U.S. patent application number 13/182003 was filed with the patent office on 2011-12-08 for tagged ligands for enrichment of rare analytes from a mixed sample.
This patent application is currently assigned to On-Q-Ity. Invention is credited to Helen Barnes, Allyn Forsyth.
Application Number | 20110300603 13/182003 |
Document ID | / |
Family ID | 40998689 |
Filed Date | 2011-12-08 |
United States Patent
Application |
20110300603 |
Kind Code |
A1 |
Forsyth; Allyn ; et
al. |
December 8, 2011 |
Tagged Ligands for Enrichment of Rare Analytes from a Mixed
Sample
Abstract
Method of enriching specific cells from cellular samples are
disclosed, comprising contacting in solution a cellular sample with
affinity-tagged ligands (ATLs) each comprising a first ligand
linked to an affinity tag, wherein the ligand selectively binds a
cellular marker of the rare cells and the affinity tag can be
selectively captured by a capture moiety, wherein the affinity tags
do not comprise a magnetic particle; and flowing the sample through
a microfluidic device comprising the capture moiety to selectively
retain ATL-bound cells. Methods for enriching circulating tumor
cells, and devices for enriching specific cells from cellular
samples are also disclosed.
Inventors: |
Forsyth; Allyn; (San Diego,
CA) ; Barnes; Helen; (San Diego, CA) |
Assignee: |
On-Q-Ity
Waltham
MA
|
Family ID: |
40998689 |
Appl. No.: |
13/182003 |
Filed: |
July 13, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12037077 |
Feb 25, 2008 |
8008032 |
|
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13182003 |
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Current U.S.
Class: |
435/188 ;
435/239; 435/243; 435/317.1; 435/371; 536/25.4 |
Current CPC
Class: |
B01L 3/502761 20130101;
G01N 2333/705 20130101; G01N 1/405 20130101; G01N 33/57492
20130101; G01N 33/54326 20130101; Y10T 436/25375 20150115; G01N
33/574 20130101; G01N 33/585 20130101; B01L 2300/0861 20130101;
Y10T 436/25125 20150115; Y10S 435/973 20130101; B01L 2400/086
20130101; Y10S 435/971 20130101; B01L 2200/0647 20130101; Y10T
436/13 20150115; Y10T 436/101666 20150115 |
Class at
Publication: |
435/188 ;
435/371; 435/243; 435/239; 435/317.1; 536/25.4 |
International
Class: |
C12N 5/09 20100101
C12N005/09; C07H 21/00 20060101 C07H021/00; C12N 9/96 20060101
C12N009/96; C12N 1/00 20060101 C12N001/00; C12N 7/02 20060101
C12N007/02 |
Claims
1. A method for enriching rare analytes from a sample comprising:
contacting in solution said sample with a plurality of
affinity-tagged ligands (ATLs) wherein said mixture of ATL's
comprises: a first ATL comprising a first ligand that selectively
binds a first marker of said rare analytes, wherein said first
ligand is linked to a first affinity tag that is selectively
captured by a first capture moiety; and a second ATL comprising a
second ligand that selectively binds a second marker of said rare
analytes, wherein said second ligand is linked to a second affinity
tag, wherein said second affinity tag is selectively captured by
said first capture moiety; and contacting said sample with said
capture moiety to selectively enrich said rare analytes.
2. The method of claim 11, wherein said first affinity tag and said
second affinity tag are identical.
3. The method of claim 11, wherein said mixture of ATL's comprises
at least 3, 4, 5, 6, 7, 8, 9, or 10 ATL's each of which comprises
an affinity tag that can be selectively captured by the first
capture moiety.
4. The method of claim 11, wherein said analytes are cells and said
mixture is a blood sample.
5. The method of claim 1, 2, or 3 wherein each of said ATL's
comprises a ratio of Ligand:Affinity Tag that is less than 1:5.
6. The method of claim 15, wherein said capture moiety is in a
microfluidic device.
7. The method of claim 16, wherein said microfluidic device
comprises an array of obstacles.
8. The method of claim 1, 2, or 3 wherein said affinity tag
comprises biotin, desthiobiotin, histidine, polyhistidine, myc,
hemagglutinin (HA), FLAG, fluorescence tag, tandem affinity
purification (TAP) tags, FLAG, glutathione S transferase (GST) or
derivatives thereof.
