[go: up one dir, main page]

WO2017040686A1 - Identification de fibroblastes circulants associés au cancer - Google Patents

Identification de fibroblastes circulants associés au cancer Download PDF

Info

Publication number
WO2017040686A1
WO2017040686A1 PCT/US2016/049730 US2016049730W WO2017040686A1 WO 2017040686 A1 WO2017040686 A1 WO 2017040686A1 US 2016049730 W US2016049730 W US 2016049730W WO 2017040686 A1 WO2017040686 A1 WO 2017040686A1
Authority
WO
WIPO (PCT)
Prior art keywords
ccaf
patient
cancer
sample
detecting
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2016/049730
Other languages
English (en)
Inventor
Marc E. Lippman
Zheng AO
Sanket H. SHAH
Ram H. DATAR
Dorraya EL-ASHRY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Miami
Original Assignee
University of Miami
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Miami filed Critical University of Miami
Publication of WO2017040686A1 publication Critical patent/WO2017040686A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/147—Microfiltration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types

Definitions

  • the present disclosure relates to the use of cancer associated fibroblasts (CAFs) as a diagnostic or prognostic tool to identify metastatic cancer and/or for evaluating a patient's response to cancer therapy.
  • CAFs cancer associated fibroblasts
  • the disclosure also provides methods for directly targeting CAFs for anticancer therapy.
  • a human tumor is a complex tissue composed of malignant cells and tumor associated stromal cells.
  • Circulating Tumor Cells are tumor cells found in cancer patients' peripheral blood.
  • Studies of CTC have revealed their promising prognostic value in several cancers including breast cancer, colorectal cancer and prostate cancer [Cristofanilli et ah, The New England Journal of Medicine 351: 781-91 (2004); Cohen et ah, Annals of Oncology:
  • the wounded epithelial cells and immune cells including monocytes and macrophages induce an activated fibroblasts phenotype by various growth factors and chemokines such as TGF-p, EGF, PDGF, FGF2, MCP-1, reactive oxygen species and ECM proteases [Kalluri et ah, Nature reviews Cancer 6: 392-401 (2006)].
  • the activated fibroblasts are identified by various markers such as a-SMA, fibroblasts specific protein (FSP), vimentin, prolyl 4-hydroxylase and fibroblasts activation protein (FAP) [Kalluri et ah, Nature reviews Cancer 6: 392-401 (2006)].
  • CAFs cancer associated fibroblasts
  • FAP is currently the most specific marker used to identify "activated fibroblasts” or CAFs, and is a type II transmembrane serine protease [Fischer et ah, Clinical Cancer Research : An Official Journal of the American Association for Cancer Research 18: 6208-18 (2012)]. Its specific function is unknown, but the enzyme activity has been implicated in tumor progression, extracellular matrix (ECM) remodeling and metastasis [Fischer et ah, Clinical Cancer Research : An Official Journal of the American Association for Cancer Research 18: 6208-18 (2012)].
  • ECM extracellular matrix
  • HGF Hepatocyte Growth Factor
  • MMPs Matrix Metalloproteinases
  • CAFs have been implicated in promoting angiogenesis through secretion of VEGF, FGF2, and SDF-1 [Kalluri et al, Nature reviews Cancer 6: 392-401 (2006)].
  • CAFs are known to induce EMT through secretion of TGF-I3 and HGF that also facilitates metastasis [Kalluri et al. , Nature Reviews Cancer 6: 392-401 (2006)].
  • CAF-secreted cytokines also support immune evasion and confer a survival advantage to tumor cells [Liao et al, PloS one. 4: e7965 (2009)].
  • CAFs collectively serve as an 'incubator' for cancer cells by providing a favorable "soil” that supports growth and proliferation of cancer cells at distant sites [Kalluri et al, Nature reviews Cancer 6: 392-401 (2006); Duda et al, Proceedings of the National Academy of Sciences of the United States of America 107: 21677-82 (2010)].
  • Depletion of CAFs has shown to decrease metastasis by altering cytokine profiling, and repressing angiogenesis and recruitment of
  • the sample is a peripheral blood sample.
  • a method of detecting a circulating cancer associated fibroblast (cCAF) in a patient comprising obtaining a peripheral blood sample from the patient; and detecting the cCAF in the sample.
