WO2017040686A1

WO2017040686A1 - Identification of circulating cancer associated fibroblasts

Info

Publication number: WO2017040686A1
Application number: PCT/US2016/049730
Authority: WO
Inventors: Marc E. Lippman; Zheng AO; Sanket H. SHAH; Ram H. DATAR; Dorraya EL-ASHRY
Original assignee: University of Miami
Current assignee: University of Miami
Priority date: 2015-09-01
Filing date: 2016-08-31
Publication date: 2017-03-09
Anticipated expiration: 2018-03-01

Abstract

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. The disclosure also provides methods for directly targeting CAFs for anticancer therapy.

Description

IDENTIFICATION OF CIRCULATING CANCER ASSOCIATED FIBROBLASTS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application Serial No.

62/212,830, filed September 1, 2015, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] 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. The disclosure also provides methods for directly targeting CAFs for anticancer therapy.

BACKGROUND OF THE INVENTION

[0003] The background description provided herein is for the purpose of generally presenting the context of the disclosure. Work of the presently named inventors, to the extent it is described in this background section, as well as aspects of the description that may not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the present disclosure.

[0004] A human tumor is a complex tissue composed of malignant cells and tumor associated stromal cells. Circulating Tumor Cells (CTCs) 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:

Official Journal of the European Society for Medical Oncology/ESMO 20: 1223-9 (2009); de Bono et ah, Clinical Cancer Research An Official Journal of the American Association for Cancer Research 14: 6302-9 (2008)]. Recently, several groups have reported existence of CTC clusters and their clinical relevance [Hou et ah, Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology 30: 525-32 (2012); Aceto et al, Cell 158: 1110-22 (2014)]. Although the prognostic value of CTC has been well validated, there are still limitations to the enumeration of CTCs and their use as a routine clinical biomarker [Alix- Panabieres et ah, Nature Reviews Cancer 14: 623-31 (2014)]. These limitations exist in using CTC as a clinical marker for early detection of cancer [Pantel et ah, Clinical Chemistry 58: 936- 40 (2012)] or using CTC as a surrogate endpoint in interventional studies [Smerage et ah, Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology 32: 3483-9 (2014)]. These findings indicate either the limitations in specificity of CTC detection assays employed, or that detecting CTC alone can be misleading or inadequate, especially when applied for early detection of metastasis. Additional biomarker assays may result in enhanced specificity and broaden the application of 'liquid biopsy.'

[0005] Tumor associated stromal cells or cells of the tumor microenvironment are made up of various types of cells, including fibroblasts, endothelial cells, immune cells, adipocytes, pericytes and extracellular matrix [Mueller et ah, Nature Reviews Cancer 4: 839-49 (2004)]. Contrary to the previous belief that cells of the tumor microenvironment are innocent bystanders, there is substantial evidence highlighting the role of all types of stromal cells in tumor progression and metastasis [Mueller et ah, Nature Reviews Cancer 4: 839-49 (2004); Pietras et ah, Experimental Cell Research 316: 1324-31 (2010)].

[0006] 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)]. These activated fibroblasts are called cancer associated fibroblasts (CAFs) [Kalluri et ah, Nature reviews Cancer 6: 392-401 (2006)]. 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)]. [0007] CAF-secreted cytokines and growth factors can act in a paracrine fashion and regulate a multitude of critical steps in tumor metastasis [Kalluri et al, Nature reviews Cancer 6: 392-401 (2006)]. CAFs are involved in breast cancer initiation by over-expression of TGF-β and

Hepatocyte Growth Factor (HGF) [Kalluri et al, Nature reviews Cancer 6: 392-401 (2006)]. CAFs promote tumor progression and invasion by secreting ECM degrading proteases, the Matrix Metalloproteinases (MMPs) [Kalluri et al, Nature reviews Cancer 6: 392-401 (2006)]. 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

immunosuppressive cells [Liao et al, PloS one. 4: e7965 (2009)].