9. The method of claim 1, 2, or 3, wherein said ligand is linked to
said affinity tag via a linker comprising one or more of modified
dextran, polyethylene glycol, polypropylene glycol, polyvinyl
alcohol, and polyvinylpyrrolidone.
10. The method of claim 1 or 2 wherein the cellular marker is a
cancer marker for one or more of breast, prostate, liver, ovary,
skin, colon, rectum, cervix, esophagus, stomach, brain, lung, or
endometrium cancer.
11. The method of claim 1, 2, or 3, wherein the ligand selectively
binds epidermal growth factor receptor (EGFR) or folic acid
receptor.
12. The method of claim 1, 2, or 3, wherein the capture moiety
comprises avidin, streptavidin, Neutravidin.TM., nickel, or
glutathione.
Description
CROSS-REFERENCE
[0001] This application is a divisional of U.S. application Ser.
No. 12/037,077 filed Feb. 25, 2008, which application is
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Analysis of a complex mixture such as blood can be difficult
but provides valuable information. For example, CD4 T cell levels
can reflect the course of disease in AIDS patients, cellular
lymphoid and myeloid markers can aid leukemia and lymphoma
diagnoses, and the presence of errant epithelial cells in the blood
may signify a metastasizing cancer.
[0003] Such complex mixtures can be analyzed by fluorescent
activated cell sorting (FACS), a technology that can quantitate
marked analytes and also separate them out from the mixtures.
Another popular method of cell separation involves magnetically
labeling a target population of cells, e.g. with ferromagnetic
beads, followed by sorting the labeled cells by passing them
through a receptacle positioned within a magnetic field, a process
also known as magnetic activated cell sorting (MACS). Despite their
widespread use, FACS and MACS technologies have disadvantages, for
example, they require machinery that is expensive and difficult to
maintain. FACS techology has the added disadvantage of having
limited portability.
[0004] Given the heavy demand, new approaches and technologies for
cell sorting are needed for medical diagnostics and other
applications.
SUMMARY OF THE INVENTION
[0005] In one aspect, methods for enriching specific cells in a
cellular sample are provided. The methods comprise a method for
enriching rare cells from a cellular sample comprising contacting
in solution a cellular sample with affinity-tagged ligands (ATLs)
each comprising a first ligand linked to an affinity tag, wherein
the ligand selectively binds a cellular marker of the rare cells
and the affinity tag can be selectively captured by a capture
moiety, wherein the affinity tags do not comprise a magnetic
particle; and flowing the sample through a microfluidic device
comprising the capture moiety to selectively retain ATL-bound
cells.
[0006] In some embodiments, the method comprises a method for
enriching rare cells from a cellular sample comprising: contacting
in solution the cellular sample with affinity-tagged ligands (ATLs)
each comprising:
a ligand linked to a plurality of affinity tags, wherein the ligand
selectively binds a cellular marker of the rare cells and the
affinity tag can be selectively captured by a capture moiety and
wherein the ratio of ligand:affinity tag of the ATL's is less than
1:1; flowing the sample through a microfluidic device comprising
the capture moiety to selectively retain ATL-bound cells.
[0007] In one aspect, methods for enriching specific cells in a
cellular sample are provided. The methods comprise a method for
enriching rare cells, wherein the rare cells are circulating tumor
cells or epithelial cells, from a cellular sample comprising
contacting in solution a cellular sample with affinity-tagged
ligands (ATLs) each comprising a first ligand linked to an affinity
tag, wherein the ligand is folic acid, Dolichos Biflorus Agglutinin
(DBA), epidermal growth factor (EGF), EGF peptide (amino acid
sequence YHWYGYTPQNVI), epi peptide (amino acid sequence
QMARIPKRLARH), transforming growth factor-alpha (TGF-alpha),
Urokinase Plasminogen Activator (UPA), FasL, MUC1/sec, catenin,
ICAM-1, plasminogen, or the like and selectively binds a cellular
marker of the rare cells and the affinity tag can be selectively
captured by a capture moiety, wherein the affinity tags comprise
biotin, desthiobiotin, histidine, polyhistidine, myc, hemagglutinin
(HA), FLAG, fluorescence tag, tandem affinity purification (TAP)
tags, FLAG, or glutathione S transferase (GST) or derivatives
thereof; and flowing the sample through a microfluidic device
comprising the capture moiety to selectively retain ATL-bound
cells; wherein the capture moiety comprises avidin, streptavidin,
Neutravidin.TM., nickel, or glutathione or other molecule capable
of binding the affinity tag; and wherein the cellular marker is a
cancer marker for one or more of breast, prostate, liver, ovary,
skin, colon, rectum, cervix, esophagus, stomach, brain, lung, or
endometrium cancer.