  • the detecting is performed via a cell-size based microfilter.
  • the detecting is performed via an antibody.
  • the antibody specifically associates with an epitope of fibroblast activation protein (FAP), alpha-Smooth Muscle Actin (a-SMA), fibroblast specific protein (FSP), vimentin, or a combination thereof.
  • FAP fibroblast activation protein
  • a-SMA alpha-Smooth Muscle Actin
  • FSP fibroblast specific protein
  • the method further comprises isolating the cCAF. In additional embodiments, the method further comprises enumerating the cCAF.
  • the disclosure provides a method of identifying metastasis in a patient, the method comprising obtaining a sample from the patient; detecting a circulating cancer associated fibroblast (cCAF) in the sample; and enumerating the cCAF; wherein the enumerating identifies whether metastasis is present in the patient.
  • the detecting is performed via a cell-size based microfilter. In some embodiments, the detecting is performed via an antibody.
  • the disclosure provides a method of determining effectiveness of cancer treatment in a patient, the method comprising obtaining a sample from the patient;
  • the sample is a peripheral blood sample.
  • the detecting is performed via a cell-size based microfilter.
  • FIG. 1A-1B shows validation of microfilter capture of CAF cells.
  • A 3,000 CAF-23 and 3,000 MCF-7 cells were spiked into 5 ml of PBS, fixed with formalin, processed through microfilter device and immunofluorescently stained with anti-FAP-Alexa 488 and anti-CK- Alexa 594 antibodies (Top Panel) Merged Picture (Bottom Panel); picture split into individual channels.
  • B 100 CAF-23 cells were spiked into 7.5 mL of blood, processed through microfilter and immunofluorescently stained for FAP. The samples were enumerated under fluorescent microscope.
  • FIG. 2 shows the identification of CTC and cCAF on microfilter.
  • Top Panel Left: cCAF identified by FAP staining in breast cancer samples.
  • CTC identified by pan-CK staining in breast cancer samples.
  • Bottom Panel Representative Picture of cCAF double stained by FAP and alpha-SMA. Picture of the same cell is split into three channels to demonstrate staining for both markers and DAPI.
  • Figure 3A-3B shows CTC and cCAF enumeration from cancer patients.
  • A Table showing cCAF and CTC enumeration from metastatic breast cancer patients (MET group), localized breast cancer patient with >5 years disease free survival (LOC group), metastatic colorectal cancer patients with metastasis to liver, localized prostate cancer patients and healthy donors.
  • B Graphical representation of the enumeration of CTC and cCAF from MET group and LOC group breast cancer patients.
  • FIG. 4 depicts the identification of CTC and cCAF clusters on microfilter.
  • Sample is labeled with DAPI (Blue), pan-CK-Alexa 488 (Green), FAP-Alexa 594 (Red) and CD45-Alexa 680 (White) (From Left to Right) Representative Picture of CTC clusters, CTC clustering with cCAF, CTC clustering with leukocytes and cCAF clusters.
  • the present disclosure discloses studies relating to CAF participation in the metastatic process and the significance of CAFs in the circulation of a patient, and provides direct evidence of circulating cancer associated fibroblasts present along with the circulating tumor cells in metastatic patients' blood.
  • the results described herein establish a strong association of the presence of cCAFs in patients' blood samples with tumor metastasis; CAFs were uniformly detectable in peripheral blood of metastatic patients but not in normal women or 'cured' patients, providing a metastasis biomarker accessible via liquid biopsy.
  • the disclosure also provides methods targeting CAFs for treatment of multiple different cancers.
  • melt means to reduce the number of, destroy, or otherwise result in a lowering in the amount of a target relative to the amount or number present in the absence of exposure to an agent and/or chemotherapeutic agent as disclosed herein.
  • the term "specifically associates” refers to association where an agent binds to a target (i.e., a CAF) without substantially associating with any other non-target. Such association is measurably different from a nonspecific interaction.