SUMMARY OF THE INVENTION

[0008] Prior to the instant disclosure there has been no evidence showing the presence of CAFs in circulation in human patients in clinical settings. Accordingly, the present disclosure is directed to the use of CAFs and their role in the process of metastasis. It is was previously assumed that 'liquid biopsies' of tumor components from blood would not only contain tumor cells but also contain other cellular components from tumor microenvironment. Yet, in spite of all the supporting literature, there has been no evidence prior to the present disclosure showing the presence of CAFs in circulation. Using cell-size based microfilter technology [see, e.g., Zheng et al, Journal of Chromatography A 1162: 154-61 (2007), incorporated herein by reference], the presence of circulating CAFs (cCAFs) along with CTCs are shown herein in breast, colorectal and prostate cancer patients' peripheral blood. In addition, the results herein show that the presence of cCAFs is highly associated with metastasis in breast cancer by comparing the enumeration data from metastatic breast cancer patients with that from patients with ductal carcinoma in situ (DCIS) or early stage 1 invasive breast cancer with no relapse for 5 years. These findings establish a significant association of cCAFs with metastasis and support cCAFs as a promising metastasis biomarker that is easily accessed minimally invasively in peripheral blood and quantified by enumeration.

[0009] The methodologies disclosed herein are useful for the isolation, enumeration, and establishment of cCAFs as viable cultures that can be studied and manipulated.

[0010] Accordingly, in some aspects, the disclosure provides a method of detecting a circulating cancer associated fibroblast (cCAF) in a patient, the method comprising obtaining a sample from the patient; and detecting the cCAF in the sample via a cell-size based microfilter.

[0011] In any embodiment of the disclosure, the sample is a peripheral blood sample.

[0012] In some embodiments, the patient has cancer. In related embodiments, the cancer is breast cancer, colorectal cancer, a sarcoma, a hematopoietic cancer, a neurological malignancy, or prostate cancer.

[0013] In some aspects, a method of detecting a circulating cancer associated fibroblast (cCAF) in a patient is provided, the method comprising obtaining a peripheral blood sample from the patient; and detecting the cCAF in the sample. In some embodiments, the detecting is performed via a cell-size based microfilter. In additional embodiments, the detecting is performed via an antibody. In some embodiments, 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.

[0014] In some embodiments, the method further comprises isolating the cCAF. In additional embodiments, the method further comprises enumerating the cCAF.

[0015] In further aspects, 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. In various embodiments, the detecting is performed via a cell-size based microfilter. In some embodiments, the detecting is performed via an antibody. In related embodiments, the antibody specifically associates with an epitope of fibroblast activation protein (FAP), cytokeratin, CD45, alpha-Smooth Muscle Actin (a-SMA), fibroblast specific protein (FSP), vimentin, or a combination thereof. [0016] In any of the embodiments of the disclosure, the diameter of the cCAF is about 10 microns (μΜ) or greater.

[0017] In further aspects, the disclosure provides a method of determining effectiveness of cancer treatment in a patient, the method comprising obtaining a sample from the patient;

detecting a circulating cancer associated fibroblast (cCAF) in the sample; enumerating the cCAF; and comparing the enumeration to an enumeration of circulating cancer associated fibroblasts (cCAFs) from the patient obtained at an earlier time point; wherein a decrease in the enumeration of cCAFs in the patient compared to the earlier time point is indicative of effective cancer treatment. In some embodiments, the sample is a peripheral blood sample. In further

embodiments, the detecting is performed via a cell-size based microfilter.

[0018] In some embodiments, the detecting is performed via an antibody, and in related embodiments the antibody specifically associates with an epitope of fibroblast activation protein (FAP), cytokeratin, CD45, alpha-Smooth Muscle Actin (a-SMA), fibroblast specific protein (FSP), vimentin, or a combination thereof.

