U.S. patent application number 17/393892 was filed with the patent office on 2021-11-25 for methods for capturing, isolation, and targeting of circulating tumor cells and diagnostic and therapeutic applications thereof.
This patent application is currently assigned to PRESIDENT AND FELLOWS OF HARVARD COLLEGE. The applicant listed for this patent is PRESIDENT AND FELLOWS OF HARVARD COLLEGE. Invention is credited to Harry Scott DRISCOLL, Donald E. INGBER, Joo-Hun KANG, Michael SUPER, Alexander L. WATTERS.
Application Number | 20210364522 17/393892 |
Document ID | / |
Family ID | 1000005756562 |
Filed Date | 2021-11-25 |
United States Patent
Application |
20210364522 |
Kind Code |
A1 |
KANG; Joo-Hun ; et
al. |
November 25, 2021 |
METHODS FOR CAPTURING, ISOLATION, AND TARGETING OF CIRCULATING
TUMOR CELLS AND DIAGNOSTIC AND THERAPEUTIC APPLICATIONS THEREOF
Abstract
The invention relates to methods of detection, capture,
isolation and targeting of cancer cells for example circulating
tumor cells (CTCs) using carbohydrate recognition domain of a
lectin. The invention relates to methods of diagnosis, prognosis
and treatment of cancer.
Inventors: |
KANG; Joo-Hun; (Boston,
MA) ; INGBER; Donald E.; (Boston, MA) ; SUPER;
Michael; (Lexington, MA) ; WATTERS; Alexander L.;
(North Andover, MA) ; DRISCOLL; Harry Scott;
(Allston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PRESIDENT AND FELLOWS OF HARVARD COLLEGE |
Cambridge |
MA |
US |
|
|
Assignee: |
PRESIDENT AND FELLOWS OF HARVARD
COLLEGE
Cambridge
MA
|
Family ID: |
1000005756562 |
Appl. No.: |
17/393892 |
Filed: |
August 4, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16092288 |
Oct 9, 2018 |
11112410 |
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PCT/US2017/026768 |
Apr 10, 2017 |
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17393892 |
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62321859 |
Apr 13, 2016 |
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62324738 |
Apr 19, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6415 20170801;
G01N 2400/00 20130101; A61K 39/0011 20130101; G01N 2333/705
20130101; G01N 2333/4724 20130101; G01N 2800/52 20130101; G01N
2333/71 20130101; G01N 2333/4725 20130101; G01N 33/57492
20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; A61K 47/64 20060101 A61K047/64; A61K 39/00 20060101
A61K039/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under
N66001-11-1-4180 awarded by U.S. Department of Defense--Defense
Advanced Research Projects Agency. The government has certain
rights in the invention.
Claims
1. A method for capturing circulating tumor cells (CTCs) from a
biological fluid of a subject who does not have a hematological
tumor, the method comprising contacting the biological fluid with a
lectin molecule attached to a surface.
2. The method of claim 1, wherein the subject has a solid
tumor.
3. The method of claim 1, wherein the lectin molecule is a C-type
lectin.
4. The method of claim 3, wherein the C-type lectin molecule
comprises a carbohydrate recognition domain (CRD).
5. The method of claim 4, wherein the carbohydrate recognition
domain (CRD) is a CRD of mannose binding lectin.
6. The method of claim 5, wherein the C-type lectin molecule
comprising a carbohydrate recognition domain (CRD) of mannose
binding lectin (MBL) lacks a MBL collagen-like domain.
7. The method of claim 5, wherein the carbohydrate recognition
domain (CRD) of mannose binding lectin (MBL) comprises the sequence
of SEQ ID NO: 3.
8. The method of claim 5, wherein the carbohydrate recognition
domain (CRD) of mannose binding lectin (MBL) does not activate
complement or coagulation.
9. The method of claim 5, wherein the lectin molecule comprises at
least 80% amino acid sequence identity to human mannose binding
lectin and retains at least 80% of the wild-type carbohydrate
binding activity.
10. The method of claim 3, wherein the C-type lectin molecule is a
carbohydrate recognition domain (CRD) of mannose binding lectin
(MBL).
11. The method of claim 1, wherein the molecule further comprises
the Fc region of an immunoglobulin.
12. The method of claim 11, wherein the molecule is FcMBL.
13. The method of claim 1, wherein the surface is a bead, hollow
fiber, porous scaffold, particle, or well.
14. The method of claim 1, wherein the surface is magnetic.
15. The method of claim 1, wherein the CTCs express mannan
carbohydrates on their cell surface.
16. The method of claim 1, wherein the CTCs express carbohydrates
containing D-mannose and L-fucose onto their cell surface.
17. The method of claim 1, wherein the biological fluid is selected
from a body fluid, such as whole blood, plasma, any cell-containing
blood fraction, cerebrospinal fluid, bone marrow, cell sample,
joint fluid, urine, tears or feces.
18. The method of claim 17, wherein the bone marrow or cell sample
is obtained before transplantation.
19. The method of claim 1, further comprising isolation of the
captured CTCs.
20. The method of claim 19, wherein the isolation comprises passing
the biological fluid containing captured CTCs through a
microfluidic magnetic separation device.
21. The method of claim 1, further comprising assaying captured
cells for CTC markers.
22. The method of claim 21, wherein the CTC markers are selected
from GlcNAc, EpCAM, EphB4, HER2, EGFR, MUC-1, or a combination
thereof.
23. A method for generating cancer vaccine, comprising: (a)
contacting a sample containing CTCs from a cancer patient not
having a hematological tumor with lectin molecule attached to a
surface, whereby the lectin molecule binds the CTCs in the sample,
thereby providing captured CTCs; (b) isolating the captured CTCs;
(c) combining the isolated CTCs or a component thereof with an
adjuvant to generate a CTC-immunogen thereby producing a cancer
vaccine.
24. A method of treating cancer, the method comprising,
administering to a subject not having a hematological tumor, a
composition comprising a lectin molecule linked to an anticancer
therapeutic molecule.
25. A composition comprising lectin molecule linked to an
anticancer therapeutic molecule.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional under .sctn. 121 of
co-pending U.S. application Ser. No. 16/092,288 filed Oct. 9, 2018,
which is a 35 U.S.C. .sctn. 371 National Phase Entry Application of
International Application No. PCT/US2017/026768 filed Apr. 10,
2017, which designates the U.S. and claims benefit under 35 U.S.C.
.sctn. 119(e) of U.S. Provisional Application No. 62/321,859 filed
Apr. 13, 2016 and 62/324,738 filed Apr. 19, 2016, the contents of
which are incorporated herein by reference in their entireties.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Apr.5, 2017, is named 002806-086332-PCT_SL.txt and is 5,901
bytes in size.
TECHNICAL FIELD
[0004] The technology disclosed herein relates to methods,
molecules, compositions and kits for detecting, capturing,
isolating and targeting of tumor cells. The technology relates to
methods for detection, prognosis, diagnosis and therapeutics of
cancer.
BACKGROUND
[0005] Circulating tumor cells (CTCs) released from primary tumor
tissues into the bloodstream or lymphatic vessels are able to
colonize distant organs giving rise to metastasis and thus play a
crucial role in tumor dissemination and progression (Baccelli, I.
et al., 2013). Detection and enumeration of CTCs is beneficial for
early metastasis detection and provides prognostic and diagnostic
information (Scher, HI. et al., 2009). (Muller, V. et al., 2005).
Therefore detection, separation and enumeration of CTCs is relevant
to understand molecular drivers of cancer and metastasis and
improving prognosis and therapeutic strategies of metastatic
cancers.
[0006] The most commonly used technique for CTC isolation is
antibody-based isolation by targeting the epithelial-specific
markers or tumor specific antigens present of the surface of CTCs.
False positive selection may occur due to expression of epithelial
markers in non-epithelial cells (Man, Y. et al., 2011). However,
detection and isolation of CTCs is also hampered by their low
numbers and the heterogeneity of CTCs, including subpopulations
which have lost the characteristic epithelial features due to the
process of Epithelial-mesenchymal transition (EMT). EMT is regarded
as a pivotal process in tumor metastasis, leading to
dedifferentiation and increased mobility of cancer cells and
eventually loss of cell adhesion and therefore plays an important
role in tumor local invasion and subsequent dissemination. Current
isolation technologies used for CTC capture that are based on
epithelial surface markers, such as Epithelial Cell Adhesion
Molecule (EpCam), could miss metastatic tumor cells circulating in
blood (Alix-Panabieres, C. & Pantel, K., 2013). Among the
EpCAM-based technologies, the CellSearch CTC test has gained
considerable attention and is the only diagnostic test that is
currently approved by the US Food and Drug Administration for the
automated detection and enumeration of circulating tumor cells
(Food and Drug Administration, 2004). However, EpCAM is not a
perfect marker for CTC selection due to the high variation in its
gene expression between tumor subtypes, its transcription in
leukocytes, and loss of expression during EMT (Soysal, S. D. et
al., 2013) (Gorges, T. M. et al., 2012). One approach to address
the concern of variable EpCAM expression, is the use of EpCAM in
addition to tumor type-specific antigens, such as MUC1 for breast
and colon cancer or PSA for prostate cancer (Gold, B. et al.,
2015); however, expression of these markers also vary between tumor
cells of the same type, and there are no known specific antigens
for most types of cancer cells. Recently, studies have suggested
that EMT markers also could be used for the detection or capture of
CTCs (Yu, M. et al., 2013). Strategies are employed using a
combination of two or three markers to increase the number of
isolated CTCs and decrease false positives. Stem cell markers and
EMT markers have been used (Wu, S. et al., 2015). Some
technologies, such as the AdnaTest technology, use a mix of
antibodies directed towards various tumor-cell associated antigens
during the enrichment step. (Visit: http://www.adnagen.com/).
However, a single marker which can overcome the limitations posed
by existing CTC capture approaches, or enhance CTC capture when
used in combination with those approaches, has not yet been
identified. As such there is an unmet need for methods of CTC
detection and isolation that overcome the limitations of current
techniques by targeting surface molecules are that are specific to
broad range of cancer cells, but not normal cells, and that remain
on the surfaces of epithelial tumor cells even after they undergo
an EMT.
SUMMARY
[0007] Described herein are methods, compositions and kits for
detection, capture and isolation of circulating tumor cells (CTCs)
and applications thereof. It is contemplated that the methods,
compositions and kits disclosed herein also can be used to detect,
capture and isolate cellular components of CTCs, for example, cell
membrane and or secreted components of CTCs, including
extracellular vesicles, exosomes, microvesicles and the like. In
one aspect, the present invention provides for use of cell surface
carbohydrate-binding proteins for example lectins, for detection,
capture and isolation of cancer cells, preferably CTCs.
[0008] Accordingly in one aspect, the technology disclosed herein
relates to a method for capturing circulating tumor cells (CTCs)
from biological fluids of a subject, comprising contacting the
biological fluid with a lectin molecule attached to a surface. In
some embodiments, the surface to which the lectin molecule is
attached can be bead, hollow fiber, porous scaffold, particle or
well. In some embodiments, the surface is magnetic. In some
embodiments, the method further comprises isolation of the captured
CTCs, e.g., by passing the biological fluid containing captured
CTCs through a microfluidic magnetic separation device.
[0009] In some embodiments, the CTCs of the present invention
express mannose-containing carbohydrates, known as mannans, on
their surfaces.
[0010] In some embodiments, the biological fluid is selected from
is selected from a body fluid, such as whole blood, plasma, any
cell-containing blood fraction, cerebrospinal fluid, bone marrow, a
cell sample (e.g., stem cells), joint fluid, urine, tears or
feces.
[0011] In some embodiments, the biological fluid, e.g., bone
marrow, is to be used in transplantation. The transplantation can
be autologous or allogeneic.
[0012] In some embodiments, the lectin molecule contains a
carbohydrate recognition domain, e.g., collectin carbohydrate
recognition domain of a collectin, carbohydrate recognition domain
of a mannose binding lectin. In some embodiments the lectin
molecule is a mannose binding lectin, ficolin, dectin, C-type
lectin, fucose-binding lectin, hemopexin, S-type lectin, Galectin.
In some embodiments, the lectin molecule is an engineered molecule,
for e.g., FcMBL. In some embodiments, the lectin molecule is of
mammalian origin, for e.g. human origin. In some embodiments, the
lectin molecule comprises at least 80% amino acid sequence identity
to human lectin and retains at least 80% of its biological
ability.
[0013] In another aspect, described herein is a method of analyzing
a CTC captured from a sample by the methods described, wherein
analyses of the captured CTCs can comprise an immunochemical
analysis, morphological analysis, genomics analysis, epigenomics
analysis, metabolomics analysis, transcriptomics analysis,
proteomics analysis, DNA mutation analysis, whole genome analysis,
protein and/or RNA expression level of a specific gene or a
combination thereof. In some embodiments, the analysis is used to
assess a risk of developing a metastatic tumor in the patient
carrying or having carried a tumor. In some embodiments, the CTC is
captured for analysis from a sample e.g., whole blood, body fluid,
any cell-containing blood fraction, a fragmented tumor, a biopsy,
aspirate, a tumor cell suspension, bone marrow or a cell culture
established from a patient's sample, or the culture supernatant or
a xenograft established from a patient's tumor.
[0014] In another aspect, described herein is a method of detecting
cancer in a subject, comprising, obtaining blood from the subject,
contacting the blood with lectin-coated magnetic beads, isolation
of the magnetic beads captured cells with a microfluidic magnetic
separation device and assaying captured cells for CTC markers for
e.g., GlcNAc, EpCAM, EphB4, HER2, EGFR, MUC-1, or a combination
thereof.
[0015] In another aspect, described herein is a method for
monitoring or assessing the effectiveness of a cancer treatment in
a patient, comprising: (a) obtaining a first sample of the patient
prior to the cancer treatment and establishing a baseline CTC count
by isolating CTC using the method described above and enumerating a
CTC count, wherein CTC count is defined as the amount of GlcNAc+
cells in the blood sample; (b) obtaining a second sample of the
patient after the cancer treatment and determining a post-treatment
level of CTC count by isolating CTC from the sample using the
method described above and enumerating a CTC count; and (c)
comparing the levels of post-treatment CTC count to the baseline
CTC count, and optionally obtaining additional samples at different
time intervals after the cancer treatment to determine a
time-series for post-treatment CTC counts, wherein if the
post-treatment CTC counts show a decreasing trend, the treatment is
said to be effective, whereas if the post-treatment CTC count shows
an increasing trend or stays at about the baseline level, the
treatment is said to be ineffective. In some embodiments the method
further comprises conducting cellular or molecular analysis on the
isolated CTCs, wherein the cellular or molecular analysis is
selected from immunochemical analysis, morphological analysis,
genomics analysis, epigenomics analysis, transcriptomics analysis,
proteomics analysis, DNA mutation analysis, whole genome analysis,
protein and/or RNA expression level of a specific gene or a
combination thereof.
[0016] In another aspect, described herein is a method for
determining a prognosis of a patient suffering from cancer
comprising:(a) obtaining a blood sample from the patient; (b)
isolating CTCs from the blood sample by applying the method
described above to the blood sample; (c) enumerating isolated CTC
count, wherein CTCs are defined as the cells that are positive for
GlcNAc expression; (d) determining a prognosis for the patient
based on the CTC count.
[0017] In another aspect, described herein is a method for early
detection of metastatic tumor in a patient, comprising: (a)
obtaining a blood sample from the patient; (b) isolating CTCs from
the blood sample by applying the method described above to the
blood sample; (c) enumerating isolated CTC count, wherein CTC is
defined as mannan-expressing cells; and (d) determining a diagnosis
based on the CTC count, wherein if the CTC count is above a
predetermined level, a likelihood of metastatic tumor is
indicated.
[0018] In another aspect, described herein is a kit for isolating
and enriching CTCs in a blood sample, comprising: a red blood cell
(RBC) lysis reagent; a lectin-coated magnetic nanobeads; a cell
culture or a nutrition medium; and an instruction insert having
encoded thereon a human readable description of the methods
embodied in the foregoing aspect. In some embodiments, the kit
further comprises a separation column, wherein said column
comprising: a body with an entry end and an exit end each having an
opening disposed thereon; and a cylindrical hollow space connecting
the openings at the entry end and the exit end to form a passage
channel, wherein said column is pre-packaged with a plurality of
spherical separation beads disposed in the passage channel, said
separation beads are comprised of a ferromagnetic material coated
with an anti-corrosion, and are capable of being magnetized to
capture a cell labeled with lectin coated magnetic nanoparticles.
In some embodiments, the kit further comprises fluorescent staining
reagents and antibodies for cancer cell markers.
[0019] In another aspect, described herein is a composition
comprising a CTC bound to a lectin molecule, further comprising the
lectin molecule attached to a surface e.g. magnetic bead.
[0020] In another aspect, described herein is a method for
generating cancer vaccine, comprising: (a) contacting a sample
containing CTCs from a cancer patient with lectin molecule attached
to surface; (b) isolating the captured CTCs; (c) combining the
isolated CTCs or a component thereof with an adjuvant to generate a
CTC-immunogen and (d) administering the CTC-immunogen to a subject,
thereby producing a cancer vaccine.
[0021] In another aspect, described herein is a method for
generating a patient-specific cancer vaccine, comprising: (a)
contacting a sample containing CTCs from a patient with lectin
molecule attached to surface; (b) isolating the captured CTCs; (c)
combining the isolated CTCs or a component thereof with a scaffold
to generate a CTC-immunogen and (d) administering the CTC-immunogen
to the patient, thereby producing a patient-specific cancer
vaccine.