  • Specific association can be measured, for example, by determining association of an agent compared to association of a control agent, which generally is an agent of similar structure that does not have associating activity.
  • specific association can be determined by competition with a control agent that is similar to the target, for example, an excess of non-labeled target. In this case, specific association is indicated if the association of the labeled target to a probe is competitively inhibited by excess unlabeled target.
  • specific association is used in relation to the interaction between the agent and the CAF.
  • specific association refers to an agent having a Kd at least 2-fold greater than that of a non-specific target, preferably an agent having a Kd at least 4-fold, 6-fold, 8-fold, 10-fold, or greater than that of a non-specific target.
  • specific association can be expressed as an agent having a Kd for the target of at least about 10 "4 M, alternatively at least about 10 "5 M, alternatively at least about 10 "6 M, alternatively at least about 10 - " 7 M, alternatively at least about 10 - " 8 M, alternatively at least about 10 "9 M, alternatively at least about 10 "10 M, alternatively at least about 10 "11 M, alternatively at least about 10 - " 12 M, or less.
  • ranges of values include the endpoints of the recited range.
  • agents that target cCAFs are contemplated for use in methods to identify, enumerate, and eliminate cCAFs, both in vivo and in vitro.
  • reagents such as antibodies, small molecules, aptamers, and DARPins are contemplated for use to detect and eliminate CAF cells.
  • agents that target a cCAF include a FAP-targeting DNA vaccine, which has been shown to specifically eliminate CAF populations and mitigate tumor metastasis in a mouse model of breast cancer [Liao et ah, PloS One. 4: e7965 (2009)].
  • Sibrotuzumab an anti-human FAP antibody
  • Sibrotuzumab has shown remarkable tumor stromal targeting properties in humans [Hofheinz et ah, Onkologie 26: 44-8 (2003); Scott et al., Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 9: 1639-47 (2003)].
  • FAP radioimmunoconjugates and antibody drug conjugates as cCAF targeting agents.
  • Methods of the disclosure surround the use of CAFs and are contemplated as in vitro and in vivo methods. Methods of isolation, identification, enumeration, and elimination of CAFs are, in various aspects, contemplated by the disclosure. In various aspects, the methods employ the use of an agent that specifically associates with a CAF. In any of the aspects or embodiments of the disclosure, the CAF is a cCAF.
  • the cCAF is identified or detected via its size.
  • the diameter of a cCAF is about 10 microns ( ⁇ ) or greater.
  • the size of a cCAF is from about 10 ⁇ to about 200 ⁇ , or from about 10 ⁇ to about 150 ⁇ , or from about 10 ⁇ to about 100 ⁇ , or from about 10 ⁇ to about 50 ⁇ , or from about 10 ⁇ to about 30 ⁇ in diameter.
  • the diameter of a cCAF is or is at least about 10 ⁇ , is or is at least about 20 ⁇ , is or is at least about 30 ⁇ , is or is at least about 40 ⁇ , is or is at least about 50 ⁇ , is or is at least about 60 ⁇ , is or is at least about 70 ⁇ , is or is at least about 80 ⁇ , is or is at least about 90 ⁇ , or is or is at least about 100 ⁇ in diameter.
  • the diameter of a cCAF is less than about 20 ⁇ , less than about 30 ⁇ , less than about 40 ⁇ , less than about 50 ⁇ , less than about 60 ⁇ , less than about 70 ⁇ , less than about 80 ⁇ , less than about 90 ⁇ , or less than about 100 ⁇ in diameter.
  • a cluster of cCAFs contains from about 2 to about 100 cells, or from about 2 to about 80, or from about 2 to about 50, or from about 2 to about 20, or from about 2 to about 10, or from about 2 to about 5 cCAF cells.
  • a cluster of cCAFs contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 50, at least 70, or at least 80 cells.
  • a cluster of cCAFs contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 cells or more.
  • the diameter of such clusters of cCAF cells is from about 20 ⁇ to about 1000 ⁇ .