[0008] In some embodiments, the method comprises a method for
enriching rare cells from a cellular sample comprising: contacting
in solution the cellular sample with affinity-tagged ligands (ATLs)
each comprising:
a ligand linked to a plurality of affinity tags, wherein the ligand
is folic acid or epidermal growth factor (EGF) and selectively
binds a cellular marker of the rare cells wherein the rare cells
are circulating tumor cells or epithelial cells, and the affinity
tag and wherein the ratio of ligand:affinity tag of the ATL's is
less than 1:1; flowing the sample through a microfluidic device
comprising the capture moiety to selectively retain ATL-bound
cells.
[0009] In one aspect, methods for enriching specific cells in a
cellular sample are provided. The methods comprise a method for
enriching rare cells, wherein the rare cells are circulating tumor
cells or epithelial cells, from a cellular sample comprising
contacting in solution a cellular sample with affinity-tagged
ligands (ATLs) each comprising a first ligand linked to an affinity
tag, wherein the ligand is an antibody that is anti-EpCam;
anti-E-Cadherin, anti-Mucin-1, anti-Cytokeratin (CK) 8,
anti-epidermal growth factor receptor (EGFR), anti-cytokeratin
(CK)19, anti-ErbB2, anti-PDGF, anti-L6, or anti-leukocyte
associated receptor (LAR) and selectively binds a cellular marker
of the rare cells and the affinity tag can be selectively captured
by a capture moiety, wherein the affinity tags comprise biotin,
desthiobiotin, histidine, polyhistidine, myc, hemagglutinin (HA),
FLAG, fluorescence tag, tandem affinity purification (TAP) tags,
FLAG, or glutathione S transferase (GST) or derivatives thereof;
and flowing the sample through a microfluidic device comprising the
capture moiety to selectively retain ATL-bound cells; wherein the
capture moiety comprises avidin, streptavidin, Neutravidin.TM.,
nickel, or glutathione or other molecule capable of binding the
affinity tag; and wherein the cellular marker is a cancer marker
for one or more of breast, prostate, liver, ovary, skin, colon,
rectum, cervix, esophagus, stomach, brain, lung, or endometrium
cancer.
[0010] In some embodiments, the method comprises a method for
enriching rare cells from a cellular sample comprising: contacting
in solution the cellular sample with affinity-tagged ligands (ATLs)
each comprising:
a ligand linked to a plurality of affinity tags, wherein the ligand
is an antibody that is anti-EpCam; anti-E-Cadherin, anti-Mucin-1,
anti-Cytokeratin (CK) 8, anti-epidermal growth factor receptor
(EGFR), anti-cytokeratin (CK)19, anti-ErbB2, anti-PDGF, anti-L6, or
anti-leukocyte associated receptor (LAR) and selectively binds a
cellular marker of the rare cells wherein the rare cells are
circulating tumor cells or epithelial cells, and the affinity tag
and wherein the ratio of ligand:affinity tag of the ATL's is less
than 1:1; flowing the sample through a microfluidic device
comprising the capture moiety to selectively retain ATL-bound
cells.
[0011] In some embodiments, the method comprises a method for
enriching rare analytes from a sample comprising:
contacting in solution said sample with a plurality of
affinity-tagged ligands (ATLs) wherein said mixture of ATL's
comprises: a first ATL comprising a first ligand that selectively
binds a first marker of rare analytes, wherein the first ligand is
linked to a first affinity tag that is selectively captured by a
first capture moiety; and a second ATL comprising a second ligand
that selectively binds a second marker of rare analytes, wherein
said second ligand is linked to a second affinity tag, wherein the
second affinity tag is selectively captured by the first capture
moiety; and contacting the sample with the capture moiety to
selectively enrich the rare analytes.