[0019] In still further aspects, a method of treating cancer in a patient is provided, comprising administering to the patient a therapeutically effective amount of an agent that eliminates a circulating cancer associated fibroblast (cCAF) from the patient. In some embodiments, the agent is an antibody that specifically targets the cCAF. In further embodiments, the antibody specifically binds to fibroblast activation protein (FAP).

[0020] In some embodiments, the method further comprises administering a chemotherapeutic agent. In further embodiments, the agent and the chemotherapeutic agent are administered sequentially, while in some embodiments the agent and the chemotherapeutic agent are administered concomitantly.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 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.

[0022] Figure 2 shows the identification of CTC and cCAF on microfilter. (Top Panel) Left: cCAF identified by FAP staining in breast cancer samples. Right: 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.

[0023] 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.

[0024] Figure 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.

DETAILED DESCRIPTION OF THE INVENTION

[0025] 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.

[0026] It is noted here that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. [0027] It is also noted that the term "about" as used herein is understood to mean approximately.

[0028] As used herein, "eliminate" 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.

[0029] 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. For example, 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. As used herein, "specific association" is used in relation to the interaction between the agent and the CAF. In particular, 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. Alternatively, 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.

[0030] As used herein, ranges of values (e.g., from about 10 μιη to about 100 μιη) include the endpoints of the recited range.

AGENTS THAT TARGET cCAFS

[0031] According to the disclosure, agents that target cCAFs are contemplated for use in methods to identify, enumerate, and eliminate cCAFs, both in vivo and in vitro.

[0032] For example and without limitation, various reagents such as antibodies, small molecules, aptamers, and DARPins are contemplated for use to detect and eliminate CAF cells. [0033] Exemplary 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, 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)]. Also contemplated herein is the use of FAP radioimmunoconjugates and antibody drug conjugates as cCAF targeting agents.

Methods

[0034] 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.

[0035] In some embodiments, the cCAF is identified or detected via its size. In various embodiments it is contemplated that the diameter of a cCAF is about 10 microns (μιη) or greater. In further embodiments, the size of a cCAF is from about 10 μιη to about 200 μιη, or from about 10 μηι to about 150 μιη, or from about 10 μιη to about 100 μιη, or from about 10 μιη to about 50 μηι, or from about 10 μιη to about 30 μιη in diameter. In still further embodiments, the diameter of a cCAF is or is at least about 10 μιη, is or is at least about 20 μιη, is or is at least about 30 μιη, is or is at least about 40 μιη, is or is at least about 50 μιη, is or is at least about 60 μιη, is or is at least about 70 μιη, is or is at least about 80 μιη, is or is at least about 90 μιη, or is or is at least about 100 μηι in diameter. In some embodiments, the diameter of a cCAF is less than about 20 μηι, less than about 30 μιη, less than about 40 μιη, less than about 50 μιη, less than about 60 μιη, less than about 70 μιη, less than about 80 μιη, less than about 90 μιη, or less than about 100 μιη in diameter.

[0036] The disclosure also contemplates that cCAFs cluster. Thus, in various embodiments, 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. In some embodiments, 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. In specific embodiments, 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 μιη to about 1000 μιη. In further embodiments, the diameter of a cluster of cCAF cells is from about 20 μιη to about 950 μιη, or from about 20 μιη to about 900 μηι, or from about 20 μιη to about 850 μιη, or from about 20 μιη to about 800 μιη, or from about 20 μηι to about 750 μιη, or from about 20 μιη to about 700 μιη, or from about 20 μιη to about 650 μηι, or from about 20 μιη to about 600 μιη, or from about 20 μιη to about 5500 μιη, or from about 20 μηι to about 500 μιη, from about 20 μιη to about 450 μιη, or from about 20 μιη to about 400 μηι, or from about 20 μιη to about 350 μιη, or from about 20 μιη to about 300 μιη, or from about 20 μηι to about 250 μιη, or from about 20 μιη to about 200 μιη, or from about 20 μιη to about 150 μηι, or from about 20 μιη to about 100 μιη, or from about 20 μιη to about 50 μιη. In specific embodiments, the diameter of a cluster of cCAF cells is about 20 μιη, about 30 μιη, about 40 μηι, about 50 μιη, about 60 μιη, about 70 μιη, about 80 μιη, about 90 μιη, about 100 μηι, about 150 μιη, about 200 μιη, about 250 μιη, about 300 μιη, about 350 μιη, about 400 μιη, about 450 μιη, about 500 μιη, about 600 μιη, about 700 μιη, about 800 μιη, about 900 μιη, about 950 μηι, about 1000 μιη or more.