[0022] In some embodiments, CTCs in the cancer vaccine can be heat
killed, inactivated, neutralized, chemically fixed, lyophilized to
generate a CTC-immunogen prior to administering administering the
CTC-immunogen to the patient, thereby producing a patient-specific
cancer vaccine. In some embodiments, the scaffold comprises a
biomaterial. The scaffold biomaterial can be selected from the
group consisting of glycosaminoglycan, silk, fibrin, MATRIGEL.RTM.,
poly-ethyleneglycol (PEG), polyhydroxy ethyl methacrylate,
polyvinyl alcohol, polyacrylamide, poly (N-vinyl pyrolidone),
poly(lactic acid), poly glycolic acid (PGA), poly
lactic-co-glycolic acid (PLGA), poly e-carpolactone (PCL),
polyethylene oxide, poly propylene fumarate (PPF), poly acrylic
acid (PAA), polyhydroxybutyric acid, hydrolysed polyacrylonitrile,
polymethacrylic acid, polyethylene amine, esters of alginic acid;
pectinic acid; and alginate, fully or partially oxidized alginate,
hyaluronic acid, carboxy methyl cellulose, heparin, heparin
sulfate, chitosan, carboxymethyl chitosan, chitin, pullulan,
gellan, xanthan, collagen, gelatin, carboxymethyl starch,
carboxymethyl dextran, chondroitin sulfate, cationic guar, cationic
starch, and combinations thereof. In some embodiments, the
biomaterial is selected from the group consisting of
poly(L-lactide-co-glycolide) acid (PLGA), mesoporous silica, and
cryogel IP, and combinations thereof. In some embodiments, the
scaffold is capable of localizing to antigen-presenting cells
(APCs) in the subject, and activating the APCs to produce high
titer antibodies against the pathogen. In some embodiments,
CTC-immunogen further comprises an adjuvant. In some embodiments,
CTC-immunogen is implanted subcutaneously. In some embodiments, of
the above noted aspect, the lectin molecule is a MBL at least
comprising the CRD. In some embodiments, the lectin molecule
comprises an antibody Fc domain (FcMBL). In some embodiments, the
lectin molecule is attached to a magnetic surface.
[0023] In another aspect, described herein is a composition
comprising a CRD region of a lectin linked to anticancer
therapeutic molecule. In another aspect, described herein is a
composition comprising an mRNA encoding a CRD region of a lectin
linked to anticancer therapeutic molecule.
[0024] In another aspect, described herein is a composition
comprising a CRD region of a lectin linked to an imaging agent. In
another aspect, described herein is a composition comprising an
mRNA encoding a CRD region of a lectin linked to an imaging
agent
[0025] In another embodiment, described herein is a method of
treating cancer, the method comprising administering to a subject,
a composition of foregoing aspects comprising a therapeutic
molecule.
[0026] In another embodiment, described herein is a method for
visualization of cancer, the method comprising administering to a
subject, a composition of foregoing aspects comprising an imaging
agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1. FcMBL-coated magnetic particles capture metastasized
tumor cells circulating in blood, which express N-acetylglucosamine
(GlcNAc) on their surface while undergoing metastasis.
FcMBL-magnetic beads selectively capture rare CTCs in blood and
enable separation by the biospleen device. This will enable
concentration of rare CTCs and deplete those cells responsible for
cancer metastasis, reduce aggressiveness or likelihood of
metastasis, and eventually improve outcome of cancer treatment.
[0028] FIG. 2. The magnetic drag velocity of the human breast tumor
cells labeled with FcMBL magnetic beads as measured in a
microfluidic channel filled with saline. MCF7, human tumorigenic
breast tumor cells, shows significantly high drag velocity,
implying that FcMBL beads capture human breast tumor cells whereas
non-tumorigenic human breast tissue cells do not interact with
FcMBL beads.
[0029] FIG. 3. Immunocytochemistry image of mammary ducts of 20
week old wild type mice (left) and 22 week old transgenic mice
(right). Carbohydrates on tumorigenic epithelial cell surface were
bound with FcMBL in the transgenic mice whereas in the wildtype
mice (left) FcMBL bound to polysaccharide-rich glycosaminoglycan
elements of ECM, not epithelial cells.
[0030] FIG. 4. The binding efficiency of FcMBL to 4T1 cells in
saline and human whole blood. The FcMBL coated 1 .mu.m magnetic
particles bind to 4T1 cells in buffer and human whole blood with
the efficiency of 97.2.+-.1.6% and 74.6.+-.4.2%, respectively. In
comparison, the control magnetic particles without FcMBL did not
show the binding affinity to 4T1 cells (1.5.+-.4.4%).
[0031] FIG. 5. The 4T1 cells implanted in mammary pads were
metastasized to the lung after 20 days (left) and 30 days (right)
post-implantation.
[0032] FIG. 6. The lysed mice blood (10 .mu.L) containing 4T1 cells
(cherry red expressing) was loaded into a hemocytometer and the
number of 4T1 cells was counted. The 4T1 cells obtained from blood
of the implanted tumor-bearing mice (30 days, left) were
significantly depleted (over 90%, right) by magnetic separation
with FcMBL-coated magnetic particles. Scale bar, 100 .mu.m.
[0033] FIG. 7. The binding efficiency of H1975 cells to
FcMBL-coated magnetic particles in saline with 5 mM calcium
chloride and heparinized human whole blood. 90.2.+-.5.0% and
80.6.+-.2.4% of H1975 cells spiked in saline and human whole blood,
respectively, were bound to 1 .mu.m FcMBL magnetic particles.
[0034] FIG. 8. Table showing the binding efficiency of cells from
different cancer lines to FcMBL: A549, H727, H358, H1975, MCF7, 4T1
(mouse) and weak binding to normal MCF10a epithelial cells.
Depletion experiments were performed by incubating cells with 1
.mu.m FcMBL coated magnetic beads and then separating the beads in
saline, whole blood, blood diluted 1:10 in saline, and blood lysed
with RBC lysis buffer.
[0035] FIG. 9. 4T1 cells (cherry red fluorescence-expressing)
spiked in blood were captured by FcMBL and isolated in the
biospleen device. The cells isolated in the device were visualized
and detected by a fluorescence microscope.
DETAILED DESCRIPTION
[0036] It should be understood that this invention is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein and as such may vary. The terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention, which
is defined solely by the claims.
[0037] As used herein and in the claims, the singular forms include
the plural reference and vice versa unless the context clearly
indicates otherwise. Other than in the operating examples, or where
otherwise indicated, all numbers expressing quantities of
ingredients or reaction conditions used herein should be understood
as modified in all instances by the term "about."
[0038] All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0039] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as those commonly understood to
one of ordinary skill in the art to which this invention pertains.
Although any known methods, devices, and materials may be used in
the practice or testing of the invention, the methods, devices, and
materials in this regard are described herein.
[0040] Described herein are methods, compositions and kits for
detection, capture and isolation of circulating tumor cells (CTCs)
and applications thereof. It is contemplated that the methods,
compositions and kits disclosed herein can be used to detect,
capture and isolate cellular components of CTCs for example cell
membrane and or secreted components of CTCs, for example
extracellular vesicles, exosomes, microvesicles. In one aspect the
present invention provides for use of carbohydrate binding proteins
for example lectins, for detection, capture and isolation of cancer
cells preferably CTCs. The term "lectin" as used herein refers to
any molecules including proteins, natural or genetically modified
(e.g., recombinant), that interact specifically with saccharides
(e.g., carbohydrates) i.e. are carbohydrate binding proteins.
Additional carbohydrate-binding proteins that can be included in
the lectin molecule described herein can include, but is not
limited to, lectins or agglutinins that are derived from a plant,
e.g., Galanthus nivalis agglutinin (GNA) from the Galanthus
(snowdrop) plant, and peanut lectin. In some embodiments, pentraxin
family members, e.g., C-reactive protein can also be used as a
carbohydrate- binding protein. Pentraxin family members can
generally bind capsulated microbes. The carbohydrate-binding
proteins can be wild-type, recombinant or a fusion protein.
[0041] The lectin molecule can comprise peptides, polypeptides,
proteins, peptidomimetic or any structural mimics mimicking the
carbohydrate binding region (e.g., Carbohydrate recognition domain
(CRD or a fragment thereof), antibodies, antibody fragments
(antigen-binding fragments of antibodies), carbohydrate binding
proteins (e.g., lectins, glycoprotein, glycoprotein
binding-molecules, amino acids, carbohydrates (including mono-,
di-, tri- and poly-saccharides), lipids, steroids, hormones,
lipid-binding molecules, cofactors, nucleosides, nucleotides,
nucleic acids (e.g., DNA or RNA, analogues and derivatives of
nucleic acids, or aptamers), peptidoglycan, lipopolysaccharide,
small molecules, and any combinations thereof
[0042] The lectins bind to carbohydrates with their carbohydrate
recognition domain (CRD). In some embodiments, the lectin molecule
comprises at least the CRD or a fragment thereof In some
embodiments, a lectin molecule can comprise a peptidomimetic that
mimics any molecule or a fragment thereof that can specifically
bind to the carbohydrate surface of a cancer cell or CTC, and/or
secreted component thereof, for example extracellular vesicles,
exosomes. For example, a lectin molecule can comprise a
peptidomimetic that mimics any carbohydrate recognition domain or a
fragment thereof, e.g., carbohydrate recognition domain of
Mannose-binding lectin or a fragment thereof; or any carbohydrate
recognition domain that is known in the art or a fragment thereof.
In some embodiments, a lectin molecule comprises the full amino
acid sequence of a carbohydrate-binding protein, a CRD or a
fragment thereof The CRD or a fragment thereof can be derived from
a carbohydrate binding protein including but not limited to lectin,
collectin, ficolin, mannose-binding lectin (MBL), maltose-binding
protein, arabinose-binding protein, and glucose-binding protein.
The respective carbohydrate recognition domains for such
carbohydrate-binding proteins are known in the art, and can be
modified for various embodiments of the lectin molecules described
herein. A lectin molecule can be full length protein comprising a
CRD, a fragment thereof comprising a CRD, a fragment of CRD. A
lectin molecule can be wild-type, recombinant or a fusion molecule.
In some embodiments, the lectin molecule can have an amino acid
sequence of about 10 to about 300 amino acid residues, or about 50
to about 150 amino acid residues. In some embodiments, the
carbohydrate binding domain can have an amino acid sequence of at
least about 5, at least about 10, at least about 15, at least about
20, at least about 30, at least about 40, at least about 50, at
least about 60, at least about 70, at least about 80, at least
about 90, at least about 100 amino acid residues or more. For any
known sequences of carbohydrate binding protein e.g. lectin and/or
a carbohydrate recognition domain e.g. CRD or a mannose-binding
lectin, one of skill in the art can determine the optimum length of
amino acid sequence for the lectin molecule.
[0043] The term "lectin" as used herein can also refer to lectins
derived from any species, including, but not limited to, plants,
animals, insects and microorganisms, having a desired carbohydrate
binding specificity. Examples of plant lectins include, but are not
limited to, the Leguminosae lectin family, such as ConA, soybean
agglutinin, peanut lectin, lentil lectin, and Galanthus nivalis
agglutinin (GNA) from the Galanthus (snowdrop) plant. Other
examples of plant lectins are the Gramineae and Solanaceae families
of lectins. Examples of animal lectins include, but are not limited
to, any known lectin of the major groups S-type lectins, C-type
lectins, P-type lectins, and I-type lectins, and galectins. In some
embodiments, the lectin molecule comprises at least of the CRD or a
fragment thereof. In some embodiments, the lectin molecule is a
C-type lectin or a carbohydrate recognition domain can be derived
from a C-type lectin, or a fragment of a carbohydrate recognition
domain derived from a C-type lectin. C-type lectin can include any
carbohydrate-binding protein that requires calcium for binding. In
some embodiments, the C-type lectin can include, but are not
limited to, collectin, DC-SIGN, and CRD and/or fragments
thereof.
[0044] Collectins are soluble pattern recognition receptors (PRRs)
belonging to the superfamily of collagen containing C-type lectins.
Exemplary collectins include, without limitations, mannose-binding
lectin (MBL) (also known as mannan-binding lectin, mannan-binding
protein, or mannose-binding protein), surfactant protein A (SP-A),
surfactant protein D (SP-D), collectin liver 1 (CL-L1), collectin
placental (CL-P1), conglutinin, collectin of 43 kDa (CL-43),
collectin of 46 kDa (CL-46). In some embodiments of the present
invention, the lectin molecule is a collectin, CRD thereof or a
fragment derived from a CRD thereof.
[0045] Mannose-binding lectin (MBL), also known as mannose binding
protein (MBP), or mannan-binding lectin or mannan-binding protein,
is a calcium-dependent serum protein that can play a role in the
innate immune response by binding to carbohydrates on the surface
of a wide range of microbes or pathogens (viruses, bacteria, fungi,
protozoa) where it can activate the complement system. MBL can also
serve as a direct opsonin and mediate binding and uptake of
pathogens by tagging the surface of a pathogen to facilitate
recognition and ingestion by phagocytes.
[0046] MBL is a member of the collectin family of proteins. A
native MBL is a multimeric structure (e.g., about 650 kDa) composed
of subunits, each of which contains three identical polypeptide
chains. Each MBL polypeptide chain (containing 248 amino acid
residues in length with a signal sequence: SEQ ID NO. 1) comprises
a N-terminal cysteine rich region, a collagen-like region, a neck
region, and a carbohydrate recognition domain (CRD). The sequence
of each region has been identified and is well known in the art.
SEQ ID NO.2 shows a full-length amino acid sequence of MBL without
a signal sequence.
[0047] The surface or carbohydrate recognition function of a native
MBL is mediated by clusters of three C-type
carbohydrate-recognition domains (CRDs) held together by
coiled-coils of a-helices. The N-terminal portion collagen- like
domain is composed of Gly-X-Y triplets. The short N-terminal domain
contains several cysteine residues that form interchain disulfide
bonds. Serum MBLs assemble into larger forms containing 2-4
trimeric subunits in rodents and as many as six subunits in humans.
All three oligomeric forms of rat serum MBP, designated MBPA, can
fix complement, although the larger oligomers have higher specific
activity. Many species express a second form of MBP. In rats, the
second form, MBP-C, is found in the liver. MBP-C does not form
higher oligomers beyond the simple subunit that contains three
polypeptides. When a native MBL interacts with carbohydrates on the
surface of microbes or pathogens, e.g., calcium-dependent binding
to the carbohydrates mannose, N-acetylglucosamine, and/or fucose,
it can form the pathogen recognition component of the lectin
pathway of complement activation. The MBL binds to surface arrays
containing repeated mannose or N-acetylglucosamine residues. It
circulates as a complex with one or more MBP-associated serine
proteases (MASPs) that autoactivate when the complex binds to an
appropriate surface. The MBL and associated MASP proteins can
activate C2/C4 convertase leading to the deposition of C4 on the
pathogen surface and opsonization for phagocytosis. The native MBL
can also activate coagulation function through MASP proteins.
[0048] In some embodiments, the lectin molecule is an engineered
molecule that binds to CTCs, comprising at least one carbohydrate
recognition domain or a fragment thereof, e.g., derived from MBL.
In some embodiments, the engineered lectin molecule can comprises
at least two, at least three or at least four carbohydrate
recognition domains or a fragment thereof. In some embodiments, the
engineered lectin molecules do not activate complement system or
coagulation side effects that are present in a native lectin.
[0049] The full-length amino acid sequence of carbohydrate
recognition domain (CRD) of MBL is shown in SEQ ID NO. 3. The
carbohydrate recognition domain of an engineered MBL described
herein can have an amino acid sequence of about 10 to about 300
amino acid residues, or about 50 to about 160 amino acid residues.
In some embodiments, the CRD can have an amino acid sequence of at
least about 5, at least about 10, at least about 15, at least about
20, at least about 30, at least about 40, at least about 50, at
least about 60, at least about 70, at least about 80, at least
about 90, at least about 100, at least about 150 amino acid
residues or more. Accordingly, in some embodiments, the
carbohydrate recognition domain of the engineered MBL molecule can
comprise SEQ ID NO. 3. In some embodiments, the carbohydrate
recognition domain of the engineered MBL molecule can comprise a
fragment of SEQ ID NO. 3. Non-limiting examples of fragments are
described in US 2014/0227723 Al, incorporated herein in its
entirety. Modifications to such CRD fragments, e.g., by
conservative substitution, are also within the scope described
herein. In some embodiments, the MBL or a fragment thereof used in
the lectin molecule described herein can be a wild- type molecule
or a recombinant molecule.
[0050] As used herein, the terms "protein", "peptide" and
"polypeptide" are used interchangeably to designate a series of
amino acid residues connected to each other by peptide bonds
between the alpha-amino and carboxy groups of adjacent residues.
The terms "protein", "peptide" and "polypeptide" refer to a polymer
of amino acids, including modified amino acids (e.g.,
phosphorylated, glycated, glycosylated, etc.) and amino acid
analogs, regardless of its size or function. "Protein" and
"polypeptide" are often used in reference to relatively large
polypeptides, whereas the term "peptide" is often used in reference
to small polypeptides, but usage of these terms in the art
overlaps. The terms "protein", "peptide" and "polypeptide" are used
interchangeably herein when referring to a gene product and
fragments thereof.
[0051] The exemplary sequences provided herein for the carbohydrate
recognition domain of the lectin molecules are not construed to be
limiting. For example, while the exemplary sequences provided
herein are derived from a human species, amino acid sequences of
the same carbohydrate recognition domain in other species such as
mice, rats, porcine, bovine, feline, and canine are known in the
art and within the scope described herein.
[0052] In some embodiments, the nucleic acid encodes a carbohydrate
recognition domain having greater than 50% homology, including
greater than 60%, greater than 70%, greater than 80%, greater than
90% homology or higher, to a fragment of at least 50, at least 60,
at least 70, at least 80, at least 90, at least 100, at least 150
contiguous amino acids or more, of any known carbohydrate-binding
molecules (e.g., mannose-binding lectins).
[0053] The term "carbohydrate recognition domain" as used herein
refers to a region, at least a portion of which, can bind to
carbohydrates on a surface of CTCs. In some embodiments, the
carbohydrate recognition domain can comprise at least about 50% of
its domain, including at least about 60%, at least about 70%, at
least about 80%, at least about 90% or higher, capable of binding
to carbohydrates on a CTC surface. In some embodiments, 100% of the
carbohydrate recognition domain can be used to bind to CTCs.
[0054] In other embodiments, in addition to carbohydrate
recognition domain, the lectin molecule can comprise additional
regions that are not capable of carbohydrate binding, but can have
other characteristics or perform other functions, e.g., to provide
flexibility to the carbohydrate recognition domain when interacting
with CTCs for example the neck region of MBL. A skilled artisan can
readily modify the identified CRD and fragments thereof to modulate
its orientation and binding performance to carbohydrates on a CTC
surface, e.g., by theoretical modeling and/or in vitro
carbohydrate-binding experiments.