[0012] In some embodiments, the method comprises a method for
enriching rare analytes from a sample comprising:
contacting in solution said sample with a plurality of
affinity-tagged ligands (ATLs) wherein said mixture of ATL's
comprises: a first ATL comprising a first ligand that selectively
binds a first marker of rare analytes, wherein the first ligand is
linked to a first affinity tag that is selectively captured by a
first capture moiety; and a second ATL comprising a second ligand
that selectively binds a second marker of rare analytes, wherein
said second ligand is linked to a second affinity tag, wherein the
second affinity tag is selectively captured by the first capture
moiety; contacting the sample with the capture moiety to
selectively enrich the rare analytes; wherein the first affinity
tag and said second affinity tag are identical; and wherein the
ligand is folic acid, (DBA), EGF; EGF peptide (amino acid sequence
YHWYGYTPQNVI), epi peptide (amino acid sequence QMARIPKRLARH),
TGF-alpha; Urokinase Plasminogen Activator (UPA), FasL, MUC1/sec,
catenin, ICAM-1, plasminogen, or the like, or wherein the ligand is
an antibody that is anti-EpCam; anti-E-Cadherin, anti-Mucin-1,
anti-Cytokeratin (CK) 8, anti-epidermal growth factor receptor
(EGFR), anti-cytokeratin (CK)19, anti-ErbB2, anti-PDGF, anti-L6, or
anti-leukocyte associated receptor (LAR).
[0013] In some embodiments, the method comprises a method for
enriching rare analytes such as cells from a sample such as a blood
sample comprising: contacting in solution said sample with a
plurality of affinity-tagged ligands (ATLs) wherein said mixture of
ATL's comprises: a first ATL comprising a first ligand that
selectively binds a first marker of rare analytes, wherein the
first ligand is linked to a first affinity tag that is selectively
captured by a first capture moiety; and a second ATL comprising a
second ligand that selectively binds a second marker of rare
analytes, wherein said second ligand is linked to a second affinity
tag, wherein the second affinity tag is selectively captured by the
first capture moiety; contacting the sample with the capture moiety
to selectively enrich the rare analytes; wherein the mixture of
ATL's comprises at least 3, 4, 5, 6, 7, 8, 9, or 10 ATL's each of
which comprises an affinity tag that can be selectively captured by
the first capture moiety;
[0014] In some embodiments, the method comprises a method for
enriching rare analytes such as cells from a sample such as a blood
sample comprising: contacting in solution said sample with a
plurality of affinity-tagged ligands (ATLs) wherein said mixture of
ATL's comprises: a first ATL comprising a first ligand that
selectively binds a first marker of rare analytes, wherein the
first ligand is linked to a first affinity tag that is selectively
captured by a first capture moiety; and a second ATL comprising a
second ligand that selectively binds a second marker of rare
analytes, wherein said second ligand is linked to a second affinity
tag, wherein the second affinity tag is selectively captured by the
first capture moiety; contacting the sample with the capture moiety
to selectively enrich the rare analytes; wherein the mixture of
ATL's comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ATL's
each of which comprises an affinity tag that can be selectively
captured by the first capture moiety and wherein each of the ATL's
comprises a ratio of Ligand:Affinity Tag that is less than 1:5; and
wherein the capture moiety is in a microfluidic device such as a
microfluidic device comprising an array of obstacles; wherein the
first or second ligand is folic acid, (DBA), EGF; EGF peptide
(amino acid sequence YHWYGYTPQNVI), epi peptide (amino acid
sequence QMARIPKRLARH), TGF-alpha; Urokinase Plasminogen Activator
(UPA), FasL, MUC1/sec, catenin, ICAM-1, plasminogen, or the like,
or wherein the ligand is an antibody that is anti-EpCam;
anti-E-Cadherin, anti-Mucin-1, anti-Cytokeratin (CK) 8,
anti-epidermal growth factor receptor (EGFR), anti-cytokeratin
(CK)19, anti-ErbB2, anti-PDGF, anti-L6, or anti-leukocyte
associated receptor (LAR); and wherein the affinity tag comprises
biotin, desthiobiotin, histidine, polyhistidine, myc, hemagglutinin
(HA), FLAG, fluorescence tag, tandem affinity purification (TAP)
tags, FLAG, or glutathione S transferase (GST) or derivatives
thereof; wherein the ligand is linked to the affinity tag either
directly or via a linker comprising modified dextran, polyethylene
glycol, polypropylene glycol, polyvinyl alcohol, or
polyvinylpyrrolidone; and wherein the capture moiety comprises
avidin, streptavidin, Neutravidin.TM., nickel, or glutathione or
other molecule capable of binding the affinity tag.