[0037] Any agent that is able to associate with a CAF is contemplated for use. Such agents include, but are not limited to, antibodies, small molecules, DARPins, and aptamers.

[0038] Antibody. Antibodies, along with fragments and derivatives thereof, 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. Antibody fragments include, but are not limited to, Fab' fragments, F(ab)2 fragments, Fv fragments, Fc fragments, one or more complementarity determining regions (CDR) fragments, individual heavy chains, individual light chain, dimeric heavy and light chains (as opposed to heterotetrameric heavy and light chains found in an intact antibody, single chain antibodies (scAb), and other antibody derivative or fragments known in the art. Antibodies include, but are not limited to, humanized antibodies (as well as antibodies modified in the manner of humanized antibodies but with the resulting antibody more closely resembling an antibody in a non-human species), chelating recombinant antibodies (CRABs), and bispecific antibodies and multispecific antibodies.

[0039] Antibodies or fragments thereof with specific effector functions are useful in various aspects of the disclosure. Such aspects include, without limitation, those in which a CAF is to be eliminated. Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

[0040] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. 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. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay (such as that described in U.S. Patent Number 6,737,056, incorporated herein by reference) may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model.

[0041] Small molecule. The term "small molecule," as used herein, 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. By "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.

[0042] 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]. General discussions of 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). Additional discussion of 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. In various aspects, an aptamer is between 10-100 nucleotides in length.

[0043] Designed Ankyrin Repeat Protein (DARPin). DARPins are derived from 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 β- turn. DARPins can be engineered to bind different target antigens by randomizing residues in the first a-helix and a β-turn of each repeat. Their binding interface can be increased by increasing the number of modules. For further details see U.S. Patent Application Publication Numbers 20120177651 and 20040132028, each of which is incorporated herein by reference in its entirety.

[0044] Therapeutic 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. Cytostatic and cytotoxic agents that target the cancer cells are specifically contemplated for combination therapy. Likewise, agents that target angiogenesis or lymphangio genesis, or immune therapies targeting checkpoint pathways are specifically contemplated for combination therapy.

[0045] As used herein, a "chemo therapeutic agent" is a chemical compound useful in the treatment of cancer. It is understood that agents of the disclosure that target and eliminate cCAFs possess, in some embodiments, chemotherapeutic properties. Chemotherapeutic agents as described herein are those agents having chemotherapeutic properties in their own right, and are in utilized in various embodiments in combination with the agents that target cCAFs.

[0046] Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and

CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and tiimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin;

callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin

(including the synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,

cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; vinca alkaloids; epipodophyllotoxins; antibiotics such as the enediyne antibiotics

(e.g. , 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 morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-f uorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitiaerine; pentostatin; phenamet; pirarubicin; losoxantione; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2 2"-trichlorotiiethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");

cyclophosphamide; thiotepa; taxoids, e.g. , TAXOL® paclitaxel (Bristol-Myers Squibb

Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16);

ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g. , CPT-11);

topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids such as retinoic acid; capecitabine; leucovorin (LV); irenotecan; adrenocortical suppressant;

adrenocorticosteroids; progestins; estrogens; androgens; gonadotropin-releasing hormone analogs; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), 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, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARTMIDEX® anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGF-A expression inhibitor (e.g. , 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; LURTOTECAN® topoisomerase 1 inhibitor; ABARELLX® rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0047] In some embodiments, the agent(s) described herein are administered in conjunction with any number of immune checkpoint inhibitors. Immune checkpoint inhibitors include antibodies, or antigen binding fragments thereof, that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, and GAL9. Exemplary immune checkpoint inhibitors include, but are not limited to, Tremelimumab (CTLA- 4 blocking antibody), anti-OX40, PD-L1 monoclonal Antibody (Anti-B7-Hl ; MEDI4736), ipilimumab, MK-3475 (PD-1 blocker) and Nivolumamb (anti-PDl antibody).