[0055] In some embodiments, in addition to the carbohydrate
recognition domain, the lectin molecule can further comprise a
portion of a carbohydrate-binding protein. However, in some
circumstances, complement or coagulation activation induced by a
carbohydrate-binding protein or a fragment thereof can be
undesirable depending on various applications, e.g., in vivo
applications. In such embodiments, the portion of the
carbohydrate-binding protein can exclude at least one of complement
and coagulation activation regions. By way of example, when the
carbohydrate-binding protein is mannose-binding lectin or a
fragment thereof, the mannose-binding lectin or a fragment thereof
can exclude at least one of the complement and coagulation
activation regions located on the collagen-like region. In such
embodiments, the mannose-binding lectin or a fragment thereof can
exclude at least about one amino acid residue, including at least
about two amino acid residues, at least about three amino acid
residues, at least about four amino acid residues, at least about
five amino acid residues, at least about six amino acid residues,
at least about seven amino acid residues, at least about eight
amino acid residues, at least about nine amino acid residues, at
least about ten amino acid residues or more, around amino acid
residue K55 or L56 of SEQ ID NO.2.
[0056] Further regarding the carbohydrate recognition domain (CRD)
or a fragment thereof, its binding characteristics can be
manipulated by directed evolution for altered binding specificity.
By way of example only, MBL can be modified so that it binds to a
more limited set of sugars or other molecular features, with the
result that the modified MBL will bind to CTCs of a specific type
of cancer to provide a capability for cancer type
identification.
[0057] The exemplary sequences provided herein for the carbohydrate
recognition domain of the lectin are not construed to be limiting.
For example, while the exemplary sequences provided herein are
derived from a human species, amino acid sequences of the same
carbohydrate recognition domain in other species such as mice,
rats, porcine, bovine, feline, and canine are known in the art and
within the scope described herein.
[0058] In some embodiments, the nucleic acid for example DNA or RNA
encoding a carbohydrate recognition domain having greater than 50%
homology, including greater than 60%, greater than 70%, greater
than 80%, greater than 90% homology or higher, to a fragment of at
least 50, at least 60, at least 70, at least 80, at least 90, at
least 100, at least 150 contiguous amino acids or more, of any
known carbohydrate-binding molecules (e.g., mannose-binding
lectins).
[0059] The CRD domain is useful for the application at hand, and
the other domains may be present or deleted depending on the needs
of the user, and can be determined by routine testing.
Modifications to CRD fragments, e.g., by conservative substitution,
are also within the scope described herein.
[0060] In some embodiments the lectin molecule can comprise at
least one CRD, including at least two, at least three, at least
four, at least five, at least six, at least seven, at least eight,
at least nine, at least ten or more CRD, can be linked together to
form a multimeric carbohydrate recognition domain. A lectin
molecule comprising multimeric CRD can have each of the individual
CRD the same. Alternatively, a multimeric CRD can have at least
one, at least two, or at least three CRD different from the rest.
In such embodiments, CRD that share a common binding specificity
for carbohydrates on a CTC surface can be used. By way of example
only, the fibrinogen-like domain of several lectins has a similar
function to the CRD of C-type lectins including MBL, and function
as pattern-recognition receptors to discriminate pathogens from
self. One of such lectins comprising the fibrinogen- like domain is
serum violins. Serum violins have a common binding specificity for
GlcNAc (N-acetylglucosamine), elastin or GalNAc
(N-acetyl-galactosamine). The fibrinogen-like domain is responsible
for the carbohydrate binding. In human serum, two types of ficolin,
known as L-ficolin (also called P35, ficolin L, ficolin 2 or
hucolin) and H-ficolin (also called Hakata antigen, ficolin 3 or
thermolabile b2-macroglycoprotein), have been identified, and both
of them have lectin activity. L-ficolin recognizes GlcNAc and
H-ficolin recognizes GalNAc. Another ficolin known as M-ficolin
(also called P3 5-related protein, ficolin 1 orficolinA) is not
considered to be a serum protein and is found in leucocytes and in
the lungs. L-ficolin and H-ficolin activate the lectin-complement
pathway in association with MASPs. M-Ficolin, L-ficolin and
H-ficolin has calcium-independent lectin activity. Accordingly, in
some embodiments, a lectin molecule, can comprise MBL and L-ficolin
carbohydrate recognition domains, MBL and H-ficolin carbohydrate
recognition domains, or a combination thereof.
[0061] Any art-recognized recombinant carbohydrate- binding
proteins or carbohydrate recognition domains can also be used in
the lectin molecules. Non limiting examples of recombinant
mannose-binding lectins, can be ones disclosed in the U.S. Pat.
Nos. 5,270,199; 6,846,649; and U.S. Patent Application No. US
2004/0229212, the contents of which are incorporated herein by
reference, can be used in constructing the lectin molecules
described herein.
[0062] In other embodiments, the lectin molecule can comprise
additional regions that are not capable of carbohydrate binding,
but can have other characteristics or perform other functions,
e.g., to provide flexibility to the carbohydrate recognition domain
when interacting with CTCs. Accordingly in some embodiments, the
lectin molecule is an engineered molecule comprising of a CRD of a
carbohydrate binding protein preferably MBL, or a fragment of the
CRD further attached to an linker for example, a Fc fragment. In
some embodiments, the lectin molecule used in the methods and
compositions disclosed herein, for example the CRD of the MBL, is
further linked to Fc portion of an immunoglobulin, preferably IgG.
In some embodiments, the lectin molecule is a genetically
engineered version of MBL (FcMBL) as described in WO 2011/090954,
US 2014/0227723 A1, US 2013/0035283 A1, WO 2013/012924 A2 the
content of both of which is incorporated herein by reference.
[0063] Amino acid sequences for MBL and engineered MBL include, but
are not limited to: MBL full length (SEQ ID NO. 1), [0064]
MSLFPSLPLL LLSMVAASYS ETVTCEDAQK TCPAVIACSS PGINGFPGKD GRDGTKGEKG
EPGQGLRGLQ GPPGKLGPPG NPGPSGSPGP KGQKGDPGKS PDGDSSLAAS ERKALQTEMA
RIKKWLTFSL GKQVGNKFFL TNGEIMTFEK VKALCVKFQA SVATPRNAAE NGAIQNLIKE
EAFLGITDEK TEGQFVDLTG NRLTYTNWNE GEPNNAGSDE DCVLLLKNGQ WNDVPCSTSH
LAVCEFPI
[0065] MBL without the signal sequence (SEQ ID NO. 2) [0066]
ETVTCEDAQK TCPAVIACSS PGINGFPGKD GRDGTKGEKG EPGQGLRGLQ GPPGKLGPPG
NPGPSGSPGP KGQKGDPGKS PDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFL
TNGEIMTFEK VKALCVKFQA SVATPRNAAE NGAIQNLIKE EAFLGITDEK TEGQFVDLTG
NRLTYTNWNE GEPNNAGSDE DCVLLLKNGQ WNDVPCSTSH LAVCEFPI
[0067] Carbohydrate recognition domain (CRD) of MBL (SEQ ID NO. 3),
VGNKFFLTNG EIMTFEKVKA LCVKFQASVA TPRNAAENGA IQNLIKEEAF LGITDEKTEG
QFVDLTGNRL TYTNWNEGEP NNAGSDEDCV LLLKNGQWND VPCSTSHLAV CEFPI
[0068] In some embodiments, the lectin molecule is attached to a
surface for example, a solid surface. The surface can be made from
a wide variety of materials and in a variety of formats. For
example, in the form of beads (including polymer microbeads,
magnetic microbeads, and the like), filters, fibers, screens, mesh,
tubes, hollow fibers, porous scaffolds, plates, channels, other
substrates commonly utilized in assay formats, and any combinations
thereof. Examples of surfaces include, but are not limited to,
nucleic acid scaffolds, protein scaffolds, lipid scaffolds,
dendrimers, microparticles or microbeads, nanotubes, microtiter
plates, medical apparatuses (e.g., needles or catheters) or
implants, dipsticks or test strips, microchips, filtration devices
or membranes, diagnostic strips, hollow-fiber reactors,
microfluidic devices, living cells and biological tissues or
organs, extracorporeal devices, mixing elements (e.g., spiral
mixers). The surface can be made of any material, including, but
not limited to, metal, metal alloy, polymer, plastic, paper, glass,
fabric, packaging material, biological material such as cells,
tissues, hydrogels, proteins, peptides, nucleic acids, and any
combinations thereof
[0069] The format and/or material of the solid substrate depend on
the application for example detection, in vivo targeting, capture,
isolation of CTCs or a combination thereof. In some embodiments,
the surface can be fabricated from or coated with a biocompatible
material. As used herein, the term "biocompatible material" refers
to any material that does not deteriorate appreciably and does not
induce a significant immune response or deleterious tissue
reaction, e.g., toxic reaction or significant irritation, over time
when implanted into or placed adjacent to the biological tissue of
a subject, or induce blood clotting or coagulation when it comes in
contact with blood. Suitable biocompatible materials include, for
example, derivatives and copolymers of polyimides, poly(ethylene
glycol), polyvinyl alcohol, poly- ethyleneimine, and
polyvinylamine, polyacrylates, polyamides, polyesters,
polycarbonates, and polystyrenes, metals for e.g. titanium and
stainless steel, or any biocompatible metal used in medical
implants. In some embodiments, biocompatible materials can include
paper substrate, e.g., as a substrate for a diagnostic strip. In
some embodiments, biocompatible materials can include peptides or
nucleic acid molecules, e.g., a nucleic acid scaffold such as a 2-D
DNA sheet or 3-D DNA scaffold. Additional materials known in the
art that can be used to coat the surface would be known to those
skilled in the art and are thereby considered within the scope of
the application. In some embodiments, the substrate surface can
encompass an outer substrate surface and/or an inner substrate
surface, e.g., with respect to a hollow structure. For example, a
hollow fiber, hollow tube, the inner surface of a needle or
catheter can be coated with the lectin molecules described herein,
e.g., for removing any CTCs from a fluid before administering the
fluid back to a subject. Devices that can be used for such
applications are known in the art for example described by Kang,
JH. et al. (2014), incorporated herein. Such devices can be easily
adapted for the technology described herein. The lectin molecule
can be coated onto the outer or inner surface of a hollow
surface.
[0070] The amount of the lectin molecules attached, conjugated or
coated on a surface can vary with a number of factors such as the
area of the surface, conjugation/coating density, types/size of
lectin molecules, and/or binding performance. A skilled artisan can
determine the optimum density of lectin molecules on a surface
using any methods known in the art. By way of example only, for
magnetic microbeads (including nanobeads) as a substrate (as
discussed in detail later), the amount of the lectin molecules used
for conjugating to or coating magnetic microbeads can vary from
about 1 wt% to about 30 wt %, or from about 5 wt % to about 20 wt
%. In some embodiments, the amount of the lectin molecules used for
conjugating to or coating magnetic microbeads can be higher or
lower, depending on a specific need.
[0071] The present invention provides for use of lectin molecule
for detection, capture and isolation of cancer cells preferably
CTCs.
[0072] The term "circulating tumor cells" or "CTCs" refers to tumor
cells found in circulation of a patient having a tumor. This term
typically does not include hematological tumors where the majority
of the tumor is found in circulation. The term "cancer cells" and
"tumor cells" are used interchangeably to refer to cells derived
from a cancer or a tumor, or from a tumor cell line or a tumor cell
culture. The term "cancer cells" and "tumor cells" and "circulating
tumor cells" or "CTCs" can be used interchangeably. The term
"metastatic cells" or "metastatic tumor cells" refers to the cells
that have the ability to produce a metastasis or are already a part
of a metastatic tumor. Accordingly, in some embodiments of the
methods and compositions disclosed herein the CTCs are contacted
with a lectin molecule. Lectin, for example MBL interact with
carbohydrates e.g., mannose, N-acetylglucosamine and/or fucose.
Accordingly, in some embodiments, the CTCs of some embodiments can
express mannan carbohydrates, mannose, fucose and/or
N-acetylglucosamine on their surface.
[0073] In some embodiments, the lectin molecule can be attached to
a microparticle comprising at least one lectin molecule on its
surface. The term "microparticle" as used herein refers to a
particle having a particle size of about 0.001 .mu.m to about 100
.mu.m, about 0.005 .mu.m to about 50 .mu.m, about 0.01 .mu.m to
about 25 .mu.m, about 0.05 .mu.m to about 10 .mu.m, or about 0.05
.mu.m to about 5 .mu.m. In one embodiment, the microparticle has a
particle size of about 0.05 .mu.m to about 1 .mu.m. In one
embodiment, the microparticle is about 0.09 .mu.m--about 0.2 .mu.m
in size. It will be understood by one of ordinary skill in the art
that microparticles usually exhibit a distribution of particle
sizes around the indicated "size. "Unless otherwise stated, the
term "size" as used herein refers to the mode of a size
distribution of microparticles, i.e., the value that occurs most
frequently in the size distribution. Methods for measuring the
microparticle size are known to a skilled artisan, e.g., by dynamic
light scattering (such as photocorrelation spectroscopy, laser
diffraction, low-angle laser light scattering (LALLS), and
medium-angle laser light scattering (MALLS)), light obscuration
methods (such as Coulter analysis method), or other techniques
(such as rheology, and light or electron microscopy). The
microparticles can be of any shape, e.g., a sphere. In general, any
biocompatible material well known in the art for fabrication of
microparticles can be used in embodiments of the microparticle
described herein. Accordingly, a microparticle comprising a lipidic
microparticle core is also within the scope described herein. An
exemplary lipidic microparticle core is, but is not limited to, a
liposome. A liposome is generally defined as a particle comprising
one or more lipid bilayers enclosing an interior, e.g., an aqueous
interior. In one embodiment, a liposome can be a vesicle formed by
a bilayer lipid membrane. Methods for the preparation of liposomes
are well described in the art.
[0074] Magnetic beads--In some embodiments, the lectin molecule is
attached to a magnetic surface for e.g. magnetic beads, wherein the
magnetic bead comprises on its surface at least one lectin molecule
disclosed herein. Such lectin molecule attached magnetic microbeads
can be used to separate CTCs from a test sample, e.g., but not
limited to, any fluid, including a biological fluid such as blood.
Attaching lectin molecules to a magnetic surface can be
advantageous because the CTC bound magnetic surface can be easily
separated from a sample fluid using a magnetic field gradient, be
examined for the presence of CTCs, and/or be used to transfer the
captured CTCs to conventional cellular and molecular assays. In
some embodiments, the captured CTCs can be isolated from sample
fluid by passing through a microfluidic magnetic separation device.
Thus, in some embodiments, lectin molecule coated magnetic
microbeads can be used to remove CTCs from any source or in any
fluid, e.g., a biological fluid (e.g., blood sample). In some
embodiments where the fluid is blood, after removal of the CTCs
from the blood collected from a subject with the lectin molecule
magnetic microbeads, the blood can be circulated back to the same
subject as a therapeutic intervention. In some embodiments, the
lectin molecule magnetic microbeads can be used in diagnostics as a
means of collecting CTCs for identification of cancer type for e.g.
in the form of liquid biopsy; Alternatively, the attachment surface
can comprise a hollow-fiber reactor or any other blood filtration
membrane or flow device (e.g., a simple dialysis tube, spiral mixer
or static mixer) or other resins, fibers, or sheets to selective
bind and sequester the CTCs. Such devices can be a component of a
kit for detection, isolation and capture of CTCs from biological
fluids e.g., blood.
[0075] The magnetic microbeads can be of any shape, including but
not limited to spherical, rod, elliptical, cylindrical, and disc.
As used interchangeably herein, the terms "magnetic microbeads" and
"magnetic beads" can refer to a nano- or micro-scale particle that
is attracted or repelled by a magnetic field gradient or has a
non-zero magnetic susceptibility. The magnetic microbeads can be
ferromagnetic, para- magnetic or super-paramagnetic. In some
embodiments, magnetic microbeads can be super-paramagnetic. In some
embodiments, magnetic microbeads can have a polymer shell for
protecting the lectin molecule from exposure to iron provided that
the polymer shell has no adverse effect on the magnetic property.
For example, biocompatible polymer-coated magnetic microbeads can
be used to remove CTCs from a test sample, e.g., a biological
fluid, such as blood.
[0076] The magnetic microbeads can range in size from 1 nm to 1 mm.
For example, magnetic microbeads can be about 2.5 nm to about 500
.mu.m, or about 5 nm to about 250 .mu.m in size. In some
embodiments, magnetic microbeads can be about 5 nm to about 100
.mu.m in size. In some embodiments, magnetic microbeads can be
about 0.01 .mu.m to about 10 .mu.m in size. In some embodiments,
magnetic microbeads can be about 0.05 .mu.m to about 5 .mu.m in
size. In some embodiments, magnetic microbeads can be about 0.08
.mu.m to about 1 .mu.m in size. In one embodiment, magnetic
microbeads can be about 10 nm to about 10 .mu.m in size. In some
embodiments, the magnetic microbeads can be magnetic nanobeads,
e.g., with a size ranging from about 1 nm to about 1000 nm, from
about 10 nm to about 500 nm, from about 25 nm to about 300 nm, from
about 40 nm to about 250 nm, or from about 50 nm to about 200 nm.
In one embodiment, the magnetic microbeads can be magnetic
nanobeads with a size of about 50 nm to about 200 nm. Magnetic
microbeads can be manipulated using magnetic field or magnetic
field gradient. Such particles commonly consist of magnetic
elements such as iron, nickel and cobalt and their oxide compounds.
Magnetic microbeads are well-known and methods for their
preparation have been described in the art. See, e.g., U.S. Pat.
Nos. 6,878,445; 5,543,158; 5,578,325; 6,676,729; 6,045,925; and
U.S. Pat. No. 7,462,446; and U.S. Patent Publications Nos.
2005/0025971; 2005/0200438; 2005/0201941; 2005/0271745;
2006/0228551; 2006/0233712; 2007/01666232; and No. 2007/0264199,
the contents of which are incorporated herein by reference.