[0015] In some embodiments, the method comprises a method of
diagnosing a condition such as cancer in a patient comprising:
enriching rare cells from the patient through any of the methods
described herein; and evaluating, identifying, or quantitating the
ATL bound cells.
[0016] In some embodiments, the invention comprises a reagent
comprising a plurality of affinity tagged ligands each of which
comprises a ligand that selectively binds a different cancer
cellular marker linked to an identical affinity tag that is
selectively captured by a capture moiety wherein the ratio of
ligand:affinity tag per ATL is less than 1:1; wherein the reagent
comprises at least 1, 2, 3, 4, or 5 different ATL's.
[0017] In some embodiments, the device comprises a microfluidic
device comprising an array of obstacles, wherein the obstacles are
functionalized with capture moieties comprising EGF-peptide,
EPI-peptide, folate or DBA.
INCORPORATION BY REFERENCE
[0018] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, and patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1A is a fluorescent image illustrating the capture of
lung cancer cells with a non-specific affinity tagged ligand
(biotinylated anti-IgG).
[0020] FIG. 1B is a fluorescent image illustrating the capture of
lung cancer cells with biotinylated anti-EpCam antibody, an
antibody that is specific for an epithelial cell marker present on
lung cancer cells.
[0021] FIG. 2A is a diagram illustrating the capture of cells with
a ligand tagged with an affinity tag such as biotin, histidine, or
a magnetic particle (top panel), and the affinity-based capture of
such cells using a microfluidic chip coated with a capture moiety
capable of interacting with the affinity tag (bottom panel).
[0022] FIG. 2B is a fluorescent image illustrating the capture of
cells bound to Biotin-tagged EGF using a microfluidic device coated
with Avidin, a capture moiety capable of interacting with the
affinity tag.
[0023] FIG. 3 is a Table of exemplary markers contemplated
herein.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present disclosure provides compositions, methods and
devices for the detection and isolation of rare analytes from a
mixed sample. Rare analytes include cells and particles, such as
eukaryotic cells, prokaryotic cells, cellular organelles, cellular
fragments, viruses, nucleic acids, proteins, and protein complexes.
A mixed sample may include a fluid sample, e.g., water, air, bodily
fluids such as blood, urine, etc. A mixed sample may also include a
solid sample that is liquefied, e.g., a tumor biopsy, food solids,
etc.
[0025] Enrichment, detection, and isolation of rare analytes is
accomplished using affinity-tagged ligands (ATLs). An ATL may
comprise Formula I:
L.sub.x-AT.sub.n, (Formula I)
wherein: L is a ligand that selectively binds a marker for a rare
analyte; x is the number of ligands within each ATL; AT is an
affinity tag which can be selectively captured using a capture
moiety; and n is the number of ATs linked to the ligand.
[0026] L and AT may be linked together directly or indirectly (via
a linker). Linkers may be straight or branched. A linker may be a
peptide, polypeptide, protein, and the like. In some embodiments,
the linker moiety is a branched polymer. Such branched polymers
include modified dextran, polyethylene glycol, polypropylene
glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene
acrylamide and combinations thereof. In some embodiments, the
linker is branched and comprises multiple ATs. For example, in some
embodiments an ATL comprises at least 5, 10, 15, 20, 25, 30, 35,
40, 50, 60, 70, 80, 90, 100 or more ATs per L. When a single ligand
is linked to multiple AT's, the AT's can each be linked to the
ligand via a single linker or via a plurality of linkers (e.g., one
linker per AT).
[0027] Examples of ligands contemplated in the methods herein
include ligands that selectively bind cell surface marker(s) (e.g.,
receptors and adhesion molecules) of rare cells of interest. A
ligand can comprise molecule(s), peptide(s), polypeptides,
particles, and the like, and/or fragments thereof. The ligand can
be naturally occurring or artificially synthesized. Rare cells
include circulating tumor cells (CTCs), circulating epithelial
cells or circulating stem cells.
[0028] In some embodiments, a ligand comprises an antibody, such as
a monoclonal antibody or a polyclonal antibody. Examples of ligand
antibodies contemplated herein include antibodies that selectively
bind CTCs, circulating epithelial cells, or circulating stem cells.