  • the diameter of a cluster of cCAF cells is from about 20 ⁇ to about 950 ⁇ , or from about 20 ⁇ to about 900 ⁇ , or from about 20 ⁇ to about 850 ⁇ , or from about 20 ⁇ to about 800 ⁇ , or from about 20 ⁇ to about 750 ⁇ , or from about 20 ⁇ to about 700 ⁇ , or from about 20 ⁇ to about 650 ⁇ , or from about 20 ⁇ to about 600 ⁇ , or from about 20 ⁇ to about 5500 ⁇ , or from about 20 ⁇ to about 500 ⁇ , from about 20 ⁇ to about 450 ⁇ , or from about 20 ⁇ to about 400 ⁇ , or from about 20 ⁇ to about 350 ⁇ , or from about 20 ⁇ to about 300 ⁇ , or from about 20 ⁇ to about 250 ⁇ , or from about 20 ⁇ to about 200 ⁇ , or from about 20 ⁇ to about 150 ⁇ , or from about 20 ⁇
  • the diameter of a cluster of cCAF cells is about 20 ⁇ , about 30 ⁇ , about 40 ⁇ , about 50 ⁇ , about 60 ⁇ , about 70 ⁇ , about 80 ⁇ , about 90 ⁇ , about 100 ⁇ , about 150 ⁇ , about 200 ⁇ , about 250 ⁇ , about 300 ⁇ , about 350 ⁇ , about 400 ⁇ , about 450 ⁇ , about 500 ⁇ , about 600 ⁇ , about 700 ⁇ , about 800 ⁇ , about 900 ⁇ , about 950 ⁇ , about 1000 ⁇ or more.
  • agent that is able to associate with a CAF is contemplated for use.
  • agents include, but are not limited to, antibodies, small molecules, DARPins, and aptamers.
  • Antibodies are contemplated for use according to the disclosure.
  • Antibodies contemplated for use in the methods and compositions of the present disclosure include without limitation antibodies that recognize and associate with a target molecule or cell (e.g., a cCAF) either in vivo or in vitro.
  • Antibodies useful as agents that target cCAFs may be polyclonal or monoclonal.
  • Antibodies derived through genetic engineering or protein engineering may be used as well.
  • An antibody or fragment thereof associates with the CAF with a higher degree of affinity (i.e., a lower K D ) relative to the affinity of the antibody or fragment for a non-target.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g. Natural Killer (NK) cells, neutrophils, and macrophages
  • NK Natural Killer
  • the antibodies “arm” the cytotoxic cells and are required for such killing.
  • the NK cells mediate ADCC, and express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991), incorporated herein by reference.
  • an in vitro ADCC assay such as that described in U.S. Patent Number 6,737,056, incorporated herein by reference
  • useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells.
  • PBMC peripheral blood mononuclear cells
  • NK cells Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model.
  • Small molecule refers to a chemical compound, for instance a peptidometic that may optionally be derivatized, or any other low molecular weight organic compound, either natural or synthetic.
  • low molecular weight is meant compounds having a molecular weight of about 1000 Daltons, typically between 300 and 700 Daltons. Low molecular weight compounds, in various aspects, are about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900 or about 1000 Daltons.
  • aptamers The production and use of aptamers is known to those of ordinary skill in the art. In general, aptamers are nucleic acid or peptide binding species capable of tightly binding to and discreetly distinguishing target ligands [Yan et ah, RNA Biol. 6(3) 316-320 (2009), incorporated by reference herein in its entirety]. Aptamers, in some embodiments, may be obtained by a technique called the systematic evolution of ligands by exponential enrichment (SELEX) process [Tuerk et al, Science 249:505-10 (1990), U.S. Patent Number 5,270,163, and U.S. Patent Number 5,637,459, each of which is incorporated herein by reference in its entirety].
  • SELEX systematic evolution of ligands by exponential enrichment
  • nucleic acid aptamers are found in, for example and without limitation, Nucleic Acid and Peptide Aptamers: Methods and Protocols (Edited by Mayer, Humana Press, 2009) and Crawford et ah, Briefings in Functional Genomics and Proteomics 2(1): 72-79 (2003).
  • aptamers including but not limited to selection of RNA aptamers, selection of DNA aptamers, selection of aptamers capable of covalently linking to a target protein, use of modified aptamer libraries, and the use of aptamers as a diagnostic agent and a therapeutic agent is provided in Kopylov et ah, Molecular Biology 34(6): 940-954 (2000) translated from Molekulyarnaya Biologiya, Vol. 34, No. 6, 2000, pp. 1097-1113, which is incorporated herein by reference in its entirety.
  • an aptamer is between 10-100 nucleotides in length.