Magnetic microbeads which can be functionalized with various
functional groups, e.g., amino groups, carboxylic acid groups,
epoxy groups, tosyl groups, or silica- like groups, are also widely
and commercially available. Suitable magnetic microbeads are
commercially available such as from AdemTech, Miltenyi, PerSeptive
Diagnostics, Inc. (Cambridge, Mass.); Invitrogen Corp. (Carlsbad,
Calif.); Cortex Biochem Inc. (San Leandro, Calif.); and Bangs
Laboratories (Fishers, Ind.). In particular embodiments, magnetic
microbeads that can be used herein can be any DYNA-BEADS.RTM.
magnetic microbeads (Invitrogen Inc.), depending on the substrate
surface chemistry.
[0077] Other surfaces: In some embodiments, the surface to which
the lectin molecule can be attached can be living cells, or a
biological tissue or organ. For example, the living cells can be
associated with an immune response, and such cells include, but are
not limited to, a phagocyte (macrophage, neutrophil, and dendritic
cell), mast cell, eosinophil, basophil, and/or natural killer cell.
Such compositions can be useful for e.g. in targeted cancer
therapy.
[0078] In some embodiments, the bottom surface of a microtiter
plate can be coated with the lectin molecule described herein,
e.g., for detecting and/or determining the number of CTCs in a
sample. After CTCs in the sample binding to the lectin molecules
bound to the microwell surface, the rest of the sample can be
removed. Detectable molecules that can also bind to CTCs (e.g., an
lectin molecule conjugated to a detectable molecule as described
herein, or antibodies to other CTC surface markers such as EpCAM
conjugated to a detectable molecule) can then be added to the
microwells with CTCs for detection of CTCs. Various signal
detection methods for determining the amount of proteins, e.g.,
using enzyme-linked immunosorbent assay (ELISA), with different
detectable molecules have been well established in the art, and
those signal detection methods can also be employed herein to
facilitate detection of the signal induced by CTC binding on the
lectin molecules.
[0079] In some embodiments, the lectin molecules can be adapted for
use in a dipstick and/or a test strip for detection and/or capture
of CTCs. For example, a dipstick and/or a test strip can include at
least one test area containing one or more lectin molecules
described herein. In some embodiments, the lectin molecules can be
conjugated or attached to a test area surface of the dipstick
and/or a test strip. Methods for conjugating a protein to a
substrate surface are known in the art, including, but not limited
to direct cross- linking, indirect cross-linking via a coupling
agent (e.g., a functional group, a peptide, a nucleic acid matrix
such as DNA matrix), absorption, or any other art-recognized
methods known in the art. The lectin molecule dipsticks and/or test
strips described herein can be used as point-of-care diagnostic
tools for CTC detection. By way of example only, a dipstick or test
strip for CTC capture (e.g., made of membrane material such as
nylon) can be brought into contact with a test sample (e.g., a
blood sample) from a patient or a subject, and incubated for a
period of time, e.g., at least about 15 seconds, at least about 30
seconds, at least about 1 min, at least about 2 mins, at least
about 5 mins, at least about 10 mins, at least about 15 mins, at
least about 30 mins, at least about 1 hour or more. In some
embodiments, the CTC-binding dipstick or test strip after contact
with a test sample (e.g., a blood sample) can be further contacted
with at least one additional agent to facilitate detection and/or
capture of CTCs. For example, some embodiments of the dipstick or
test strip after contact with a test sample (e.g., a blood sample)
can be further contacted with a detectable label that is conjugated
to a molecule that binds to a CTC component . Examples of such
components can include, but are not limited to, one or more
embodiments of the lectin molecule described herein, an antibody
specific for the other known CTC surface markers e.g. EpCAM to be
detected, a protein, a peptide, a carbohydrate or a nucleic acid
that is recognized by the CTCs to be detected, and any combinations
thereof.
[0080] The present invention discloses methods of isolation of CTCs
using a lectin molecule from a biological fluid. As described
herein, the lectin molecules attached to a surface, for example a
magnetic surface can be used to detect, capture and isolate CTCs
from a variety of samples, non-limiting examples of which include;
body fluids such as whole blood plasma, plasma, any cell containing
blood fraction, cerebrospinal fluid, joint fluid, urine, tears,
feces, a fragmented tumor, a tumor cell suspension, a cell sample,
cell culture established from a patients sample bone marrow (e.g.,
before transplantation), the culture supernatant or a xenograft
established from a patients sample.
[0081] The lectin molecule attached to a surface, for example a
magnetic surface can bind to a CTCs from a wide array of cancer
types including but not limited to breast cancer, prostate cancer,
colorectal cancer, lung cancer. Markers, or tumor-specific antigens
can be used to confirm the identity of captured cells as tumor
cells are well known in the tart and include, but are not limited
to, binding agents to cell surface epitopes available from Miltengi
Biotec GmbH.
[0082] As used herein, the terms "tumor-specific antigens" or
"cancer antigens" or "cancer specific antigens" refer to a receptor
polypeptide (e.g. a polypeptide that binds specifically to a
molecule in the extracellular environment) that is present on the
surface of a cancer cell and/or differentially expressed by cancer
cells and can thereby be exploited in order to identify cancer
cells. A tumor specific antigen can be a receptor displayed
exclusively on cancer cells, a receptor displayed at a higher level
on cancer cells than normal cells of the same or different tissue
types, or a receptor displayed on both cancerous and normal cell
types. In some embodiments, a tumor-specific can be a receptor
that, in cancer cells, has altered (e.g. higher or lower than
normal) expression and/or activity. In some embodiments, a tumor
specific antigen can be a receptor that is implicated in the
disease process of cancer. In some embodiments, a tumor specific
antigen can be a receptor that is involved in the control of cell
death and/or apoptosis. Some examples of cancer antigens include
the cancer-testis (CT) antigens BAGE, GAGE, MAGE-1 and MAGE-3,
NY-ESO-1, SSX. These antigens are found in melanoma, lymphoma,
lung, bladder, colon, and breast carcinomas. Cancer antigens
normally found in melanocytes, epithelial tissues, prostate, and
colon also include the differentiation antigens Gp100,
Melan-A/Mart-1, Tyrosinase, PSA, CEA, and Mammaglobin-A. These
antigens are found in melanoma, prostate cancer, and in colon and
breast carcinomas. Some cancer antigens are shared antigens that
are ubiquitously expressed at low levels but overexpressed in
cancers. Examples of overexpressed cancer antigens include p53,
HER-2/neu, livin, and survivin, found in esophagus, liver,
pancreas, colon, breast, ovary, bladder, and prostate carcinomas.
Other cancer antigens are unique, such as .beta.-catenin-m,
.beta.-Actin/4/m, Myosin/m, HSP70-2/m, and HLA-A2-R170J, which are
associated with one or more of melanoma, non-small cell lung
cancer, and renal cancer. Still other cancer antigens are the
tumor-associated carbohydrate antigens that are normally found in
epithelia tissues such as renal, intestinal, and colorectal
tissues. These cancer antigens include GM2, GD2, GD3, MUC-1, sTn,
abd globo-H, which can be found in melanoma, neuroblastoma,
colorectal, lung, breast, ovarian, and prostate cancers. Additional
tumor antigens, peptide epitopes, and descriptions thereof are
described in U.S. Pat. Nos. 7,906,620; 7,910,692; 8,097,242;
7,935,531; 8,012,468; 8,097,256; 8,003,773; Tartour et al., Immunol
Lett 2000; 74(1): 1-3, the contents of which are herein
incorporated by reference in their entireties.
[0083] In an exemplary method for capture and/or detection of CTC
in a test sample, the test sample is contacted with a lectin
molecule attached to a surface for example a magnetic surface and
CTC bound to lectin molecule are isolated using a microfluidic
device, for example a magnetic microfluidic separation device. Use
of a microfluidic device can automate the methods disclosed herein
and/or allow analysis of multiple samples at the same time. One of
skill in the art is well aware of methods in the art for
collecting, handling and processing biological fluids which can be
used in the practice of the present disclosure. The process
described herein can allow sample analysis in short time periods.
For example, the process can be completed in less than 6 hours,
less than 5 hours, less than 4 hours, less than 3 hours, less than
2 hours, less than 1 hour, less than 30 minutes. In some
embodiments, presence and identity of a CTC in the sample can be
done within 10 minutes to 60 minutes of starting the process. The
methods described herein can be utilized to detect the presence of
CTCs in a sample of any given volume. In some embodiments, sample
volume is about 0.25 ml to about 50 ml, about 0.5 ml to about 25
ml, about 1 ml to about 15 ml, about 2 ml to about 10 ml. In some
embodiments, sample volume is about 5 ml. In one embodiment, sample
volume is 8 ml. In some embodiments, prior to contacting with the
lectin molecules, the sample can be preprocessed. The preprocessing
can serve a number of purpose for example hemolyzing blood cells,
dilution of samples, etc. A preprocessing agent can be present in
the sample container or can be added to the sample in the
container. When the sample is a biological fluid, the sample
container can be a VACUTAINER.RTM., e.g., a heparinized
VACUTAINER.RTM..
[0084] The preprocessing reagents include, but are not limited to,
surfactants and detergents, salts, cell lysing reagents,
anticoagulants, degradative enzymes (e.g., proteases, lipases,
nucleases, lipase, collagenase, cellulases, amylases and the like),
and solvents, such as buffer solutions. In some embodiments, a
preprocessing reagent is a surfactant or a detergent. In one
embodiment, the preprocessing reagent is Triton X100. Amount of
preprocessing reagent to be added can depend on a number of
factors. Generally, the preprocessing reagent is added to a final
concentration of about 0.1 mM to about 10 mM. If a liquid, the
preprocessing reagent can be added so as to dilute the sample at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 60%, at least 80%, at least 90%, at least
1-fold, at least 2-fold, at least 3-fold, or at least 5-fold. One
or more preprocessing agents can be a component of a kit to isolate
and/or detect CTCs using the technology described herein. After
preprocessing step, the sample is subjected to CTC capturing
process, which can comprise mixing lectin molecules attached to a
surface such as beads for e.g. lectin coated magnetic microbeads or
lectin coated fluorescent microbeads. Amount of lectin molecule
coated-microbeads added to the sample can be dependent on a number
of different factors, such as, number of affinity molecules on each
microbead, size of the microbead, binding affinity of the affinity
molecule to the CTC, and concentration of the CTCs in the sample.
Additionally, amount of coated-microbeads added to the sample can
be adjusted to optimize the capture of CTCs. In some embodiments,
amount of coated-microbeads added to the sample is such that a
microbead binds with one CTC. However, each CTC can be bound to
more than one coated-microbeads or conversely each microbead is
bound to more than one CTC. In some embodiments, a plurality of
coated-micro-beads can be contacted with a test sample. The
plurality of coated-microbeads can comprise at least two subsets
(e.g., 2, 3, 4, 5, or more subsets), wherein each subset of
coated-microbeads have a pre-determined dimension. In some
embodiments, the plurality of coated-microbeads can comprise a
first subset of the coated-microbeads and a second subset of the
coated-microbeads. In such embodiments, the first subset of the
coated-microbeads each has a first predetermined dimension; and the
second subset of the coated-microbeads each has a second
predetermined dimension. The pre-determined dimension of a
coated-micro-bead depends, in part, on the dimension of a microbead
described herein to which the lectin molecules are conjugated. For
example, the microbead can have a size of about 10 nm to 10 .mu.m,
about 20 nm to about 5 .mu.m, about 40 nm to about 1 .mu.m, about
50 nm to about 500 nm, or about 50 nm to about 200 nm.
Additionally, each subset of the coated-microbeads can comprise on
their surfaces substantially the same density or different
densities of the affinity molecules (e.g., FcMBL or lectin
molecules described herein).
[0085] Different subsets of the plurality of the coated-microbeads
can be brought into contact with a test sample in any manner. For
example, in some embodiments, the plurality of the
coated-microbeads can be provided as a single mixture comprising at
least two subsets of the coated-microbeads to be added into a test
sample. In some embodiments, in order to distinguish among
different subsets of the coated-micro- beads, the coated-microbeads
in each subset can have a distinct detection label, e.g., a
distinctly-fluorescent label that can be sorted afterward, for
example, by flow cytometry.
[0086] In other embodiments, the plurality of the coated-
microbeads can be brought into contact with a test sample in a
sequential manner. For example, a test sample can be contacted with
a first subset of the coated-microbeads, followed by a contact with
at least one more subsets of the coated- microbeads. The previous
subset of the coated-microbeads can be removed from the test sample
before addition of another subset of the coated-microbeads into the
test sample. After addition of the lectin molecule
coated-microbeads, they can be mixed in the sample to allow CTC to
bind with the microbeads. This can be simply accomplished by
agitating the sample, e.g., shaking or vortexing the sample and/or
moving the sample around in a microfluidic device.
[0087] The volume of a test sample required for contacting the
lectin molecule attached to a surface can vary with, e.g., the
selection of the surface to which the lectin molecule is attached
(e.g., microbeads, fibers, filters, filters, fibers, screens, mesh,
tubes, hollow fibers), the concentration of CTCs present in a test
sample, and/or the platform used to carry out the methods (e.g., a
microfluidic device or a blood collection tube, a microtiter
plate). In some embodiments, the test sample volume used to perform
the assay described herein, e.g., in a microfluidic platform, can
range from about 1 .mu.L to about 500 .mu.L, from about 5 .mu.L to
about 250 .mu.L, or from about 10 .mu.L to about 100 .mu.L. In
other embodiments, the test sample volume used to perform the assay
described herein, e.g., in a tube platform, can range from about
0.05 mL to about 50 mL, from about 0.25 ml to about 50 ml, about
0.5 ml to about 25 ml, about 1 ml to about 15 ml, or about 2 ml to
about 10 mi. In some embodiments, the test sample volume used to
perform the assay described herein can be about 1 mL to about 5 mL.
In one embodiment, the test sample volume used to perform methods
described herein is about 5 ml to about 10 mL.
[0088] The sample mixture can be incubated for a period of time to
allow the CTCs to bind onto the lectin molecule attached to a
surface, e.g., incubation for at least one minute, at least two
minutes, at least three minutes, at least four minutes, at least
five minutes, at least ten minutes, at least fifteen minutes, at
least about twenty minutes, at least thirty minutes, at least
forty-five minutes, or at least one hour. In one embodiment, the
sample mixture can be incubated for a period of about 10-20
minutes. Such incubation can be performed at any appropriate
temperature, In some embodiments, the incubation can be performed
at a temperature ranging from about room temperature to about
37.degree. C.
[0089] The captured CTCs bound to the lectin molecule are then
subjected to an isolation process. The isolation can be carried out
by passing the sample containing the CTCs bound to a lectin
molecule through a microfluidic device. In some embodiments, where
the lectin beads are bound to a magnetic surface, the separation
can be carried out by using magnetic separation device for example
microfluidic magnetic separation. Microfluidic devices including,
those enabling magnetic separation are well known in the art
US20130035630A1, CN202912946U, WO2010124227A3, US20070026469A1, US
2011/0039280, US 6,365,362. 13., Kang, J H. et al. (2014). In
alternative embodiments, the lectin molecule is immobilized onto a
microfluidic device and the sample is passed through the device for
obtaining, sample free of CTCs. In some embodiments, the CTC-bound
lectin molecule after isolated from the test sample or processing
buffer can be washed with a buffer (e.g., TBST) to remove any
residues of test sample, solution comprising the chelating agent or
any unbound CTCs. The number of wash steps can range from 1 to
many, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more wash steps. In
one embodiments, the CTC bound to a lectin molecule after isolated
from the solution comprising the chelating agent e.g. EDTA and/or
the test sample can be washed with a buffer (e.g., TBST) for about
at least 1-3 times.
[0090] In some embodiments, the release can be accomplished either
by cleaving the bond tethering the lectin molecule to the solid
surface, or by displacing the captured cells from the lectin
molecule.
[0091] In one embodiment, the method of the invention utilizes
cleavable bi-functional linkers for the capturing lectin molecule,
e.g., photo-cleavable or chemical groups contained within the
bi-functional linkers to enable release of the captured cells.
Several photo-cleavable linkers are available to one skilled in the
art, see, e.g. Kan oh, N., et al. (2010) Cleavable linker for
photo-cross-linked small-molecule affinity matrix, Bioconjug. Chem.
21:182-186 and references cited therein. These bi-functional
linkers can consist of different lengths and composition, such as
single-stranded oligonucleotides that contain the photo-cleavable
residue or an abasic site that can be cleaved enzymatically.
[0092] In another embodiment, the invention utilizes an avidin
compound, e.g., avidin, streptavidin, nitroavidin or neutravidin,
interacting with a biotinylated capturing lectin molecule to enable
capture and release of cells. The avidin compound dissociates from
the biotin part of the biotinylated antibody upon a change in pH of
the solution from neutral to alkaline in the case of nitroavidin.
The advantage of this embodiment is the ability to regenerate the
binding surface.
[0093] In yet another embodiment, where cells are captured using
peptide inhibitors, ligands or other binding partners to enable
release of captured cells, the cells are made to dissociate from
the matrix by adding excess of the binding agent in soluble
form.
[0094] The captured CTC can remain bound on the lectin molecule
during detection and/or analysis, or be isolated from it prior to
detection and/or analysis. In some embodiments, a composition
comprising a peptide comprising the CRD region of a lectin is
linked to a detectable label for example an "imaging agent" or a
"contrast agent". As used herein, the term "detectable label"
refers to a agent capable of producing a detectable signal
indicative of the presence of a target for example a cancer cells,
CTCs, solid tumor, metastatic tumor. Detectable labels include any
composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Suitable labels include fluorescent molecules, radioisotopes,
nucleotide chromophores, enzymes, substrates, chemiluminescent
moieties, bioluminescent moieties, and the like. As such, a label
is any composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means
needed for the methods and compositions described herein.