In some embodiments, such antibodies selectively bind an antigen
such as EpCam; E-Cadherin, Mucin-1, Cytokeratin (CK) 8, epidermal
growth factor receptor (EGFR), cytokeratin (CK)19, ErbB2, PDGF, L6,
or leukocyte associated receptor (LAR). In certain embodiments, the
ligand may be an antibody to a marker listed in FIG. 3.
[0029] In other embodiments, a ligand is not an antibody. Examples
of non-antibody ligands include folic acid, Dolichos Biflorus
Agglutinin (DBA), epidermal growth factor (EGF), EGF peptide (amino
acid sequence YHWYGYTPQNVI), epi peptide (amino acid sequence
QMARIPKRLARH), transforming growth factor-alpha (TGF-alpha),
Urokinase Plasminogen Activator (UPA), FasL, MUC1/sec, catenin,
ICAM-1, plasminogen, or the like. In some embodiments, the ligand
activates a cellular process, and in other embodiments, the ligand
may not activate a cellular process.
[0030] An ATL may have a single ligand or multiple ligands (e.g.,
x>1, or x>2, 5, 10, 20, or more). When an ATL comprises
multiple ligands, each ligand may be identical to the other
ligands, different from the other ligands, or a combination
thereof.
[0031] Affinity tags (AT) can include molecules, peptides,
polypeptides, particles, and/or other substances capable of being
captured by a capture moiety or enrichment device. Examples of ATs
include biotin, desthiobiotin, histidine, poly-histidine,
glutathione S transferase (GST), myc, hemagglutinin (HA), FLAG,
fluorescence tag, tandem affinity purification (TAP) tags, or
derivatives thereof. The TAP tag may comprise calmodulin-binding
protein, TEV, and protein A or G subunit. In some embodiments, the
affinity tag comprises a magnetic particle. In other embodiments,
the affinity tag is a molecule or particle other than a magnetic
particle. In some instances, an affinity tag does not comprise an
antibody.
[0032] As described above, an ATL may comprise one AT, or, for
example, may comprise at least 2, 5, 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, or more ATs.
[0033] To detect rare analytes (or single analyte) in a fluid
sample, the sample is mixed with a homogenous or heterogeneous set
of ATLs. The sample is incubated with the ATLs for a sufficient
amount of time to enable binding of the ligands to the receptors or
rare analytes. The sample and ATLs are then applied to one or more
capture moieties that selectively bind the affinity tags, thereby
allowing enrichment of the bound rare analytes. The capture moiety
can be coupled to a solid support (e.g. a tube, a flat substrate,
or an array of obstacles).
[0034] To detect rare analytes in a solid or semi-solid sample, the
sample is first solubilized and then mixed with the mixture of ATLs
as described above. Once the sample is incubated with the ATLs for
a sufficient period of time to allow binding between the ligands
and the rare analyte markers, the sample is applied to one or more
capture moieties that selectively binds to the affinity tag.
[0035] Capture moieties may be inorganic molecules, organic
molecules, peptides, polypeptides, and/or antibodies, and the like,
or may include fragments of, or derivatives, of same. The capture
moieties are capable of binding to, or interacting with, an
affinity tag. Examples of capture moieties include, but are not
limited to, a magnet or magnetized substrate, avidin, streptavidin,
Neutravidin.TM., glutathione, nickel, calcium/calmodulin, or IgG.
Other examples of capture moieties include magnetic or magnetized
surfaces, avidin and avidin-coated particles.
[0036] In some instances, capture moieties are part of a
microfluidic device (e.g., a microfluidic gap or channel coated
with capture moieties). A microfluidic device contemplated herein
can comprise an array of obstacles that form a network of
microfluidic channels.
[0037] The affinity tags can bind irreversibly or reversibly to the
capture moiety(ies). In some instances, an affinity tag may be
releasable from the capture moiety. For example, desthio-biotin may
be used as an affinity tag with avidin as the capture moiety. The
bond between biotin and avidin can then be released by applying
desphio-biotin or biotin to the sample.