[0048] The treatment methods described herein optionally include monitoring the effect of the therapeutic composition on the tumor. For example, the size of the tumor can be determined, as can the presence of metastases. Also contemplated is measurement of the degree of metastasis, e.g. , by measuring the number of metastatic modules or by measurement of ascites associated with metastasis. [0049] The agent that specifically targets cCAFs according to the disclosure and other drugs/therapies can be administered in combination either simultaneously in a single

composition or in separate compositions. Alternatively, the 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). Alternatively, 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

[0050] The disclosure also provides kits for carrying out the methods of the disclosure. In various embodiments, 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. , by injection), including without limitation reconstituting a lyophilized formulation in a syringe for injection or for diluting concentrate to a lower concentration. Furthermore, extemporaneous injection solutions and suspensions can be prepared from, e.g. , sterile powder, granules, or tablets comprising a composition comprising an inhibitor as described herein. The kits can also include dispensing devices, such as aerosol or injection dispensing devices, pen injectors, autoinjectors, needleless injectors, syringes, and/or needles. In various embodiments, the kit also provides an oral dosage form, e.g. , a tablet or capsule or other oral formulation described herein, of the inhibitor for use in the method. The kit also provides instructions for use.

[0051] The invention will be more fully understood by reference to the following examples which detail exemplary embodiments of the invention. They should not, however, be construed as limiting the scope of the invention. All citations throughout the disclosure are hereby expressly incorporated by reference.

EXAMPLES

[0052] It is disclosed herein that the presence of cCAFs is 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.

[0053] Using Fibroblast Activation Protein (FAP) as a marker for CAFs, cCAFs have been identified in the peripheral blood of 3 different cancer types: breast cancer, colorectal cancer and prostate cancer. The presence of cCAFs strongly associates with the presence of metastases in these cancers. cCAF enumeration in metastatic breast cancer patients and localized breast cancer patients with long term disease free survival revealed a sharp contrast between the two groups. The cCAF level was shown to be consistently low (0-1, with 5/6 having zero) in localized breast cancer patients' peripheral blood samples, which is more consistent as compared with CTC enumeration data, while in metastatic breast cancer patient's samples the range is (0-117) with 16/19 samples having cCAFs. cCAFs were found in 6/6 colorectal cancer with metastasis samples where 2/6 had no CTCs. Taken together, the findings described below enable the use of cCAFs as a biomarker for metastasis.

Methods

[0054] Tissue Culture. 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% C0₂ forced air incubator and passaged continuously by detachment using TrypLE™ Express (Gibco-Life Technologies). Cell Cultures were checked routinely using Myco Alert™ Mycoplasma Detection Kit (Lonza).

[0055] Blood Collection and Processing. Blood samples were collected from breast cancer patients under approved IRB protocol 20130312, and from prostate cancer patients under approved IRB protocol 20100635 and 20101056 following informed consent. Blood samples from colorectal cancer patients were obtained from University of Miami Tissue Bank Core Facility. These colorectal cancer patient samples as well as healthy donors' blood samples were determined as non-human subject research by IRB. Blood was collected via venipuncture into EDTA tubes (BD). The first tube for blood was discarded or used for other analyses to avoid potential contamination of epithelial cells and stromal cells during venipuncture. 7.5 mL blood was diluted 1: 1 with 1XPBS (Gibco) and fixed with final concentration of 1% formalin (Sigma Aldrich) for 10 minutes. Post-fixation, blood was processed through a microfilter at a flow rate of 200 mL/hour using the protocol as reported previously for CTC capture [Zheng et ah, Journal of Chromatography A 1162: 154-61 (2007)].