[0095] As used herein, the term "imaging agent" refers to an
element or functional group in a molecule that allows for the
detection, imaging, and/or monitoring of the presence and/or
progression of a condition(s), pathological disorder(s), and/or
disease(s) for example cancer. The imaging agent can be an
echogenic substance (either liquid or gas), non-metallic isotope,
an optical reporter, a boron neutron absorber, a paramagnetic metal
ion, a ferromagnetic metal, a gamma-emitting radioisotope, a
positron-emitting radioisotope, or an x-ray absorber. As used
herein the term "contrast agent" refers to any molecule that
changes the optical properties of tissue or organ containing the
molecule. Optical properties that can be changed include, but are
not limited to, absorbance, reflectance, fluorescence,
birefringence, optical scattering and the like. In some
embodiments, the detectable labels also encompass any imaging agent
(e.g., but not limited to, a bubble, a liposome, a sphere, a
contrast agent, or any detectable label described herein) that can
facilitate imaging or visualization of a tissue or an organ in a
subject, e.g., for detection of CTCs and/or for diagnosis of
cancer.
[0096] Suitable optical reporters include, but are not limited to,
fluorescent reporters and chemiluminescent groups. A wide variety
of fluorescent reporter dyes are known in the art. Typically, the
fluorophore is an aromatic or heteroaromatic compound and can be a
pyrene, anthracene, naphthalene, acridine, stilbene, indole,
benzindole, oxazole, thiazole, benzothiazole, cyanine,
carbocyanine, salicylate, anthranilate, coumarin, fluorescein,
rhodamine or other like compound.
[0097] Exemplary fluorophores include, but are not limited to, 1,5
IAEDANS; 1,8-ANS ; 4-Methylumbelliferone;
5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);
5-Carboxynapthofluorescein (pH 10); 5-Carboxytetramethylrhodamine
(5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5-Hydroxy Tryptamine
(HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA
(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G;
6-JOE; 7- Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);
7-Hydroxy-4-methylcoumarin; 9- Amino-6-chloro-2-methoxyacridine;
ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine);
Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin;
Acriflavin Feulgen SITSA; Aequorin (Photoprotein); Alexa Fluor
350.TM..
[0098] Suitable radioisotopes include, but are not limited to,
99mTc, 95Tc, 111In, 62Cu, 64Cu, Ga, 68Ga, and 153Gd. Suitable
paramagnetic metal ions include, but are not limited to, Gd(III),
Dy(III), Fe(III), and Mn(II). Suitable X-ray absorbers include, but
are not limited to, Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Au,
Yb, Dy, Cu, Rh, Ag, and Ir.
[0099] In some embodiments, the radionuclide is bound to a
chelating agent or chelating agent-linker attached to the lectin
molecule. Suitable chelating agents include, but are not limited
to, DOTA, BAD, TETA, DTPA, EDTA, NTA, HDTA, their phosphonate
analogs, and mixtures thereof. One of skill in the art will be
familiar with methods for attaching radionuclides, chelating
agents, and chelating agent-linkers to molecules such as the lectin
molecules coated surfaces and carrier scaffolds disclosed
herein.
[0100] Means of detecting such labels are well known to those of
skill in the art. Thus, for example, radiolabels can be detected
using photographic film or scintillation counters, fluorescent
markers can be detected using a photo-detector to detect emitted
light. Enzymatic labels are typically detected by providing the
enzyme with an enzyme substrate and detecting the reaction product
produced by the action of the enzyme on the enzyme substrate, and
calorimetric labels can be detected by visualizing the colored
label. Exemplary methods for in vivo detection or imaging of
detectable labels include, but are not limited to, radiography,
magnetic resonance imaging (MRI), Positron emission tomography
(PET), Single-photon emission computed tomography (SPECT, or less
commonly, SPET), Scintigraphy, ultrasound, CAT scan, photoacoustic
imaging, thermography, linear tomography, poly tomography,
zonography, orthopantomography (OPT or OPG), and computed
Tomography (CT) or Computed Axial Tomography (CAT scan).
[0101] In some embodiments, the detectable label can include an
enzyme. Exemplary enzymes for use as detectable labels include, but
are not limited to, horseradish peroxidase (HRP), alkaline
phosphastase (AP), or any combinations thereof. In some
embodiments, the detectable label is a fluorophore or a quantum
dot. Without wishing to be bound by a theory, using a fluorescent
reagent can reduce signal-to-noise in the imaging/readout, thus
maintaining sensitivity. Accordingly, in some embodiments, prior to
detection, the CTCs isolated from or remained bound on the lectin
molecule can be stained with at least one stain, e.g., at least one
fluorescent staining reagent comprising a CTC-binding molecule,
wherein the CTC-binding molecule comprises a fluorophore or a
quantum dot. Examples of fluorescent stains include, but are not
limited to, any CTC-binding molecule (e.g., antibodies targeting
known surface markers of CTC, cancer cell markers, EMT markers) or
any cancer cell binding proteins or peptides or oligonucleotides)
typically conjugated with a fluorophore or quantum dot, and any
fluorescent stains used for detection as described herein. In some
embodiments, the detectable label is a gold particle. In some
embodiments, the labeling molecule can comprise MBL or a lectin
binding molecule described herein. In one embodiment, the labeling
molecule comprises FcMBL. Without wishing to be bound by a theory,
labeling molecules based on MBL, and FcMBL in particular, attach
selectively to a broad range of CTCs, and so they enable the
methods described herein to detect the CTCs from a variety of
samples from an array of cancer types with high sensitivity and
specificity.
[0102] Any method known in the art for detecting the particular
label can be used for detection. Exemplary methods include, but are
not limited to, spectrometry, fluorometry, microscopy imaging,
immunoassay, and the like. While the CTC capture step can
specifically capture CTCs, it can be beneficial to use a labeling
molecule that can enhance this specificity. If imaging, e.g.,
microscopic imaging, is to be used for detecting the label, the
staining can be done either prior to or after the CTCs have been
laid out for microscopic imaging. Additionally, imaging analysis
can be performed via automated image acquisition and analysis. In
some embodiments, the methods disclosed herein can be used to
detect cancer cells by imaging biopsy sections and or detect in
vivo by injecting the compositions disclosed herein into the
subject.
[0103] For optical detection, including fluorescent detection, more
than one stain or dye can be used to enhance the detection or
identification of the cancer cell for example CTC. For example, a
first dye or stain can be used that can bind with a cancer cell
and/or CTC, and a second dye or strain can be used that can bind
with all cells for e.g. a DNA satin such as DAPI. Colocalization of
the two dyes then provides enhanced detection or identification of
the cancer cells by reducing false positive detection.
[0104] In some embodiments, microscopic imaging can be used to
detect signals from label on the labeling agent. Generally, the
cancer cells and/or CTCs in the subsample are stained with a
staining reagent and one or more images taken from which an artisan
can easily count the number of cells present in a field of view. In
particular embodiments, CTCs can be detected through use of one or
more enzyme assays, e.g., enzyme- linked assay (ELISA). In some
embodiments, enzyme assays can be configured such that an enzyme
will catalyze a reaction involving an enzyme substrate that
produces a fluorescent product. Enzymes and fluorescent enzyme
substrates are known and are commercially available (e.g.,
Sigma-Aldrich, St. Louis, Mo.). In some embodiments, enzyme assays
can be configured as binding assays that provide for detection of
CTCs. For example, in some embodiments, a labeling molecule for
e.g., FcMBL, lectin molecule can be conjugated with an enzyme for
use in the enzyme assay. An enzyme substrate can then be introduced
to the one or more immobilized enzymes such that the enzymes are
able to catalyze a reaction involving the enzyme substrate to
produce a detectable signal. Similarly, a variety of enzymes can be
used, with either colorimetric or fluorogenic substrates. In some
embodiments, the reporter-enzyme produces a calorimetric change
which can be measured as light absorption at a particular
wavelength. Exemplary enzymes include, but are not limited to,
beta-galactosidases, peroxidases, catalases, alkaline phosphatases,
and the like. In some embodiments, the enzyme is a horseradish
peroxidase (HRP). In some embodiments, the enzyme is an alkaline
peroxidase (AP). Methods of conducting an ELISA assay are well
known to those skilled in the art.
[0105] In some embodiments, the isolated CTCs can be further
cultured in an in vitro culture system. In some embodiments, the
CTCs are subjected to immunochemical analysis. The "immunochemical
analysis" can include for example immunostaining the captured CTCs
with one or more CTC markers known in the art. Non-limiting
examples of CTC and/or tumor specific markers (also referred to as
"tumor specific antigens") useful in the embodiments disclosed
herein include cytokeratin, prostate-specific antigen (PSA),
prostate specific membrane antigen (PSMA), mucin-1 (MUC-1), human
epidermal growth factor receptor 2 (HER2), AFP (a-fetoprotein),
N-cadherin, epithelial cell adhesion molecule (EpCAM), EphB4or
carcinoembryonic antigen (CEA) or a combination thereof analysis of
these markers can be done using methods such as flowcytometry or
Fluorescence in situ hybridization (FISH). Accordingly the isolated
CTCs can also be used for downstream immunocytochemical analysis,
RT-PCR, PCR, FISH, flowcytometry, or other types of image
cytometry.
[0106] It should be noted that a number of different cell analysis
platforms can be used to identify and enumerate the captured CTCs.
Examples of such analytical platforms are Immunicon's
CellSpotter.RTM. system, a magnetic cell immobilization and
analysis system, using microscopic detection for manual observation
of cells described in Example II, and the CellTracks system, an a
more advanced automatic optical scanning system, described in U.S.
Pat. Nos. 5,876,593; 5,985,153 and 6,136,182 respectively. All of
the aforementioned U.S. Patent Applications are incorporated by
reference herein as disclosing the respective apparatus and methods
for manual or automated quantitative and qualitative cell analysis.
A decrease in the number of circulating tumor cells is indicative
of an improvement in patient status or efficacy of treatment,
whereas an increase indicates a worsening of the disease. Such
devices may be used to advantage in the diagnostic and monitoring
kits of the present invention. Using methods described herein, a
composition comprising CTC cell population can obtained. A
composition comprising the CTC population can be used for
downstream analysis, for example, physical, chemical (e.g.,
biochemical), and/or molecular analysis. Various techniques can be
used to conduct these studies to analyze physical, chemical and/or
molecular features (e.g., DNA, RNA, microRNA, DNA methylation, and
protein) of the CTCs. Examples of the analysis include, but are not
limited to cytomorphological analysis, genomics analysis,
proteomics analysis, transcriptomics analysis, epigenomics
analysis, and any combinations thereof In some embodiments, the
analysis is performed on a single CTC. In some embodiments, the
analysis is performed on a substantially pure population of CTCs.
As used herein, non- limiting examples of cellular analysis include
counting the number of the CTCs, cytomorphological analysis of the
CTCs, and other techniques available for studying cellular details
of cells. In some embodiments, one or more molecular features of
the CTCs are analyzed. Examples of the molecular features include,
but are not limited to, nucleic acid composition, protein
composition, DNA methylation profile, protein glycosylation, and
phosphorylation pattern. In some embodiments, nucleic acids (e.g.,
DNAs and RNAs) of the CTCs are isolated and analyzed. In some
embodiments, whole genome amplification is performed before the
molecular analysis. In some embodiments, the DNA sequence in cancer
mutation hotspots in the CTCs is determined. Non-limiting examples
of cancer mutation hotspots include mutation hotspots in genes such
as Ras, p53, Braf, Pten, Egfr, Erccl , Rrml, Elm4, Alk, and Her2
gene. In some embodiments, the CTCs are analyzed for the presence
or absence of gene amplification or translocation. For example, the
CTCs can be analyzed to determine the presence or absence of
Elm4-Alk translocation. Examples of methods that can be used for
downstream analyses to characterize and/or analyze the cells
include, but are not limited to, biochemical analysis;
immunochemical analysis; image analysis; cytomorphological
analysis; molecule analysis such as PCR, sequencing, determination
of DNA methylation; proteomics analysis such as determination of
protein glycosylation and/or phosphorylation pattern; genomics
analysis; epigenomics analysis; transcriptomics analysis; and any
combination thereof. In some embodiments, molecular features of the
CTCs are analyzed by image analysis, PCR (including the standard
and all variants of PCR), microarray (including, but not limited to
DNA microarray, MMchips for microRNA, protein microarray, cellular
microarray, antibody microarray, and carbohydrate array),
sequencing, biomarker detection, or methods for determining DNA
methylation or protein glycosylation pattern.
[0107] In some embodiments, the methods allow obtaining CTCs
without significant disruption of the cells. Therefore, these
methods allow preservation of cytologic details of the cells and
detailed downstream analysis of the CTCs. Any suitable methods
known in the art can be used to determine the structural integrity
of the rare cells. Non-limiting examples of such methods include
immunocytochemical procedures, fluorescence in situ hybridization
(FISH), flow cytometry, image cytometry, and any combinations
thereof. The methods disclosed herein allow studying the
distribution of the markers of interest for example Epithelial to
mesenchymal transition markers (EMT) (for example, mutation, gene
expression, protein, DNA methylation, regulatory RNA (e.g., miRNA
and siRNA), and etc.) among the CTCs.
[0108] Genomics, epigenomics, transcriptomics, and proteomics
analysis of CTCs obtained by the methods described herein will
provide a real-time window into the biology of a tumor and
facilitate an understanding of tumor biology in real-time. For
example, the condition of a cancer patient can be evaluated by
analyzing sequence information obtained from a CTC. The sequence
information can include insertion/deletion/mutation of the genomic
sequence, methylation pattern of the DNA, and epigenetic
characteristic of the DNA. In some embodiments, the condition of a
cancer patient can be evaluated by analyzing biochemistry
information obtained from a CTC. The biochemistry information can
include information regarding protein glycosylation, protein
phosphorylation and other post-translational modification on
proteins.
[0109] In some embodiments, one or more gene mutations in the CTCs
are determined. The types of gene mutation are not particularly
limited. Non-limiting examples of gene mutation include insertions,
deletions, substitutions, translocations, gene amplifications, and
any combinations thereof. In some embodiments, the gene mutation is
located in KRAS, BRAF, PTEN, EGFR, ERCC1 , RRM1, ELM4, HER2, or ALK
gene. In some embodiments, the DNA mutation is an EML4-ALK fusion
or a gene amplification in Her2. In some embodiments, whole-genome
analysis of the CTCs is performed.
[0110] In some embodiments, protein expression level of a cancer
specific gene of the CTCs is determined. In some embodiments, RNA
expression level of a cancer specific gene of the CTCs is
determined. Examples of cancer specific gene include, but are not
limited to, cytokeratin, prostate-specific antigen (PSA), prostate
specific membrane antigen (PSMA), mucin- 1 (MUC-1), human epidermal
growth factor receptor 2 (HER2), AFP (a-fetoprotein), N-cadherin,
epithelial cell adhesion molecule (EpCAM), epidermal growth factor
receptor (EGFR), ERCC 1 , androgen receptor (AR), human
equilibrative nucleoside transporter 1 (hENT1), RRM1, and
carcinoembryonic antigen (CEA). Other non-limiting examples of the
cancer specific gene include epithelial mesenchymal transition
(EMT) markers are cancer stem cell (CSC) markers. Non-limiting
examples of EMT markers include N-cadherin, vimentin, B-catenin
(nuclear localized), Snail-1, Snail-2 (Slug), Twist, EF1/ZEB1,
SIP1/ZEB2, and E47. Examples of CSC markers include, but are not
limited to, CD 133 and CD44.
[0111] The embodiments disclosed herein also include methods for
assessing or predict response of a patient suffering from cancer to
a treatment, where the methods include providing a circulating
tumor cell (CTC) or a substantially pure population of CTCs from
the patient and performing one or more cellular or molecular
analyses on the CTCs to determine treatment response in the
patient. For example, expression levels of HER2 protein was found
to correlate significantly with patients' response to anti-cancer
drug lapatinib. Single CTCs obtained from a cancer patient using
the methods disclosed herein can be analyzed for HER2 protein
expression, and the HER2 protein expression level can be used to
predict or assess the patient's response to lapatinib treatment and
thus can be used in the development of an appropriate treatment
regimen. As another example, the presence of cancer stem cell
markers such as ALDH, CD44, CD 133, and CD 166 correlates with poor
prognosis for colorectal cancer patients. However, certain
therapies, i.e., dasatinib and curcumin combination therapy, has
been shown to significantly reduce the number of cancer stem cells.
Accordingly, the isolation and analysis of CTCs for cancer stem
cell markers can be used to determine whether it is appropriate to
treat a patient with certain chemotherapeutics. As such, methods
disclosed herein for isolating single CTCs can be used to develop
targeted therapies for cancer patients. As another example,
molecular features (e.g., sequence and biochemistry information)
obtained from the CTCs can be used to evaluate the patient's
response to a cancer treatment, patient prognosis, patient
diagnosis, or remission state of a patient.
[0112] The results obtained from the physical, chemical, and
molecular analysis of CTCs can provide valuable information for
various applications including, but not limited to, evaluating
condition of the cancer patient, assessing or predicting cancer
progression, assessing or predicting treatment response of the
cancer patient, cancer prognosis, screening targets for cancer
drugs, predicting treatment outcome, discovering novel biomarkers,
and understanding response of cancer cell to therapeutic
pressure.
[0113] The methods and compositions disclosed herein can be useful
for cancer diagnosis and therapeutics. In some embodiments, the
invention relates to a method of assessing a risk of developing a
metastatic tumor in the patient. In some embodiments, the patient
is carrying a tumor or had tumor a tumor. The patients sample can
comprise whole blood, body fluid, any cell-containing blood
fraction, a fragmented tumor, a tumor cell suspension, or a cell
culture established from a patient's sample, or the culture
supernatant or a xenograft established from a patient's tumor. In
variations of this embodiment, the method further comprises a step
of detecting one or more of the following biomarkers: EpCAM, CD146,
CK5, CK7, CK18, CK19, CD44, Cd44v6, EphB4, IGF-1R, BCL2, HER2,
HER3, CA19-9, CEA, CD133, MUC1, N-cadherin, Survivin, EGFR, KRAS,
BRAF, p53, Pi3KCA, PTEN, KRT19, CD34, CD24, ACT2, VIM, NANOG, CXCR4
and TWIST1 in the captured cells. Other cancer epitopes (also
referred to as "tumor specific antigens") are known in one art (and
mentioned above) and can be used alone or in combination with the
above-noted markers. In variations of this embodiment, the method
further comprises analysis of the isolated CTCs using one or more
of cellular and/or molecular methods described above.