  • DARPin Designed Ankyrin Repeat Protein
  • Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton.
  • a single ankyrin repeat is a 33 residue motif consisting of two a-helices and a ⁇ - turn.
  • DARPins can be engineered to bind different target antigens by randomizing residues in the first a-helix and a ⁇ -turn of each repeat. Their binding interface can be increased by increasing the number of modules.
  • U.S. Patent Application Publication Numbers 20120177651 and 20040132028 each of which is incorporated herein by reference in its entirety.
  • compositions described herein can be administered in therapeutically effective dosages alone or in combination with adjunct cancer therapy such as surgery, chemotherapy, radiotherapy, immunotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g. , reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, sensitizing tumors to cancer treatments, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.
  • adjunct cancer therapy such as surgery, chemotherapy, radiotherapy, immunotherapy, thermotherapy, and laser therapy
  • a beneficial effect e.g. , reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, sensitizing tumors to cancer treatments, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.
  • Cytostatic and cytotoxic agents that target the cancer cells are specifically contemplated for combination therapy.
  • agents that target angiogenesis or lymphangio genesis, or immune therapies targeting checkpoint pathways are specifically contemplated for combination therapy.
  • chemotherapeutic agents include: alkylating agents such as thiotepa and
  • callystatin including its adozelesin, carzelesin and bizelesin synthetic analogues
  • cryptophycins particularly cryptophycin 1 and cryptophycin 8
  • dolastatin duocarmycin
  • calicheamicin especially calicheamicin gammall and calicheamicin omegall; L- asparaginase; anthracenedione substituted urea; methyl hydrazine derivatives; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo- 5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholin
  • cyclophosphamide thiotepa
  • taxoids e.g. , TAXOL® paclitaxel (Bristol-Myers Squibb
  • ifosfamide mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g. , CPT-11);
  • adrenocorticosteroids adrenocorticosteroids
  • progestins include estrogens; androgens; gonadotropin-releasing hormone analogs; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • SERMs selective estrogen receptor modulators
  • tamoxifen including NOLVADEX® tamoxifen
  • raloxifene raloxifene, droloxifene, 4- hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON-toremifene
  • aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMAS L® exemestane, formest
  • ANGIOZYME® ribozyme and a HER2 expression inhibitor
  • vaccines such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine
  • PROLEUKIN® rJL-2 PROLEUKIN® rJL-2
  • LURTOTECAN® topoisomerase 1 inhibitor ABARELLX® rmRH
  • pharmaceutically acceptable salts, acids or derivatives of any of the above are examples of any of the above.
  • the treatment methods described herein optionally include monitoring the effect of the therapeutic composition on the tumor.
  • the size of the tumor can be determined, as can the presence of metastases.
  • measurement of the degree of metastasis e.g. , by measuring the number of metastatic modules or by measurement of ascites associated with metastasis.
  • the agent that specifically targets cCAFs according to the disclosure and other drugs/therapies can be administered in combination either simultaneously in a single
  • compositions or in separate compositions are sequential.
  • administration is sequential.
  • Simultaneous administration is achieved by administering a single composition or
  • pharmacological protein formulation that includes both the inhibitor and other therapeutic agent(s).
  • the other chemotherapeutic agent(s) are taken separately at about the same time as a pharmacological formulation (e.g. , tablet, injection or drink) of the inhibitor.
  • kits for carrying out the methods of the disclosure.
  • the kit contains, e.g. , bottles, vials, ampoules, tubes, cartridges and/or syringes that comprise a liquid (e.g. , sterile injectable) formulation or a solid (e.g. , lyophilized) formulation.