[0056] Immunofluorescence Staining. To enumerate for both CTC and cCAF on the same microfilter, the sample was subjected to triple staining with pan-cytokeratin, CD45 and FAP. The sample was blocked with blocking buffer (5% Normal Goat Serum (Life Technologies) and 0.3% Triton X-100 (Sigma Aldrich)) at room temperature for 1 hour. Then, the sample was incubated with primary antibody: mouse anti-human CD45 (Dako) and rabbit anti-human FAP (Millipore) at 4°C overnight. 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).

[0057] For the parallel sample collected from breast cancer patients at same time point, doubling staining of alpha-SMA and FAP was performed by incubation with primary antibodies consisting of mouse anti-human aSMA (Santa Cruz Biotechnology) and rabbit anti-human FAP (Millipore) at 4°C overnight after the same blocking procedure as described above, followed by incubation with Goat anti-mouse Alexa 488 and Goat anti-rabbit Alexa 594 (Life Technologies) incubation at room temperature for 1 hour. The sample was then coverslipped with DAPI as described above.

Example 1

Validation of cCAF enumeration using microfilter technology

[0058] In this example, 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. To test this in a model system, 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 sample was then processed through a microfilter using the same protocol as previously reported for CTC capture [Lin et ah, Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 16: 5011-8 (2010)]. Following cell capture, the microfilter was subjected to a double immunofluorescence staining for pan-cytokeratin and FAP to identify epithelial MCF-7 and fibroblastic CAF23 cells, respectively. The results showed that CAF23 cells were captured on the filter based on their larger size (approximately 20 μιη - 40 μιη) (Figure 1A). Further evaluation of CAF capture efficiency was also performed by spiking 100 CAF-23 cells into 7.5 mL of healthy donor's blood. An average capture efficiency of

95.0%+2.8% was demonstrated (Figure IB).

Example 2

cCAF Identification and Enumeration From Human Patients.

[0059] Having demonstrated that CAFs were captured from human blood with a very high efficiency, 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. Utilizing a triple staining for pan- CK, FAP and the lymphocyte marker CD45, 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). As shown in 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. These data highlight the importance of 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. During analysis for CTCs and cCAFs in cancer patient's peripheral blood, occasional clusters captured by the microfilter were also identified. CTCs clustering with CTCs, CTCs forming clusters with cCAFs, CTCs clustering with leukocytes and cCAFs alone clusters were also observed (Figure 4).

[0060] It has been widely documented that 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

Oncology/ESMO 20: 1223-9 (2009); de Bono et al, Clinical Cancer Research An Official Journal of the American Association for Cancer Research 14: 6302-9 (2008)]. However, using CTCs for early detection of solid tumors is not established. This is primarily due to the 'false positive' detection of circulating epithelial cells in circulation caused by other diseases, such as benign colon disease [Pantel et al., Clinical Chemistry 58: 936-40 (2012)]. In addition, CTCs are detected in both early stage and metastatic breast cancer [Nakagawa et al. , Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 13: 4105-10 (2007)] making it difficult to use their presence or number as a standalone biomarker for metastasis. A companion biomarker such as cCAFs will benefit the early detection of a solid tumor and an efficient biomarker for metastasis. The results described herein demonstrate that cCAFs were only detected in 2 of the 10 samples analyzed from the LOC group, and in each of these the cCAF number was 2 or fewer, while in the MET group, cCAFs were detected in 17 of 20 samples with only 5 of the 17 having 2 or fewer. Similarly, cCAF number were significantly higher in metastatic colorectal cancer samples compared with localized prostate cancer samples. Also, in 2 metastatic colorectal cancer patients and 1 localized prostate cancer patient, cCAFs were detected but not CTCs, thus further highlighting the importance of this CAF population in circulation and their clinical relevance. According to the disclosure, cCAF enumeration is a biomarker for early detection of cancer metastasis, either alone or in combination with other biomarker s.