[0114] In another embodiment, the invention is a method of
detecting the presence of a malignant tumor or assessing metastatic
potential of an existing or excised tumor in a patient by detecting
mannan carbohydrates expressing CTCs in a patient's sample. The
sample may comprise whole blood, body fluid, any cell-containing
blood fraction, a fragmented tumor, a tumor cell suspension, or a
cell culture established from a patient's sample, or the culture
supernatant. In this embodiment, the captured cells may be further
characterized as CTCs and assessed for their numbers and gene
expression profile comprising e.g. one or more of the biomarkers ,
for example ACT2, IGF-1R, BCL2, HER2, EphB4, CA19-9, CEA, CD24,
CD44, CD133, CD146, CXCR4, TWIST1, VIM, NANOG, KRT19, MUC1,
Survivin, EGFR, KRAS, BRAF, p53, Pi3KCA and PTEN. In variations of
this embodiment, the method further comprises analysis of the
isolated CTCs using one or more of cellular and/or molecular
methods described above.
[0115] In yet another embodiment, the invention relates to a method
of determining prognosis for a patient having a tumor, the method
comprising determining the metastatic potential of the tumor by
assessing the number of detected CTCs defined as mannan expressing
cells within a patient sample e.g. blood, wherein the CTC count
higher than that defined in the art for various cancer types is
used as an indicator of poor outcome. The poor outcome can be for
example in terms of progression free survival of the patient.
According to the method, the prognosis may be formed for any
malignant solid tumor known to have metastatic potential, including
without limitation, lung cancer (e.g., non-small cell lung cancer
(NSCLC), bone cancer, pancreatic cancer, cancer of the head or
neck, melanoma, uterine cancer, ovarian cancer, cervical cancer,
colorectal cancer, gastric cancer, breast cancer, endometrial
cancer, thyroid cancer, prostate cancer, bladder cancer, kidney
cancer (e.g., renal cell carcinoma), liver cancer (e.g.,
hepatocellular carcinoma), and cancers of the central nervous
system (CNS), (e.g., glioma, glioblastoma multiforme or
astrocytoma).
[0116] In some embodiments, the technology described herein relates
to a method for assessing or monitoring the effectiveness of a
cancer treatment for e.g. chemotherapy, radiation therapy, in a
patient. When the level of CTC before a cancer treatment is
compared to the level of CTC after the treatment, the comparison
may serve as a benchmark to assess whether the particular treatment
method is effective on the particular patient. Monitoring or
assessment may be done by single point comparison or by taking a
time-series of the CTC level and then analyzing the trend to make a
diagnostic determination. In some embodiments of the method, the
sample is collected from the patient prior to starting a
therapeutic regimen, and at regular intervals post-treatment onset.
In some embodiments of the method, the CTCs are captured and
enumerated using the methods disclosed herein. The CTCs can be
further analyses using one or more of the methods described above.
The treatment is said to be effective if the number of captured
CTCs are enumerated to be lower upon treatment onset and
demonstrates a decreasing number in sample obtained at intervals
after the onset. Accordingly, an increase in the number of captured
CTCs in the sample obtained post-treatment onset and/or an
increasing number in the samples obtained at regular intervals
post-treatment onset indicates the treatment of choice is
ineffective.
[0117] In some embodiments, the technology disclosed herein relates
to a method for diagnosing metastatic tumor in a patient. The
patient may be carrying a benign tumor or had been previously
diagnosed with a benign tumor. The method encompasses capturing and
enumerating the CTCs. A diagnosis of a metastatic tumor can be made
based on a CTC count higher than a predetermined level.
[0118] In some embodiments, the binding of cancer cells and/or CTCs
to the lectin molecule can facilitate detection and localization of
cancer cells in a tissues and biological fluids of a subject. The
binding further facilitates isolation and removal of cancer cells
from a biological fluid of a subject, for e.g., blood. Accordingly,
in one aspect the technology disclosed herein is a method of
treatment of cancer. In some embodiments, the invention relates to
a composition comprising, the lectin molecule attached to a surface
linked to an imaging agent. The lectin molecule can be a CRD region
of a lectin. The lectin molecule can be peptide of the lectin
and/or the CRD region of the lectin or a nucleic acid for e.g. mRNA
coding for the peptide of the lectin and/or the CRD region of the
lectin. Thus in some embodiments, the compositions comprising the
lectin molecule disclosed herein can detect and localize a tumor
and/or tumor cells in a subject, which can then be removed by
surgery or treated for example by targeted radiation therapy. In
some embodiments, the surface substrate can be labeled for specific
imaging of tumor sites. Non-limiting examples of radioisotopes
tracers used for imaging e.g. in positron emission tomography
include Carbon-11, Fluorine-18, Copper-64, Gallium-68, Bromine-76,
Zirconium-89, Iodine-124. In some embodiments, the invention
relates to a composition comprising, the lectin molecule attached
to a surface linked to a therapeutic agent. The lectin molecule can
be a CRD region of a lectin. The lectin molecule can be peptide of
the lectin and/or the CRD region of the lectin or a nucleic acid
for e.g. mRNA coding for the peptide of the lectin and/or the CRD
region of the lectin. Thus, in these embodiments the compositions
can be used to therapeutically target and treat a tumor in a
subject. Non-limiting example of a therapeutic agents include
Paclitaxel, Doxorubicin, Anastrozole, Everolimus, Melphalan,
Rituximab, Bevacizumab, Trastuzumab, Imatinib. In some embodiments,
the composition can further comprise at least one of an therapeutic
agent and a drug delivery vehicle. As used herein, the term "drug
delivery vehicle" generally refers to any material that can be used
to carry an active agent to a target site. Examples of drug
delivery vehicles includes, but are not limited to, a cell, a
peptide particle, a polymeric particle, a dendrimer, a vesicle, a
liposome, a hydrogel, a nucleic acid scaffold, an aptamer, and any
combinations thereof The term "therapeutic agent" as used herein
refers to any entity with anti-cancer activity, i.e. the ability to
inhibit or reduce the growth of a tumor and/or kill cancer cells,
e.g., by at least about 30%, at least about 40%, at least about
50%, at least about 75%, at least about 90% or more, as compared to
in the absence of an therapeutic agent. For therapeutic
application, the compositions disclosed herein can be formulated or
configured as per different applications using the methods known in
the art.
[0119] In one aspect, the invention relates to methods for
generating a cancer vaccine by capturing and isolating the CTCs
from a sample. The method comprises of isolating the CTCs using a
lectin molecule and combining the isolated CTCs or a component
thereof for example cell membrane with a scaffold to generate a
cancer vaccine composition. In some embodiments, the scaffold can
comprise a biomaterial. Non limiting examples of a biomaterial are
glycosaminoglycan, silk, fibrin, MATRIGEL.RTM., poly-ethyleneglycol
(PEG), polyhydroxy ethyl methacrylate, polyvinyl alcohol,
polyacrylamide, poly (N-vinyl pyrolidone), poly(lactic acid), poly
glycolic acid (PGA), poly lactic-co-glycolic acid (PLGA), poly
e-carpolactone (PCL), polyethylene oxide, poly propylene fumarate
(PPF), poly acrylic acid (PAA), polyhydroxybutyric acid, hydrolysed
polyacrylonitrile, polymethacrylic acid, polyethylene amine, esters
of alginic acid; pectinic acid; and alginate, fully or partially
oxidized alginate, hyaluronic acid, carboxy methyl cellulose,
heparin, heparin sulfate, chitosan, carboxymethyl chitosan, chitin,
pullulan, gellan, xanthan, collagen, gelatin, carboxymethyl starch,
carboxymethyl dextran, chondroitin sulfate, cationic guar, cationic
starch, poly(L-lactide-co-glycolide) acid (PLGA), mesoporous
silica, and cryogel IP, an adjuvant, and combinations thereof. In
some embodiments, the scaffold composition can further comprise an
adjuvant. In some embodiments, the composition, further comprises a
factor that recruits cells of immune response for example dendritic
cells, macrophages. Examples of factors that can recruit immune
cells include but are not limited to cytokines, for e.g. GM-CSF.
The scaffold can further comprise, of lectin molecules disclosed
herein. In some embodiments, the lectin molecules comprise of the
CRD region of a lectin. In some embodiments, the lectin molecule
also comprises of the complement activation domain which can aid in
activation of the complement cascade at the tumor site. In some
embodiments, the cancer vaccine compositions are administered to a
patient for e.g. subcutaneously to elicit an immune response in the
patient. In some embodiments, the CTCs are isolated from a patient
and then administered to the same patient in the form a patient
specific cancer vaccine formulation. In some embodiments, the CTCs
isolated using the lectin molecule can be preprocessed prior to
administration to a subject. In some embodiments, the cells are
subjected to radiation, are heat killed, lyophilized, subjected to
physical degradation to separate its components, for example cell
membrane, organelles Methods of preparation of cancer vaccine
scaffolds are known in the art as shown in reference WO 2009/102465
A2, WO 2007/070660. The methods to determine the effectiveness of
immune response can include imaging of tumor to detect a decrease
in tumor size, Isolation and enumeration of CTCs in the blood
post-administration of the vaccine. A decrease in cell numbers or a
reduction of tumor size can indicate effective treatment. In one
aspect, the technology described herein relates to a method to
removing CTCs from blood using a process similar to apheresis.
Exemplary method can comprise, a hollow fiber, hollow tube, the
inner surface of a which can be coated with the lectin molecules
described herein, e.g., for removing any CTCs from a fluid before
administering the fluid back to a subject. Devices that can be used
for such applications are known in the art for example described by
Kang, J H. et al. (2014), incorporated herein. Such devices can be
easily adapted for the technology described herein. The lectin
molecule can be coated onto the outer or inner surface of a hollow
surface.
[0120] As used herein, the term "administering," refers to the
placement of compositions disclosed herein, for e.g., in form of a
cancer vaccine to elicit an cancer specific immune response in a
patient or in form of a composition for therapeutically targeting
cancer, detecting or localizing cancer or visualizing cancer as
disclosed herein into a subject by a method or route that results
in at least partial delivery of the composition at a desired site.
Non limiting example of administration can be subcutaneous
injection of the composition described herein.
[0121] The terms "increased" ,"increase", "increasing" or "enhance"
are all used herein to generally mean an increase by a statically
significant amount; for the avoidance of doubt, the terms
"increased", "increase", or "enhance", mean an increase of at least
10% as compared to a reference level, for example an increase of at
least about 10%, at least about 20%, or at least about 30%, or at
least about 40%, or at least about 50%, or at least about 60%, or
at least about 70%, or at least about 80%, or at least about 90% or
up to and including a 100% increase or any increase between 10-100%
as compared to a reference level, or at least about a 2-fold, or at
least about a 3-fold, or at least about a 4-fold, or at least about
a 5-fold or at least about a 10-fold increase, or any increase
between 2-fold and 10-fold or greater as compared to a reference
level.
[0122] The terms, "decrease", "reduce", "reduction", "lower" or
"lowering," or "inhibit" are all used herein generally to mean a
decrease by a statistically significant amount. For example,
"decrease", "reduce", "reduction", or "inhibit" means a decrease by
at least 10% as compared to a reference level, for example a
decrease by at least about 20%, or at least about 30%, or at least
about 40%, or at least about 50%, or at least about 60%, or at
least about 70%, or at least about 80%, or at least about 90% or up
to and including a 100% decrease (e.g., absent level or
non-detectable level as compared to a reference level), or any
decrease between 10-100% as compared to a reference level. In the
context of a marker or symptom, by these terms is meant a
statistically significant decrease in such level. The decrease can
be, for example, at least 10%, at least 20%, at least 30%, at least
40% or more, and is preferably down to a level accepted as within
the range of normal for an individual without a given disease.
[0123] The term "adjuvant" as used herein refers to any agent or
entity which increases the antigenic response or immune response by
a cell to a cancer antigen. Examples of adjuvants include, but are
not limited to mineral gels such as aluminum hydroxide; surface
active substances such as lysolecithin, pluronic polyols,
polyanions; other peptides; oil emulsions; and potentially useful
human adjuvants such as BCG and Corynebacterium parvum. QS-21,
Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide,
PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026,
Adjuvax, CpG ODN, Betafectin, Alum, and MF59.
[0124] In some embodiments, the composition as described herein
further comprises an adjuvant. Adjuvants are a heterogeneous group
of substances that enhance the immunological response against an
antigen that is administered simultaneously. In some instances,
adjuvants are added to a vaccine to improve the immune response so
that less vaccine is needed. Adjuvants serve to bring the
antigen--the substance that stimulates the specific protective
immune response--into contact with the immune system and influence
The type of immunity produced, as well as the quality of the immune
response (magnitude or duration). Adjuvants can also decrease the
toxicity of certain antigens; and provide solubility to some
vaccine components.
[0125] In one embodiment, a vaccine composition as described herein
further comprise an adjuvant. Examples of adjuvants include, but
are not limited to QS- 21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G,
CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF,
B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and
MF59.
[0126] In some embodiments, suitable adjuvants include, but are not
limited to, alum, MF59, LTR72 (a mutant of E. coli heat-labile
enterotoxin with partial knockout of ADP-ribosyltransferase
activity), polyphosphazine adjuvant, interleukins such as IL-1,
IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12, interferons such as
alpha-interferon and gamma-interferon, tumor necrosis factor (TNF),
platelet derived growth factor (PDGF), GCSF, granulocyte-macrophage
colony-stimulating factor (GM-CSF), epidermal growth factor (EGF),
and the like. Examples of adjuvants capable of stimulating cellular
immune responses include cytokines secreted by helper T cells
called Th1 cells, e.g., interleukin-2 (IL-2), interleukin-4,
interleukin-12 (IL-12) and interleukin-18, fusion proteins having
one of such Th1 type cytokines (e.g., IL-2) fused to the Fc portion
of immunoglobulin G (IgG), interferons such as alpha-interferon,
beta-interferon and gamma-interferon, and chemokines that attract T
cells to infected tissues. Non-coding, ISS-enriched plasmid DNAs or
ISS oligonucleotides (ISS-ODNs) can also be used in the present
invention as adjuvants to enhance cellular immunity.
[0127] Using particulate systems as adjuvants, the antigens are
associated or mixed with or into a matrix, which has the
characteristics of being slowly biodegradable. Care must be taken
to ensure that that the matrices do not form toxic metabolites.
Preferably, the main kinds of matrices used are mainly substances
originating from a body. These include lactic acid polymers,
poly-amino acids (proteins), carbohydrates, lipids and
biocompatible polymers with low toxicity. Combinations of these
groups of substances originating from a body or combinations of
substances originating from a body and biocompatible polymers can
also be used. Lipids are the preferred substances since they
display structures that make them biodegradable as well as the fact
that they are a critical element in all biological membranes.
[0128] Adjuvants for vaccines are well known in the art. Examples
include, but not limited to, monoglycerides and fatty acids (e. g.
a mixture of mono-olein, oleic acid, and soybean oil); mineral
salts, e.g., aluminium hydroxide and aluminium or calcium phosphate
gels; oil emulsions and surfactant based formulations, e.g., MF59
(microfluidised detergent stabilised oil-in-water emulsion), QS21
(purified saponin), AS02 [SBAS2] (oil-in-water emulsion+MPL+QS-21),
Montanide ISA-51 and ISA-720 (stabilised water-in-oil emulsion);
particulate adjuvants, e.g., virosomes (unilamellar liposomal
vehicles incorporating influenza haemagglutinin), ASO4 ([SBAS4] A1
salt with MPL), ISCOMS (structured complex of saponins and lipids),
polylactide co-glycolide (PLG); microbial derivatives (natural and
synthetic), e.g., monophosphoryl lipid A (MPL), Detox (MPL+M. Phlei
cell wall skeleton), AGP [RC-529] (synthetic acylated
monosaccharide), DC_Chol (lipoidal immunostimulators able to self
organize into liposomes), OM-174 (lipid A derivative), CpG motifs
(synthetic oligonucleotides containing immunostimulatory CpG
motifs), modified LT and CT (genetically modified bacterial toxins
to provide non-toxic adjuvant effects); endogenous human
immunomodulators, e.g., hGM-CSF or hIL-12 (cytokines that can be
administered either as protein or plasmid encoded), Immudaptin (C3d
tandem array) and inert vehicles, such as gold particles. Adjuvants
are further described in U.S. Pat. No. 6,890,540, U. S. Patent
Application No. 2005/0244420, and PCT/SE97/01003, the contents of
which are incorporated herein by reference in their entirety.
[0129] An aspect of the present invention is directed to a kit for
capturing, detecting, isolating and/or enriching CTCs from a
biological sample e.g. a blood sample. Kits in accordance with this
aspect of the invention are designed to facilitate performance of
the cell isolation method and CTC-based assays as described above.
Thus, they generally include reagents required for performing the
cell capture and isolation method and/or CTC enumeration step of
the assays. They may also include containers, vials, and other
tools for facilitating the manipulation of reagents and blood
samples. Exemplary tools that may be included in the kit may
include but are not limited to needles, blood collection vials, RBC
lysis solution and reagent, depletion device and reagents
(including but not limiting to separation beads, separation column,
anti-CD45 depletion reagents, and nutrition medium in the presence
of 1-20% FBS), enumeration reagents (including but not limiting to
lectin molecule conjugated to detection label, anti-CD45 antibody,
anti-EpCAM antibody, anti-PDPN antibody, anti-thyroid hormone
receptor antibody, fluorescence-conjugated secondary antibodies,
and DNA binding dye).
[0130] In some embodiments, the kit can comprise: (a) one or more
containers containing a population of lectin molecules described
herein; and (b) at least one reagent. In these embodiments, a user
can generate their own composition of lectin molecule attached to a
surface by conjugating the provided lectin molecules to their
desired substrate, e.g., using any art-recognized conjugation
chemistry and/or methods described herein. In such embodiments, the
reagent can include, but is not limited to, a coupling agent for
conjugation of lectin molecules to a surface. In some embodiments,
the kit can further comprise one or more surface substrates (e.g.,
microbeads such as magnetic microbeads) to which the lectin
molecules described herein are conjugated. In such embodiments, a
user can further modify the surface chemistry of the provided
substrate prior to conjugation of the lectin molecules to the
substrate. In some embodiments, the kit can provide lectin
molecules attached to a surface which are ready to use.