  • the kits can also contain pharmaceutically acceptable vehicles or carriers (e.g. , solvents, solutions and/or buffers) for reconstituting a solid (e.g. , lyophilized) formulation into a solution or suspension for administration (e.g.
  • cCAFs are significantly associated with metastasis: cCAF population was observed in 17 of 20 patients with metastatic breast cancer and in 2 of 10 patients with localized breast cancer with long term disease free survival.
  • the findings outlined below enable the use of cCAFs as, in various aspects, a minimally invasive liquid biopsy biomarker for metastasis, a biomarker for the effectiveness of cancer treatment, and a target for the treatment of cancer.
  • MCF-7 cells were obtained from American Type Culture Collection (ATCC) and maintained in phenol red DMEM supplemented with heat inactivated 10% FBS.
  • CAF cells were previously developed and characterized [Drews-Elger et ah, Breast Cancer Research and Treatment 144: 503-17 (2014)] and maintained in phenol red IMEM supplemented with heat inactivated 10% FBS. Both cells were grown in 37°C, 5% C0 2 forced air incubator and passaged continuously by detachment using TrypLETM Express (Gibco-Life Technologies). Cell Cultures were checked routinely using Myco AlertTM Mycoplasma Detection Kit (Lonza).
  • the sample was then subjected to secondary antibody incubation using Goat anti-mouse Alexa 680 and Goat anti-rabbit Alexa 594 (Life Technologies) at room temperature for 1 hour. Then the sample was further labeled with pre-conjugate CK with Alexa 488. CK antibody (Dako) was pre-labeled with Alexa 488 using Alexa 488 antibody labeling kit (Life Technologies). Finally, the sample was coverslipped with Prolong Gold Antifade mounting media with DAPI (Life Technologies).
  • cell-size -based CTC isolation microfilter [Zheng et ah, Journal of Chromatography A 1162: 154-61 (2007)] was used to isolate cCAFs that are large relative to hematopoietic cells.
  • CAF23 cells were used, which were previously established from a triple negative breast cancer patient primary tumor [Drews-Elger et ah, Breast Cancer Research and Treatment 144: 503-17 (2014)]. Three thousand CAF23 cells were spiked together with 3,000 MCF-7 breast cancer cells resuspended into 1XPBS.
  • the microfilter capture process was employed to attempt identification of cCAFs in cancer patients' peripheral blood. Seven and a half milliliters of peripheral blood samples were collected from a total of 30 breast cancer patients— 20 patients with metastatic breast cancer (Stage IV, MET group) and 10 patients with localized breast cancer with > 5 years of long term disease free survival (Stage I patients treated with curative therapy, LOC group). This latter group of patients was predominantly composed of individuals who will not recur although a small number of these patients will develop recurrences late.
  • cCAFs were identified as CK-, FAP+, CD45-cells whereas CTCs are identified as CK+, CD45- cells ( Figure 1).
  • cCAF identification was also confirmed in parallel samples by a FAP/a-Smooth Muscle Actin staining ( Figure 2 Bottom panel).
  • Figure 3A cCAFs can be detected in 17/20 (85%) patients from the MET group but in only 2/10 (20%) of patients from the LOC group and at very low level ( ⁇ 2 cCAFs). It is noted that CTCs are detected at a much higher incidence (80%) in the LOC group.
  • cCAFs as a biomarker, either as a standalone or in combination with, e.g., CTCs, for metastasis.
  • cCAFs were also analyzed in colorectal cancer patients with metastasis to liver as well as patients with localized prostate cancer. It was demonstrated that cCAFs were detectable in both colorectal and prostate cancer setting, and not detectable in healthy donor's blood (Figure 3A). cCAFs were also detected at high levels in metastatic colorectal patients and low levels in localized prostate cancer patients. It is also noted that in 2 metastatic colorectal cancer patients and 1 localized prostate cancer patient, cCAFs, but not CTCs, were detected.
  • CTCs are of important prognostic value in several cancer types [Cristofanilli et al., The New England Journal of Medicine 351: 781-91 (2004); Cohen et al., Annals of Oncology: Official Journal of the European Society for Medical