[0061] While the present invention has been described in terms of various embodiments and examples, it is understood that variations and improvements will occur to those skilled in the art.

Therefore, only such limitations as appear in the claims should be placed on the invention.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting a circulating cancer associated fibroblast (cCAF) in a patient, the method comprising: obtaining a sample from the patient; and detecting the cCAF in the sample via a cell- size based microfilter.

2. The method of claim 1, wherein the sample is a peripheral blood sample.

3. The method of claim 1 or claim 2, wherein the patient has cancer.

4. The method of any one of claims 1-3, wherein the cancer is breast cancer, colorectal cancer, a sarcoma, a hematopoietic cancer, a neurological malignancy, or prostate cancer.

5. A method of detecting a circulating cancer associated fibroblast (cCAF) in a patient, the method comprising: obtaining a peripheral blood sample from the patient; and detecting the cCAF in the sample.

6. The method of claim 5, wherein the detecting is performed via a cell- size based microfilter.

7. The method of claim 5, wherein the detecting is performed via an antibody.

8. The method of claim 7, wherein the antibody specifically associates with an epitope of fibroblast activation protein (FAP), alpha-Smooth Muscle Actin (a-SMA), fibroblast specific protein (FSP), or Vimentin.

9. The method of any one of claims 1-8, further comprising isolating the cCAF.

10. The method of any one of claims 1-9, further comprising enumerating the cCAF.

11. 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.

12. The method of claim 11, wherein the diameter of the cCAF is about 10 microns (μΜ) or greater.

13. The method of claim 11 or claim 12, wherein the sample is a peripheral blood sample.

14. The method of any one of claims 11-13, wherein the detecting is performed via a cell-size based microfilter.

15. The method of any one of claims 11-13, wherein the detecting is performed via an antibody.

16. The method of claim 15, wherein the antibody specifically associates with an epitope of fibroblast activation protein (FAP), cytokeratin, CD45, alpha-Smooth Muscle Actin (a-SMA), fibroblast specific protein (FSP), vimentin, or a combination thereof.

17. A method of determining effectiveness of cancer treatment in a patient, the method comprising: obtaining a sample from the patient; detecting a circulating cancer associated fibroblast (cCAF) in the sample; enumerating the cCAF; and comparing the enumeration to an enumeration of circulating cancer associated fibroblasts (cCAFs) from the patient obtained at an earlier time point; wherein a decrease in the enumeration of cCAFs in the patient compared to the earlier time point is indicative of effective cancer treatment.

18. The method of claim 17, wherein the diameter of the cCAF is about 10 microns (μΜ) or greater.

19. The method of claim 17 or claim 18, wherein the sample is a peripheral blood sample.

20. The method of any one of claims 17-19, wherein the detecting is performed via a cell-size based microfilter.

21. The method of any one of claims 17-19, wherein the detecting is performed via an antibody.

22. The method of claim 21, wherein the antibody specifically associates with an epitope of fibroblast activation protein (FAP), cytokeratin, CD45, alpha-Smooth Muscle Actin (a-SMA), fibroblast specific protein (FSP), vimentin, or a combination thereof.

23. A method of treating cancer in a patient comprising administering to the patient a therapeutically effective amount of an agent that eliminates a circulating cancer associated fibroblast (cCAF) from the patient.

24. The method of claim 23, wherein the agent is an antibody that specifically targets the cCAF.

25. The method of claim 24, wherein the antibody specifically binds to fibroblast activation protein (FAP).

26. The method of any one of claims 23-25, further comprising administering an chemotherapeutic agent.

27. The method of claim 26, wherein the agent and the chemotherapeutic agent are administered sequentially.

28. The method of claim 26, wherein the agent and the chemotherapeutic agent are administered concomitantly.