Accordingly, in these embodiments, the kit can comprise: (a) one or
more compositions of lectin molecules attached to a surface
disclosed herein; and (b) at least one reagent. In some
embodiments, the lectin molecule attached to a surface can include
one or more CTC-binding dipsticks, e.g., as described herein. In
other embodiments, the lectin molecule attached to a surface can
include a population of CTC capturing microbeads (including, but
not limited to, polymeric microbeads and magnetic microbeads). In
some embodiments, the lectin molecule attached to a surface can
include a population of magnetic microbeads coated with lectin
molecules disclosed herein. The microbeads or magnetic microbeads
can be provided in one or more separate containers, if desired. In
some embodiments, the population of the microbeads or magnetic
microbeads coated with lectin molecule contained in one or more
containers can be lyophilized.
[0131] In some embodiments of any aspects of the kits described
herein, the population of the microbeads or CTC-capturing
microbeads can comprise at least one distinct subset of the
microbeads For example, each distinct subset of the microbeads can
be provided in a separate container. In some embodiments, the
distinct subset of the microbeads can have a distinct size. In some
embodiments, the distinct subset of microbeads can comprise on
their surfaces a different density of lectin molecules from the
rest of the population. In some embodiments, the distinct subset of
lectin molecules coated microbeads, can comprise a different
carbohydrate recognition domain from the others.
[0132] In some embodiments, the kit can comprise a separation
column, for e.g., a cylindrical hollow space. The cylindrical
hollow space can have an entry end for e.g., to inject the sample
and an exit end, for example to remove the sample depleted of CTCs.
The said column can be prepackaged with a plurality of separation
beads, for example microbeads coated with one or more lectin
molecules. The microbeads for example can comprise of ferromagnetic
material coated with an anticorrosion (detailed above). The beads
are capable of being magnetized to capture a cell labeled with
lectin molecule attached to a magnetic bead. In some embodiments,
the kit further comprises fluorescent staining reagents and
antibodies for cancer cell markers.
[0133] In some embodiments of any aspects of the kits described
herein, the surface substrate (e.g., microbeads) or lectin molecule
attached to a surface (e.g., lectin molecule coated microbeads) can
further comprise a detection label. By way of example only,
depending on the choice of detection methods, each distinct subset
of the microbeads can comprise a unique detection label or the same
detection label. For example, if each distinct subset of the lectin
molecule coated microbeads is used in a different sampling well,
the same detection label can be used on the lectin molecule coated
microbeads. Detectable labels suitable for use in any kits provided
herein include any composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical or
chemical means. Any art-recognized detectable labels or the ones
described herein can be included in the kits described herein.
Means of detecting such labels are well known to those of skill in
the art and exemplary detection methods are described herein. For
example, radiolabels can be detected using photographic film or
scintillation counters, fluorescent markers can be detected using a
photo-detector to detect emitted light. Enzymatic labels are
typically detected by pro- viding the enzyme with an enzyme
substrate and detecting the reaction product produced by the action
of the enzyme on the enzyme substrate, and calorimetric labels can
be detected by visualizing the colored label.
[0134] In some embodiments of any aspects described herein, the
kits can further comprise one or more containers containing a
population of detectable labels, wherein the detectable label is
conjugated to a molecule. In some embodiments, at least one of the
containers can contain a distinct population of detectable labels.
The molecule conjugated to a detectable label can be any molecule
that binds to a CTC. For example, in some embodiments, the molecule
conjugated to a detectable label can comprise the same carbohydrate
recognition domains as used in the lectin molecule coated
microbeads (e.g., lectin molecule coated magnetic microbeads). In
such embodiments, at least one population of the
molecule-detectable label conjugate can comprise at least one
carbohydrate recognition domain or a fragment thereof, e.g.,
derived from mannose-binding lectin or at least a portion of the
CRD domain, e.g., encoded by SEQ ID NO. 3, or a fragment thereof In
some embodiments, the molecule conjugated to a detectable label can
further comprise a Fc region of an immunoglobulin. In alternative
embodiments, the molecule conjugated to a detectable label can
comprise an antibody specific to an additional known marker of CTC
for e.g., EpCAM, antibody specific to targets recognized by the
lectin molecules described herein for e.g., N-Acetylglucosamine, or
an antibody specific to at least one type of carbohydrate
recognition domain (e.g., C-type lectins vs. S-type lectins)
employed in the lectin molecules described herein. However, the
antibody can also be a common antibody that binds to all cancer
cells. Without limitations, a molecule attached to a detectable
label can also include any ligand targeting microbial cell surface
proteins or receptors, including carbohydrates, lipids, lectins,
aptamers, protein, peptides, nucleic acid, polynucleotides,
antibody or a portion thereof, an antibody-like molecule,
peptidomimetic, and any combinations thereof.
[0135] Depending on the configuration/combination of the
molecule-detectable label conjugates provided in the kit, different
populations of the microbeads or magnetic microbeads can be mixed
together with a test sample in a single reaction, or different
populations each can be applied separately to different aliquots of
the same test sample. After contacting the test sample with the
lectin molecule coated microbeads or lectin molecule coated
magnetic microbeads, any cancer cell and/or CTC recognized by the
lectin molecules will bind to the microbeads or magnetic
microbeads. In some embodiments where the kits comprise lectin
molecule coated microbeads, the kits can further comprise a magnet
adapted for use with the assay for isolation of the CTC bound to
from a test sample. For example, if the assay is carried out in a
blood collection tube, the magnet can be adapted for use with the
blood collection tube, e.g., a magnet can be designed to be a
magnet collar surrounding the blood collection tube to immobilize
or isolate the CTC capturing magnetic microbeads from a test sample
or an assay buffer. In any aspects of the kits provided herein, the
kits can further comprise a portable readout machine or device,
e.g., to determine and display the signal produced from the assay
performed with the kit. For example, the readout machine or device
can detect a colorimetric signal and/or a fluorescent signal
produced from the assay of CTC detection performed with the kits
described herein. In any aspects of the kits provided herein, when
the detection label includes an enzyme (e.g., horseradish
peroxidase, alkaline phosphatase and any others commonly used for
colorimetric detection), the kits can further comprise one or more
containers containing an enzyme substrate that produces a color
change in the presence of the enzyme. One of skill in the art can
readily recognize an appropriate enzyme substrate for any
art-recognized enzymes used for colorimetric detection. By way of
example only, an exemplary substrate for alkaline phosphatase can
include BLIP/NOT or PAPP (p-Nitro phenyl Phosphate, Disodium Salt);
an exemplary substrate for horseradish peroxidase can include
TOMB.]. In any aspects of the kits provided herein, the kits can
further comprise at least one microtiter plate, e.g., for
performing the reaction and the detection. In some embodiments, the
kits described herein can be used to screen a pharmaceutical
product (e.g., a drug, a therapeutic agent, or an imaging agent),
and/or a medical device (including, but not limited to, implantable
devices) for the presence or absence of cancer cells.
[0136] Preferably, kits of the present invention will also include
an instruction insert with instructions for performing the
isolation method or assays as described above. The instruction
insert may be in any human understandable format, including but not
limited to a written booklet, an instructional DVD, an audio
recording, and a printed link to an instructional website.
[0137] As used herein, a "subject", "patient", "individual" and
like terms are used interchangeably and refers to a vertebrate,
preferably a mammal, more preferably a primate, still more
preferably a human. Mammals include, without limitation, humans,
primates, rodents, wild or domesticated animals, including feral
animals, farm animals, sport animals, and pets. Primates include,
for example, chimpanzees, cynomologous monkeys, spider monkeys, and
macaques, e.g., Rhesus. Rodents include, for example, mice, rats,
woodchucks, ferrets, rabbits and hamsters. Domestic and game
animals include, for example, cows, horses, pigs, deer, bison,
buffalo, feline species, e.g., domestic cat, and canine species,
e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich,
and fish, e.g., trout, catfish and salmon. The terms, "individual,"
"patient" and "subject" are used interchangeably herein. A subject
can be male or female.
[0138] Preferably, the subject is a mammal. The mammal can be a
human, non-human primate, mouse, rat, dog, cat, horse, or cow, but
is not limited to these examples. Mammals other than humans can be
advantageously used as subjects that represent animal models of
conditions or disorders associated with cancer
[0139] A subject can be one who has been previously diagnosed with
or identified as suffering from or under medical supervision for a
cancer. The cancer can be metastatic or benign. A subject can be
one who is diagnosed and currently being treated for, or seeking
treatment, monitoring, adjustment or modification of an existing
therapeutic treatment, or is at a risk of developing such a
disorder. A subject can be one who has undergone chemotherapy or
radiation therapy.
[0140] In some embodiments, the biological fluid sample for
detection, capture and/or isolation of CTCs are from a patient
suffering from cancer. In some embodiments, the biological fluid
sample are from a subject suspected of cancer. In some embodiment,
the cancer patient is receiving or has been treated with cancer
treatment(s). In some embodiments, the CTCs are obtained from a
blood sample. In some embodiments, the CTCs are from body fluid.
The types of cancer for which the methods and compositions
disclosed herein can be used for diagnosis and prognosis and
therapeutics are not particularly limited. The cancer can be, for
example, lung cancer, esophageal cancer, bladder cancer, gastric
cancer, colon cancer, skin cancer, papillary thyroid carcinoma,
colorectal cancer, breast cancer, lymphoma, pancreatic cancer,
prostate cancer, ovarian cancer, pelvic cancer, and testicular
cancer.
[0141] The terms "disease", "disorder", or "condition" are used
interchangeably herein, refer to any alternation in state of the
body or of some of the organs, interrupting or disturbing the
performance of the functions and/or causing symptoms such as
discomfort, dysfunction, distress, or even death to the person
afflicted or those in contact with a person. A disease or disorder
can also be related to a distemper, ailing, ailment, malady,
disorder, sickness, illness, complaint, or affectation.
[0142] The term "in need thereof" when used in the context of a
therapeutic or prophylactic treatment, means having a disease,
being diagnosed with a disease, or being in need of preventing a
disease, e.g., for one at risk of developing the disease. Thus, a
subject in need thereof can be a subject in need of treating or
preventing a disease.
[0143] In accordance with the various embodiments, described
herein, a test sample or sample, including any biological fluid,
body fluid, which can be processed or preprocessed, that is
suspected of comprising a cancer cell and/or CTC can be subjected
to methods, assay, kits and system disclosed herein. The sample can
be aqueous or non-aqueous. Non-limiting example of biological
fluids include from a body fluid, such as whole blood, plasma, any
cell-containing blood fraction, cerebrospinal fluid, joint fluid,
urine, tears or feces. In some embodiments, the biological fluid
sample obtained from a subject, e.g., a mammalian subject such as a
human subject or a domestic pet such as a cat or dog, can contain
cells from the subject. In other embodiments, the biological fluid
sample can contain non-cellular biological material, such as
non-cellular fractions of blood, saliva, or urine, which can be
used to measure plasma/serum biomarker expression levels. The
biological fluid sample can be freshly collected from a subject or
a previously collected sample. In some embodiments, the biological
fluid sample used in the assays and/or methods described herein can
be collected from a subject no more than 24 hours, no more than 12
hours, no more than 6 hours, no more than 3 hours, no more than 2
hours, no more than 1 hour, no more than 30 mins or shorter. In
some embodiments, the biological fluid sample or any fluid sample
described herein can be treated with a chemical and/or biological
reagent described herein prior to use with the assays and/or
methods described herein. In some embodiments, at least one of the
chemical and/or biological reagents can be present in the sample
container before a fluid sample is added to the sample container.
For example, blood can be collected into a blood collection tube
such as VACUTAINER.RTM., which has already contained heparin.
Examples of the chemical and/or biological reagents can include,
with- out limitations, surfactants and detergents, salts, cell
lysing reagents, anticoagulants, degradative enzymes (e.g., pro-
teases, lipases, nucleases, collagenases, cellulases, amylases),
and solvents such as buffer solutions. In some embodiments, the
sample can be a fragmented tumor, a tumor cell suspension, or a
cell culture established from a patients sample, or the culture
supernatant, or a xenograft established from a patients tumor or a
tumor biopsy or tissue section comprising tumor.
[0144] Some embodiments of the technology described herein can be
defined according to any of the following numbered paragraphs:
[0145] 1. A method for capturing circulating tumor cells (CTCs)
from biological fluids of a subject, comprising; contacting the
biological fluid with a lectin molecule attached to a surface.
[0146] 2. The method of paragraph 1, wherein the surface is a bead,
hollow fiber, porous scaffold, particle, or well. [0147] 3. The
method of paragraph 1, wherein the surface is magnetic. [0148] 4.
The method of paragraph 1, further comprising isolation of the
captured CTCs. [0149] 5. The method of paragraph 4, wherein the
isolation comprises passing the biological fluid containing
captured CTCs through a microfluidic magnetic separation device.
[0150] 6. The method of paragraphs 1-5, wherein the CTCs express
mannan carbohydrates on their cell surface. [0151] 7. The method of
paragraphs 1-5, wherein the CTCs express carbohydrates containing
D-mannose and L-fucose onto its cell surface. [0152] 8. The method
of paragraphs 1-7, wherein the biological fluid is selected from a
body fluid, such as whole blood, plasma, any cell-containing blood
fraction, cerebrospinal fluid, bone marrow (e.g., before
transplantation), cell sample (e.g., before transplantation), joint
fluid, urine, tears or feces. [0153] 9. The method of paragraphs
1-8, wherein the lectin molecule contains a collectin carbohydrate
recognition domain (CRD). [0154] 10. The method of paragraphs 1-9,
wherein the lectin molecule is a Mannose binding lectin (MBL).
[0155] 11. The method of paragraph 1, wherein the lectin molecule
is a ficolin. [0156] 12. The method of paragraph 1, wherein the
lectin molecule is a dectin. [0157] 13. The method of paragraph
1-10, wherein the lectin molecule is a C-type lectin. [0158] 14.
The method of paragraph 1, wherein the lectin molecule is a
fucose-binding lectin. [0159] 15. The method of paragraph 14,
wherein the fucose-binding lectin is Hemopexin. [0160] 16. The
method of paragraphs 1-15, wherein the lectin molecule comprises of
carbohydrate recognition domain (CRD). [0161] 17. The method of
paragraph 16, wherein the CRD is that of a mannose binding lectin.
[0162] 18. The method of paragraph 1, wherein the lectin is an
S-type lectin. [0163] 19. The method of paragraph 18, wherein the
S-type lectin is Galectin. [0164] 20. The method of paragraphs
1-19, wherein the lectin molecule is an engineered molecule. [0165]
21. The method of paragraphs 1-20, wherein the engineered molecule
further comprises the Fc region of an immunoglobulin. [0166] 22.
The method of paragraph 21, wherein the engineered molecule is
FcMBL. [0167] 23. The method of paragraphs 1-22, wherein the lectin
molecule is of mammalian origin. [0168] 24. The method of paragraph
23, wherein the lectin molecule is of human origin. [0169] 25. The
method of paragraphs 1-24, wherein the lectin molecule comprises at
least 80% amino acid sequence identity to human lectin and retains
at least 80% of its biological ability. [0170] 26. A method of
analyzing a CTC captured from a sample by the method of paragraphs
1-5, wherein the analysis comprises cell culture, an immunochemical
analysis, morphological analysis, genomics analysis, metabolomics,
epigenomics analysis, transcriptomics analysis, proteomics
analysis, DNA mutation analysis, whole genome analysis, protein
and/or RNA expression level of a specific gene or a combination
thereof [0171] 27. A method of paragraph 26, wherein the analysis
is used to assess a risk of developing a metastatic tumor in a
patient carrying or having carried a tumor. [0172] 28. The method
of paragraph 26, wherein the sample comprises whole blood, body
fluid, any cell-containing blood fraction, a fragmented tumor,
biopsy, aspirate, a tumor cell suspension, or a cell culture
established from a patient's sample, or the culture supernatant or
a xenograft established from a patient's tumor. [0173] 29. A method
of detecting cancer in a subject, comprising, obtaining a
biological fluid or cell sample from the subject, contacting the
sample with lectin-coated magnetic beads, isolation of the magnetic
beads captured cells with a microfluidic magnetic separation device
and assaying captured cells for CTC markers. [0174] 30. A method of
paragraph 29, wherein the CTC markers are selected from GlcNAc,
EpCAM, EphB4, HER2, EGFR, MUC-1, or a combination thereof. [0175]
31. A method for monitoring or assessing the effectiveness of a
cancer treatment in a patient, comprising: [0176] (a) obtaining a
first sample of the patient prior to the cancer treatment and
establishing a baseline CTC count by isolating CTC using the method
of paragraphs 1-5 and enumerating a CTC count, wherein CTC count is
defined as the number of cells in the blood sample expressing
mannan on their surface; [0177] (b) obtaining a second sample of
the patient after the cancer treatment and determining a
post-treatment level of CTC count by isolating CTC from the sample
using the method of paragraphs 1-5 and enumerating a CTC count; and
[0178] (c) comparing the levels of post-treatment CTC count to the
baseline CTC count, and optionally obtaining additional samples at
different time intervals after the cancer treatment to determine a
time-series for post-treatment CTC counts, wherein if the
post-treatment CTC counts show a decreasing trend, the treatment is
said to be effective, whereas if the post-treatment CTC count shows
an increasing trend or stays at about the baseline level, the
treatment is said to be ineffective. [0179] 32. The method of
paragraph 31 further comprising conducting cellular or molecular
analysis on the isolated CTCs, wherein the cellular or molecular
analysis is selected from cell culture, immunochemical analysis,
morphological analysis, genomics analysis, metabolic analysis,
epigenomics analysis, transcriptomics analysis, proteomics
analysis, DNA mutation analysis, whole genome analysis, protein
and/or RNA expression level of a specific gene or a combination
thereof. [0180] 33. A method for determining a prognosis of a
patient suffering from cancer comprising: [0181] (a) obtaining a
blood sample from the patient; [0182] (b) isolating CTCs from the
blood sample by applying the method of paragraphs 1-5 to the blood
sample; [0183] (c) enumerating isolated CTC count, wherein CTCs are
defined as the cells that are positive for mannan expression;
[0184] (d) determining a prognosis for the patient based on the CTC
count. [0185] 34. A method for early detection of metastatic tumor
in a patient, comprising: [0186] (a) obtaining a blood sample from
the patient; [0187] (b) isolating CTCs from the blood sample by
applying the method of paragraphs 1-5 to the blood sample; [0188]
(c) enumerating isolated CTC count, wherein CTC is defined as
mannan-expressing cells; and [0189] (d) determining a diagnosis
based on the CTC count, wherein if the CTC count is above a
predetermined level, a likelihood of metastatic tumor is indicated.