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Water Supply & Treatment (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Physiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne l'utilisation de fibroblastes circulants associés au cancer (CAF) en tant qu'outil de diagnostic ou de pronostic pour identifier un cancer métastatique et/ou pour évaluer la réponse d'un patient à une cancérothérapie. L'invention concerne également des procédés pour cibler directement des CAF pour un traitement anti-cancéreux.
PCT/US2016/049730 2015-09-01 2016-08-31 Identification de fibroblastes circulants associés au cancer Ceased WO2017040686A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201562212830P 2015-09-01 2015-09-01
US62/212,830 2015-09-01

Publications (1)

Publication Number Publication Date
WO2017040686A1 true WO2017040686A1 (fr) 2017-03-09

Family

ID=58189072

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2016/049730 Ceased WO2017040686A1 (fr) 2015-09-01 2016-08-31 Identification de fibroblastes circulants associés au cancer

Country Status (1)

Country Link
WO (1) WO2017040686A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020118216A1 (fr) * 2018-12-08 2020-06-11 Board Of Regents, The University Of Texas System Identification et ciblage de fibroblastes associés aux carcinomes favorisant les tumeurs pour le diagnostic et le traitement du cancer et d'autres maladies
US12473598B2 (en) 2018-03-28 2025-11-18 Board Of Regents, The University Of Texas System Identification of epigenetic alterations in DNA isolated from exosomes

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999041613A1 (fr) * 1998-02-12 1999-08-19 Immunivest Methodes et reactifs pour l'isolation rapide et efficace de cellules cancereuses circulantes
WO2004076643A2 (fr) * 2003-02-27 2004-09-10 Immunivest Corporation Cellules tumorales circulantes (ctc): evaluation precoce du temps d'evolution, de la survie et de la reaction aux therapies des patients cancereux metastasiques
US7632814B2 (en) * 2006-09-07 2009-12-15 University Of South Florida HYD1 peptides as anti-cancer agents
WO2013138522A2 (fr) * 2012-03-16 2013-09-19 Genelux Corporation Méthodes d'évaluation de l'efficacité et de la surveillance d'un traitement viral oncolytique
US20140315295A1 (en) * 2013-03-15 2014-10-23 Creatv Microtech, Inc. Polymer microfilters, devices comprising the same, methods of manufacturing the same, and uses thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999041613A1 (fr) * 1998-02-12 1999-08-19 Immunivest Methodes et reactifs pour l'isolation rapide et efficace de cellules cancereuses circulantes
WO2004076643A2 (fr) * 2003-02-27 2004-09-10 Immunivest Corporation Cellules tumorales circulantes (ctc): evaluation precoce du temps d'evolution, de la survie et de la reaction aux therapies des patients cancereux metastasiques
US7632814B2 (en) * 2006-09-07 2009-12-15 University Of South Florida HYD1 peptides as anti-cancer agents
WO2013138522A2 (fr) * 2012-03-16 2013-09-19 Genelux Corporation Méthodes d'évaluation de l'efficacité et de la surveillance d'un traitement viral oncolytique
US20140315295A1 (en) * 2013-03-15 2014-10-23 Creatv Microtech, Inc. Polymer microfilters, devices comprising the same, methods of manufacturing the same, and uses thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AO, Z ET AL.: "Identification of Cancer-Associated Fibroblasts in Circulating Blood from Patients with Metastatic Breast Cancer.", CANCER RESEARCH, vol. 75, no. 22, 15 October 2015 (2015-10-15), pages 4681 - 4687, XP055366231 *
CHIAPPINI, F: "Circulating Tumor Cells Measurements in Hepatocellular Carcinoma.", INTERNATIONAL JOURNAL OF HEPATOLOGY., vol. 2012, no. 684802, 28 May 2012 (2012-05-28), XP055366213 *
GUNAYDIN, G ET AL.: "Cancer Associated Fibroblasts have Phenotypic and Functional Characteristics Similar to the Fibrocytes that Represent a Novel MDSC Subset.", ONCOLMMUNOLOGY., vol. 4, no. 9, 27 May 2015 (2015-05-27), pages 1 - 10, XP055366218 *
WANG, H ET AL.: "Detection and Enumeration of Circulating Tumor Cells Based on their Invasive Property.", ONCOTARGET., vol. 6, no. 29, 11 July 2015 (2015-07-11), pages 27304 - 27311, XP055366222 *
XING, F ET AL.: "Cancer Associated Fibroblasts (CAFs) in Tumor Microenvironment.", FRONTIERS IN BIOSCIENCE., vol. 15, 1 January 2011 (2011-01-01), pages 166 - 179 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12473598B2 (en) 2018-03-28 2025-11-18 Board Of Regents, The University Of Texas System Identification of epigenetic alterations in DNA isolated from exosomes
WO2020118216A1 (fr) * 2018-12-08 2020-06-11 Board Of Regents, The University Of Texas System Identification et ciblage de fibroblastes associés aux carcinomes favorisant les tumeurs pour le diagnostic et le traitement du cancer et d'autres maladies
CN113412130A (zh) * 2018-12-08 2021-09-17 得克萨斯州大学系统董事会 用于诊断和治疗癌症及其他疾病的对促肿瘤癌症相关成纤维细胞的识别和靶向

