5 MCSCs and P7 MCSC cultures. *P ⁇ 0.05. This proteome comparison (mean pixel density) of ICAM-1 between supernatants from P7 MCSC and P5 MCSC culture revealed almost a 53-fold difference. *P ⁇ 0.05. Large amount of ICAM-1 (53-fold increase in P7 MCSCs versus P5 MCSCs, FIG. 7) appears to compensate the loss of CD44.
  • the anti-CD44 monoclonal antibody inhibited the proliferation of MCSCs, and demonstrated significant inhibition of the proliferation and cluster formation of early MCSCs with lower ICAM-1 protein levels.
  • An embodiment of a method of treatment of cancer includes administering a therapeutically effective amount of inhibitors of CD44 and ICAM-1 to the patient and inhibiting proliferation of the mesenchymal cancer stem cells.
  • the inhibitor of ICAM-1 can be an agent that interferes with expression of the ICAM-1 gene.
  • the inhibitor of ICAM-1 can be an agent that interferes with modification or activity of the ICAM-1 protein or its variants.
  • the inhibitor of ICAM-1 can be an agent that interferes with interactions of ICAM-1.
  • the inhibitor of ICAM-1 can be an anti- ICAM-1 antibody.
  • therapeutically effective amount of inhibitors of CD44 and ICAM-1 are part of the therapeutic regimen for AML.
  • anti-CD44 with anti-ICAM-1 antibodies are provided to inhibit MCSCs’ tumorigenic proliferation and cytokine release.
  • primary MCSCs are treated with anti-CD44 (clone# BJ18, BioLegend) in combination with anti-ICAM-1 (clone# BBIG-I1, R&D systems) antibodies ex vivo.
  • the experimental groups include: Group 1: MCSCs with no treatment; Group 2: MCSCs treated with anti-CD44 alone; Group 3: MCSCs treated with anti-ICAM-1 alone; Group 4: MCSCs treated with both anti-CD44 and anti-ICAM-1.
  • the tumorigenic clusters are quantified and apoptosis assays are conducted to detect early vs.
  • MCSCs are the cellular origin of angiogenesis and cytokine release in an AML patient’s BM. Embodiments include a method of isolating these MCSCs from bone marrow of AML patients.
  • the AML bone marrow sample is incubated in a lysing buffer containing ammonium chloride and potassium salts (such as ACK Lysing Buffer (Catalog# A1049201, Thermo Fisher Scientific)) at room temperature for 5 minutes.
  • the lysing buffer is used for the lysis of red blood cells in samples, such as bone marrow.
  • the residual cells were washed by phosphate buffered saline and collected by centrifugation at 800 rpm for 5 minutes at room temperature.
  • the cell pellet was mixed with the culture medium from a defined culture kit for derivation and expansion of mesenchymal progenitor cells (STEMdiffTM Mesenchymal Progenitor Kit, STEMCELL Technologies Inc.) and prepared for further plating.
  • These bone marrow cells (2 x 105 cells/ml) were plated in a 24-well plate for one week to allow MSCs to attach to the plastic bottom. Medium change was performed every 2-3 days. Instead of discarding the non-adherent floating cells according to protocols in the art, the floating cells were transferred to a new 24-well plate for continuous culture for another week to allow MCSCs to attach to the plastic bottom of the plate.
  • the target of AML therapy regimen is the MCSC population with pro-angiogenic properties, including vessel structure formation and the release of both cytokines and growth factors that are essential for the proliferation and metastasis of cancer cells (Right Panel, FIG. 3).
  • CD90 CD44 + cancer stem cells have been reported in gastric and breast cancers, which grew in floating spheres in vitro and exhibited mesenchymal features and high metastatic capabilities in vivo.
  • the MCSCs create a conducive environment to support LSCs relapse and metastasis and are a good target to manage or treat AML malignancy (Left Panel, FIG.3).
  • Therapeutic strategies include targeting MCSC to inhibit CD44 and release of cytokine to prevent AML progression and relapse.
  • Targeting MCSCs can be part of treatment regimens for other cancers as well.
  • An embodiment of a method of treatment of cancer includes administering a therapeutically effective amount of inhibitors of CD44 and ICAM-1 to the patient and inhibiting proliferation of the mesenchymal cancer stem cells. Certain embodiments include evaluation of the level of ICAM-1 levels in a patient to monitor the progression and detect any relapse of AML. In certain embodiments, anti-CD44 pre-treatment is supplemented with an inhibitor of ICAM-1 to inhibit the tumorigenic proliferation of MCSCs that express higher amount of ICAM-1 proteins than normal. Therapeutically effective amount of inhibitors of CD44 and ICAM-1 are provided to the patient to inhibit progression and relapse of AML. An embodiment includes a new immunotherapy to combine anti-CD44 and anti-ICAM-1 antibodies to target key adhesion molecules of MCSCs to prevent AML progression and relapse.

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Abstract

La présente invention concerne la prise en charge ou le traitement de cancers, tels que la leucémie aiguë myéloïde, le cancer du sein ou le cancer de l'estomac, par l'inhibition de la prolifération de cellules souches cancéreuses mésenchymateuses par l'administration d'inhibiteurs de CD44. L'invention concerne également la prise en charge ou le traitement de cancers, tels que la leucémie aiguë myéloïde, le cancer du sein ou le cancer de l'estomac, par l'inhibition de la libération de cytokine par les cellules souches cancéreuses mésenchymateuses. L'invention concerne également la prise en charge ou le traitement de cancers, tels que la leucémie aiguë myéloïde, le cancer du sein ou le cancer de l'estomac, par l'inhibition de la prolifération de cellules souches cancéreuses mésenchymateuses par l'administration au patient d'un inhibiteur angiogénique. Le régime de traitement peut comprendre un ou plusieurs éléments parmi un inhibiteur angiogénique, un inhibiteur de CD44, un inhibiteur d'ICAM-1 ou un composé permettant d'inhiber la libération de cytokine par des cellules souches cancéreuses mésenchymateuses dans une moelle osseuse du patient.
PCT/US2020/070905 2019-12-12 2020-12-12 Méthodes et compositions pour le traitement d'une leucémie aiguë myéloïde WO2021119669A1 (fr)

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