[0190] 35. A kit for isolating and enriching CTCs in a blood
sample, comprising: [0191] a red blood cell (RBC) lysis reagent;
[0192] lectin-coated magnetic nanobeads; [0193] cell culture or a
nutrition medium; and [0194] an instruction insert having encoded
thereon a human readable description of the method of paragraphs
1-5. [0195] 36. The kit of paragraph 35, further comprising a
separation column, wherein said column comprising: [0196] a body
with an entry end and an exit end each having an opening disposed
thereon; and [0197] a cylindrical hollow space connecting the
openings at the entry end and the exit end to form a passage
channel, [0198] wherein said column is pre-packaged with a
plurality of spherical separation beads disposed in the passage
channel, said separation beads are comprised of a ferromagnetic
material coated with an anti-corrosion, and are capable of being
magnetized to capture a cell labeled with lectin coated magnetic
nanoparticles. [0199] 37. The kit of paragraph 35-36, further
comprising fluorescent staining reagents and antibodies for cancer
cell markers. [0200] 38. A composition comprising a CTC bound to a
lectin molecule. [0201] 39. The composition of paragraph 38,
further comprising the lectin molecule attached to a surface,
wherein the surface is magnetic bead. [0202] 40. A method for
generating cancer vaccine, comprising: [0203] (a) contacting a
sample containing CTCs from a cancer patient with lectin molecule
attached to surface; [0204] (b) isolating the captured CTCs; [0205]
(c) combining the isolated CTCs or a component thereof with an
adjuvant to generate a CTC-immunogen and [0206] (d) administering
the CTC-immunogen to a subject, thereby producing a cancer vaccine.
[0207] 41. A method for generating a patient-specific cancer
vaccine, comprising: [0208] (a) contacting a sample containing CTCs
from a patient with lectin molecule attached to surface; [0209] (b)
isolating the captured CTCs; [0210] (c) combining the isolated CTCs
or a component thereof with an adjuvant to generate a CTC-immunogen
and (d) administering the CTC-immunogen to the patient, thereby
producing a patient-specific cancer vaccine. [0211] 42. A method
for generating a patient-specific cancer vaccine, comprising:
[0212] (a) contacting a sample containing CTCs from a patient with
lectin molecule attached to surface; [0213] (b) isolating the
captured CTCs; [0214] (c) combining the isolated CTCs or a
component thereof with a scaffold to generate a CTC-immunogen and
(d) administering the CTC-immunogen to the patient, thereby
producing a patient-specific cancer vaccine. [0215] 43. The method
of paragraphs 40-41, wherein the captured CTCs are heat killed,
inactivated, neutralized, chemically fixed, lyophilized to generate
a CTC-immunogen prior to step (d) [0216] 44. The method of
paragraphs 42, wherein the scaffold comprises a biomaterial. [0217]
45. The method of paragraphs 44, wherein the biomaterial is
selected from the group consisting of glycosaminoglycan, silk,
fibrin, MATRIGEL.RTM., poly-ethyleneglycol (PEG), polyhydroxy ethyl
methacrylate, polyvinyl alcohol, polyacrylamide, poly (N-vinyl
pyrolidone), poly(lactic acid), poly glycolic acid (PGA), poly
lactic-co-glycolic acid (PLGA), poly e-carpolactone (PCL),
polyethylene oxide, poly propylene fumarate (PPF), poly acrylic
acid (PAA), polyhydroxybutyric acid, hydrolysed polyacrylonitrile,
polymethacrylic acid, polyethylene amine, esters of alginic acid;
pectinic acid; and alginate, fully or partially oxidized alginate,
hyaluronic acid, carboxy methyl cellulose, heparin, heparin
sulfate, chitosan, carboxymethyl chitosan, chitin, pullulan,
gellan, xanthan, collagen, gelatin, carboxymethyl starch,
carboxymethyl dextran, chondroitin sulfate, cationic guar, cationic
starch, and combinations thereof. [0218] 46. The method of
paragraph 44, wherein the biomaterial is selected from the group
consisting of poly(L-lactide-co-glycolide) acid (PLGA), mesoporous
silica, and cryogel IP, and combinations thereof. [0219] 47. The
method of paragraphs 42-46, wherein the scaffold is capable of
localizing to antigen-presenting cells (APCs) in the subject, and
activating the APCs to produce high titer antibodies against the
pathogen. [0220] 48. The method of paragraphs 42, wherein the
CTC-immunogen further comprises an adjuvant. [0221] 49. The method
of paragraphs 42, wherein the CTC-immunogen is implanted
subcutaneously. [0222] 50. The method of paragraphs 42, wherein the
lectin molecule is a MBL at least comprising the CRD. [0223] 51.
The method of paragraphs 42, wherein the lectin molecule comprises
an antibody Fc domain (FcMBL). [0224] 52. The method of paragraphs
42, wherein the lectin molecule is attached to a magnetic surface.
[0225] 53. A composition comprising a CRD region of a lectin linked
to an anticancer therapeutic molecule. [0226] 54. A composition
comprising an mRNA encoding a CRD region of a lectin linked to
anticancer therapeutic molecule. [0227] 55. A composition
comprising a CRD region of a lectin linked to an imaging agent.
[0228] 56. A composition comprising an mRNA encoding a CRD region
of a lectin linked to an imaging agent. [0229] 57. A method of
treating cancer, the method comprising, administering to a subject,
the composition of paragraphs 53-56. [0230] 58. A method for
visualization of cancer, the method comprising administering to a
subject, the composition of paragraphs 53-56 and imaging cancer.
[0231] 59. The method of paragraph 31 or 32, wherein the CTC is
confirmed with a tumor-specific marker (e.g., GlcNAc or EpCam).
EXAMPLES
[0232] The following examples illustrate some embodiments and
aspects of the invention. It will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be performed without altering the
spirit or scope of the invention as defined in the claims which
follow. The technology described herein is further illustrated by
the following examples which is no way should be construed as being
further limiting.
Example 1
Binding of FcMBL-Magnetic Beads to Tumorigenic Human Breast Cancer
cells
[0233] Specific interaction of FcMBL only with tumorigenic human
breast cancer cells (MCF7), not with non-tumorigenic breast cells
(MCF10A) was validated. MCF7 and MCF10A cells (10.sup.4 cells/1 mL)
were incubated with 25 .mu.g/ml of 128 nm (or 1 um) FcMBL coated
magnetic nanoparticles blocked with PEG (polyethylene
glycol)-biotin in TBS (Tries buffered saline) with 2 mM calcium for
20 min. The sample was flowed through the microfluidic channels
disclosed in Kang, J H. et al. (2012), and the drag velocity was
measured of the cells driven by the magnetic flux density gradients
as described in Kang, J H. et al. (2012). The magnetic drag
velocity of MCF7 was determined about 40 .mu.m/sec and 120
.mu.m/sec when they were bound to 128 nm and 1 um FcMBL magnetic
beads, respectively, while those of MCF10A had -0.95 .mu.m/sec and
2.3 .mu.m/sec of magnetic drag velocity when they were incubated
with FcMBL magnetic beads of 128 nm and 1 um sizes, respectively.
(FIG. 2).
Example 2
Quantifying CTC-Isolation Efficiency using Human Breast Tumor Cell
Line
[0234] To determine the quantitative efficiency of cell isolation
of the method, MCF7 cells (.about.10.sup.5 cells in total) stained
with calcein AM (green fluorescent when viable) and labeled with 1
um FcMBL magnetic beads were spiked either into 10 mL of buffer or
pig whole blood. The sample was flowed through the biospleen device
at each flow rate of 10 mL/h, 100 mL/h, 500 mL/h, 1 L/h, and 2 L/h,
and then isolation efficiency was measured by counting the cell
numbers from the inlet and the outlet. The spiked cells were
quantitated in buffer and blood using disposable hemocytometers as
described in Kang, J H. et al. (2012). The isolation efficiency of
the 1 um FcMBL magnetic bead captured MCF7 cells was above 95% at
flow rates from 10 mL/h.about.500 mL/h, and it decreased down to
.about.66% as the flow rates go up to 2.0 L/h.
Example 3
FcMBL Capturing CTCs in a Tumor Bearing Mice Model
[0235] Staining Breast Tumor Cells of Transgenic Mice with
FcMBL
[0236] Preferential binding of FcMBL to tumor cells, compared to
normal cells was examined, within tissues in vivo. Primary breast
tumors that spontaneously developed in FIB C3(1)-SV40 T-antigen
mammary tumor-bearing transgenic mice was used for these studies.
Mammary tissue samples were surgically removed from 22 and 20 week
old transgenic mice or wild type mice, and cryo-sectioned for
immunohistochemistry (IHC) staining with fluorescently-labeled
FcMBL. Epithelial tumor cells in mammary duct of transgenic mice
(22 week) (blue: DAPI) bound FcMBL (green) strongly, whereas wild
type epithelial cells did not show any evidence binding to FcMBL.
Fibrillar fluorescence staining in the wild type mammary duct is
most likely due to polysaccharide-rich glycosaminoglycan elements
of the extracellular matrix (ECM) (FIG. 3).
Example 4
Capturing Circulating Tumor Cells (4T1 Cells) in Blood Collected
from Tumor Bearing Mice Using FcMBL Magnetic Particles.
[0237] To test the use of FcMBL to capture tumor cells circulating
in blood of an animal, we implanted mouse 4T1 (mouse mammary
carcinoma, cherry red expressing) breast tumor cells in mice and
whole blood was obtained over time to assess the number of 4T1
cells that invaded into the bloodstream. Before validating the
binding of FcMBL to 4T1 cells in vivo, a binding affinity test was
carried out in vitro using 4T1 cells (10.sup.5 cells/mL) suspended
in saline with 5 mM calcium chloride and heparinized human whole
blood. The 4T1 cell concentrations before and after performing a
bead capture experiment were measured by a hemocytometer to
calculate the binding efficiency of FcMBL-coated particles to 4T1
cells. The experimental results show that 1 .mu.m FcMBL magnetic
particles capture 97.2% and 74.6% of 4T1 cells spiked in saline and
human whole blood, respectively, whereas control (uncoated)
magnetic particles did not exhibit any binding (FIG. 4).
[0238] 4T1 cells were implanted within mammary fat pads of mice.
Methods for implantation are described in ref (2) and whole blood
of the mice was collected after 10 days, 20 days and 30 days
post-implantation. Metastasis of the implanted breast tumor to the
lung was apparent after 20 days (FIG. 5) and the mice at 30 days
post-implantation showed difficulty in breathing due to
metastasized tumors in the lung.
[0239] At each time point (10, 20, 30 days post-implantation),
whole blood (0.5.about.1.0 mL) was drawn from mice into heparin
Vacutainer vials. The blood volume was measured to normalize the
number of CTCs per unit volume of blood. 4T1 cells were not
detected in blood collected at the 10 day time point; however, the
blood collected after 20 days contained about .about.102 4T1
cells/mL and the concentration increased up to about 10.sup.6
cells/mL at 30 days post-implantation. The blood drawn from mice
was mixed with Roche RBC lysis buffer (Roche 10202500) to remove
RBCs, and the pellets containing white blood cells and 4T1 cells
were collected by centrifugation at 300 RCF for 5 min. The pellets
were resuspended in 1 mL of saline with 5 mM calcium chloride and
15 .mu.L of 1 .mu.m FcMBL magnetic particles were then added and
incubated for 20 min in a rotating mixer. The cells captured by
FcMBL magnetic particles were removed by a magnetic separator and
their numbers were counted using a hemocytometer to assess the
binding efficiency of FcMBL magnetic particles to 4T1 cells. The
experimental results show that over 90% of 4T1 cells circulating in
mice blood were captured by FcMBL magnetic particles, which
corresponds to the in vitro results described above.
Example 5
FcMBL Binding Affinity to Various Human Cancer Cells
[0240] The binding ability of FcMBL to various human cancer cells,
including H1975 (human lung adenocarcinoma), A549 (adenocarcinomic
human alveolar basal epithelial cells), H358 (human
bronchioloalveolar carcinoma cells), and H727 (human bronchial
carcinoid cells) was tested in vitro. A binding test with H1975 was
carried out using the methods described above. The experimental
results (FIG. 7) show that 1.mu.m FcMBL magnetic particles could
capture 90.2% and 80.6% of H1975 cells spiked in saline and human
whole blood, respectively. Specific FcMBL binding was confirmed for
all tested cancer cell types (FIG. 8). Weak binding was observed
for MCF10a normal cells. These results demonstrate that the
technology disclosed herein can be used to detect, capture and
isolate CTCs from broad range of carcinomas.
REFERENCES
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Alix-Panabieres, C. & Pantel, K. (2013) Technologies for
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Sequence CWU 1
1
31248PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Met Ser Leu Phe Pro Ser Leu Pro Leu Leu Leu
Leu Ser Met Val Ala1 5 10 15Ala Ser Tyr Ser Glu Thr Val Thr Cys Glu
Asp Ala Gln Lys Thr Cys 20 25 30Pro Ala Val Ile Ala Cys Ser Ser Pro
Gly Ile Asn Gly Phe Pro Gly 35 40 45Lys Asp Gly Arg Asp Gly Thr Lys
Gly Glu Lys Gly Glu Pro Gly Gln 50 55 60Gly Leu Arg Gly Leu Gln Gly
Pro Pro Gly Lys Leu Gly Pro Pro Gly65 70 75 80Asn Pro Gly Pro Ser
Gly Ser Pro Gly Pro Lys Gly Gln Lys Gly Asp 85 90 95Pro Gly Lys Ser
Pro Asp Gly Asp Ser Ser Leu Ala Ala Ser Glu Arg 100 105 110Lys Ala
Leu Gln Thr Glu Met Ala Arg Ile Lys Lys Trp Leu Thr Phe 115 120
125Ser Leu Gly Lys Gln Val Gly Asn Lys Phe Phe Leu Thr Asn Gly Glu
130 135 140Ile Met Thr Phe Glu Lys Val Lys Ala Leu Cys Val Lys Phe
Gln Ala145 150 155 160Ser Val Ala Thr Pro Arg Asn Ala Ala Glu Asn
Gly Ala Ile Gln Asn 165 170 175Leu Ile Lys Glu Glu Ala Phe Leu Gly
Ile Thr Asp Glu Lys Thr Glu 180 185 190Gly Gln Phe Val Asp Leu Thr
Gly Asn Arg Leu Thr Tyr Thr Asn Trp 195 200 205Asn Glu Gly Glu Pro
Asn Asn Ala Gly Ser Asp Glu Asp Cys Val Leu 210 215 220Leu Leu Lys
Asn Gly Gln Trp Asn Asp Val Pro Cys Ser Thr Ser His225 230 235
240Leu Ala Val Cys Glu Phe Pro Ile 2452228PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Glu Thr Val Thr Cys Glu Asp Ala Gln Lys Thr Cys Pro Ala Val Ile1 5
10 15Ala Cys Ser Ser Pro Gly Ile Asn Gly Phe Pro Gly Lys Asp Gly
Arg 20 25 30Asp Gly Thr Lys Gly Glu Lys Gly Glu Pro Gly Gln Gly Leu
Arg Gly 35 40 45Leu Gln Gly Pro Pro Gly Lys Leu Gly Pro Pro Gly Asn
Pro Gly Pro 50 55 60Ser Gly Ser Pro Gly Pro Lys Gly Gln Lys Gly Asp
Pro Gly Lys Ser65 70 75 80Pro Asp Gly Asp Ser Ser Leu Ala Ala Ser
Glu Arg Lys Ala Leu Gln 85 90 95Thr Glu Met Ala Arg Ile Lys Lys Trp
Leu Thr Phe Ser Leu Gly Lys 100 105 110Gln Val Gly Asn Lys Phe Phe
Leu Thr Asn Gly Glu Ile Met Thr Phe 115 120 125Glu Lys Val Lys Ala
Leu Cys Val Lys Phe Gln Ala Ser Val Ala Thr 130 135 140Pro Arg Asn
Ala Ala Glu Asn Gly Ala Ile Gln Asn Leu Ile Lys Glu145 150 155
160Glu Ala Phe Leu Gly Ile Thr Asp Glu Lys Thr Glu Gly Gln Phe Val
165 170 175Asp Leu Thr Gly Asn Arg Leu Thr Tyr Thr Asn Trp Asn Glu
Gly Glu 180 185 190Pro Asn Asn Ala Gly Ser Asp Glu Asp Cys Val Leu
Leu Leu Lys Asn 195 200 205Gly Gln Trp Asn Asp Val Pro Cys Ser Thr
Ser His Leu Ala Val Cys 210 215 220Glu Phe Pro
Ile2253115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 3Val Gly Asn Lys Phe Phe Leu Thr Asn Gly Glu
Ile Met Thr Phe Glu1 5 10 15Lys Val Lys Ala Leu Cys Val Lys Phe Gln
Ala Ser Val Ala Thr Pro 20 25 30Arg Asn Ala Ala Glu Asn Gly Ala Ile
Gln Asn Leu Ile Lys Glu Glu 35 40 45Ala Phe Leu Gly Ile Thr Asp Glu
Lys Thr Glu Gly Gln Phe Val Asp 50 55 60Leu Thr Gly Asn Arg Leu Thr
Tyr Thr Asn Trp Asn Glu Gly Glu Pro65 70 75 80Asn Asn Ala Gly Ser
Asp Glu Asp Cys Val Leu Leu Leu Lys Asn Gly 85 90 95Gln Trp Asn Asp
Val Pro Cys Ser Thr Ser His Leu Ala Val Cys Glu 100 105 110Phe Pro
Ile 115
* * * * *
References