Similar Documents

Publication Publication Date Title
US10966998B2 (en) Cancer therapy via a combination of epigenetic modulation and immune modulation
US20230266324A1 (en) Predicting response to pd-1 axis inhibitors
US20200123258A1 (en) Targeting b cells to enhance response to immune checkpoint blockade
AU2014364520A1 (en) Methods and assays relating to circulating tumor cells
US20210030703A1 (en) Use of caloric restriction mimetics for potentiating chemo-immunotherapy for the treatment of cancers
JP2016536303A (ja) 末梢循環腫瘍細胞クラスターおよび癌処置に関する方法
IL269718B1 (en) Combination of anti-PD-L1 antibody and DNA-PK inhibitor for cancer treatment
US20220047596A1 (en) Combination of parp inhibitor and brd4 inhibitor for the treatment of cancer
US20200164034A1 (en) Methods for improving sex-dimorphic responses to targeted therapy in melanoma
Baker et al. IL-36 expression is increased in NSCLC with IL-36 stimulation of lung cancer cells promoting a pro-tumorigenic phenotype
WO2017040686A1 (fr) Identification de fibroblastes circulants associés au cancer
US20210156866A1 (en) Detection of immune checkpoint molecules by deglycosylation
WO2024108114A2 (fr) Procédés de sélection de patients atteints d'un cancer réceptifs à un traitement avec un inhibiteur de point de contrôle immunitaire
US20220016205A1 (en) Methods of overcoming resistance to immune checkpoint inhibitors
TW202241455A (zh) 使用akr1c3活化化合物與免疫檢查點抑制劑之組合療法
US20250340648A1 (en) Combination therapy for treatment of cancer comprising anti-pd-l1 and anti-cd73 antibodies
EP3976040A1 (fr) Antagoniste de liaison à l'axe pd-1 pour traiter le cancer avec des mutations génétiques dans des gènes spécifiques
EP3787634A1 (fr) Immunothérapies à base d'inositol
US12485149B2 (en) Methods and compositions for the treatment of HPV-related cancer
US20220313748A1 (en) Methods and compositions for the treatment of hpv-related cancer
WO2024218697A1 (fr) Méthodes et systèmes pour améliorer des thérapies anticancéreuses
US20220390456A1 (en) Small extracellular vesicle-associated vegf as a predictor for therapeutic responses
JP2025520098A (ja) Ｎｓｃｌｃを処置するためのバイオマーカーおよび方法
WO2022056043A1 (fr) Immunoconjugués cpg de thérapie anti-cancéreuse
WO2020150208A1 (fr) Compositions et méthodes de traitement du cancer utilisant des inhibiteurs de la voie de signalisation de l'il-17 et des inhibiteurs de points de contrôle immunitaire

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16842922

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16842922

Country of ref document: EP

Kind code of ref document: A1