[0038] In one example, the ATL's herein can be used to enrich rare
analytes such as cells from a mixed sample such as blood. Such
method comprises the step of contacting in solution a test sample
with affinity-tagged ligands (ATLs). Each ATL comprises: at least
one ligand linked to at least one affinity tag. The ligand and
affinity tag are optionally liked via a linker comprising one or
more of modified dextran, polyethylene glycol, polypropylene
glycol, polyvinyl alcohol, or polyvinylpyrrolidone.
[0039] The ligands of the ATL's selectively bind a one or more
markers of the rare analytes. The affinity tags of the ATLs are
ones that can be selectively captured by a single capture moiety or
a plurality of capture moieties. Examples of capture moieties
contemplated for any of the embodiments herein include avidin,
streptavidin, Neutravidin.TM., nickel, or glutathione. The affinity
tags may be one that does not comprise a magnetic particle.
Examples of affinity tags include biotin, desthiobiotin, histidine,
polyhistidine, myc, hemagglutinin (HA), FLAG, fluorescence tag,
tandem affinity purification (TAP) tags, FLAG, glutathione S
transferase (GST) or derivatives thereof. After incubation for a
sufficient amount of time to allow the sample and ATL's to mix, the
mixed sample is contacted with one or more capture moieties or is
flowed through a microfluidic device comprising the one or more
capture moieties. This selectively retains ATL-bound analytes or
cells.
[0040] In some instances, each ATL comprises a ratio of ligand to
affinity tag such that there are more affinity tags for each
ligand. In some instances the ligand:affinity tag ratio is less
than 1:1 or less than 1:5 or less than 1:10 or less than 1:100.
Furthermore, a single ATL can have at least 2, 3, 4, 5, 6, 7, 8, 9
or 10 different ligands. Each of the ligands can be specific to a
different marker, such as a cancer marker.
[0041] Cellular cancer markers contemplated herein include markers
for a cancer such as breast, prostate, liver, ovary, skin, colon,
rectum, cervix, esophagus, stomach, brain, lung, pancreatic, or
endometrium cancer. In some instances a cellular marker is
epidermal growth factor receptor (EGFR), EPCAM, or folic acid
receptor. In some instances, the cellular marker is E-Cadherin,
Mucin-1, Cytokeratin (CK) 8, cytokeratin (CK)19, ErbB2, PDGF, L6,
or leukocyte associated receptor (LAR) In other instances it is any
marker listed in the table of FIG. 3.
[0042] To enrich rare analytes such as rare cells (epithelial cells
or CTC) found in bodily fluid (e.g., blood), the ligands in the
ATL's can be one or more of the following: folic acid, epidermal
growth factor (EGF), transforming growth factor-alpha (TGF-alpha),
Urokinase Plasminogen Activator (UPA), FasL, MUC1/sec, catenin,
ICAM-1, plasminogen, or fragments thereof. In some embodiments, the
ligands comprise haptens. In some instances, the ligand is an
antibody and in others it is not an antibody.
[0043] In some instances, a reagent used to enrich/detect rare
analytes comprises a plurality of affinity-tagged ligands (ATLs)
wherein each ATL comprises: a first ATL comprising a first ligand
that selectively binds a first marker of the rare analytes linked
to a first affinity tag that is selectively captured by a first
capture moiety; and a second ATL comprising a second ligand that
selectively binds a second marker linked to a second affinity tag,
wherein the second affinity tag is selectively captured by the
first capture moiety. In such embodiments, the reagent described
above is mixed with a mixed sample of analytes. After allowing the
ATL's and sample to mix for a sufficient amount of time for binding
to occur, the sample/ATL mixture is contacted with the first
capture moiety to selectively enrich the rare analytes. The capture
moiety can be avidin and the capture moiety can be in a
microfluidic gap or channel. The first and second affinity tags can
be identical (both biotin) or different (biotin and desphio-biotin)
but both selectively captured by the same capture moiety. In some
instances, a mixed sample such as the one described above is
contacted with at least 3, 4, 5, 6, 7, 8, 9, or 10 different ATL's
each of which comprises an affinity tag that can be selectively
captured by a single capture moiety or the same affinity tag.
[0044] A sample, such as a blood sample, can be mixed with one or
more different ATLs. In certain embodiments, ATLs of similar
configuration may be contacted with the targeted analytes. In other
embodiments, a mixture of different ATLs may be used. In still
other embodiments, step-wise purification of the analyte may occur
through sequential use of the same or different ATLs.
[0045] An exemplary ATL can have a single ligand comprising
anti-EpCAM antibody linked to a plurality of ATs, each of which
comprises a biotin molecule. The ATs are linked to the ligand via a
branched linker such that each ligand is coupled to at least five
ATs. In some embodiments, the ligand may bind the marker (EpCAM) at
more than one site. In other embodiments, e.g., EGF, the ligand
binds the marker only once. Once the sample is mixed with the ATLs,
the sample flows through a microfluidic device, optionally
comprising an array of obstacles, coated with avidin capture
moieties. Enriched rare cells (e.g., circulating epithelial cells)
can then be visualized/detected using any means known in the art,
including fluorescent in situ hybridization (FISH) staining.
[0046] Some applications may require analysis downstream of the
capture of the ATL-bound cells. In some embodiments, the ATLs may
be conjugated to one or more detectable labels. Such detectable
labels comprise fluorescent markers, HRP-alkaline phosphatase
detection or quantum dots. In some embodiments, in addition to an
ATL, the cells may be contacted with a detectably-labeled ligand
specific for a cellular marker. Such detectable labels may comprise
one or more of fluorescent probes, magnetic particles, or quantum
dots. The detectably-labeled ligands comprise the ligands described
herein relating to ATLs. An example of a detectably-labeled ligand
is a fluorescently-labeled antibody.
[0047] Varying the number of affinity tags and/or ligands in a
single ATL may affect the ability to capture target analytes. Thus,
the disclosure provides ATLs with more than one affinity tag and/or
more than one ligand. The affinity tags may be identical tags,
similar tags, or different tags. Since higher
affinity-tag-to-ligand ratios may result in enhanced or more
efficient capture of cells, the ratio of affinity tag to ligand in
a single ATL may be greater than or equal to 1, for example, 2:1,
3:1, 4:1, 5:1, 10:1, 20:1, or greater.
[0048] In some embodiments, the marker is a marker for one or more
of the following: cancer of the breast, lung, prostate, liver,
ovary, skin, colon, rectum, cervix, esophagus, stomach, brain,
bladder, kidney, testicular, or endometrium. In some embodiments,
the marker is for a blood cancer such as leukemia or lymphoma.
[0049] In some embodiments, vascular disease is monitored by
enriching for cells with endothelial markers. Other cancers may
also be monitored by enriching for endothelial cells. In some
embodiments, fetal markers (e.g. Y chromosome proteins, gamma
globin) are used for capturing fetal cells in the maternal
circulation. In some embodiments, stem cell markers (CD44) are used
to capture stem cells.
[0050] In certain embodiments, the methods provided comprise
contacting a cellular sample with one or more ATLs to enable
binding to one or more cellular markers, followed by capture of the
bound cells by an enrichment device. The contacting of the cellular
sample with one or more ATLs may occur under conditions
pre-determined to maximize binding efficiency. In some embodiments,
the conditions may be optimized to minimize non-specific binding.
For example, the timing of the contacting between the cellular
sample and the one or more ATLs may be optimized. Such optimized or
predetermined amount of time may be used for the contacting step.
Likewise, conditions for the temperature of the contacting step may
be optimized to maximize binding efficiency and/or to minimize
internalization of cellular markers. A pre-determined or optimized
temperature or temperatures may be used for the contacting step. In
some embodiments, the composition of the solution containing the
cellular sample and/or the ATL may be optimized for more efficient
binding. Blocking reagents or serums may be added to aid in binding
efficiency. Examples of such blocking reagents include bovine serum
albumin (BSA), fetal calf serum (FCS), milk, and other
commercially-obtainable reagents.
[0051] The methods may further comprise a wash step in which
unbound (or free) ATL is removed from the cellular sample prior to
capture by the enrichment device. For example, the wash step could
be accomplished by one or more rounds comprising centrifugation of
the cellular sample, aspiration of the supernatant, and
re-suspension of the cellular sample in wash buffer. The
composition of the wash buffer may comprise buffered solutions and
may be optimized to maximize binding efficiency. The optimized wash
buffer may comprise one or more blocking reagents or serums
described herein.
[0052] In some embodiments, the cells are added to an enrichment
device that comprises a surface functionalized with a capture
moiety recognizing the affinity tag or a cellular marker, such as a
receptor. In certain embodiments, a surface of the enrichment
device is coated or treated with one or more capture moieties
(e.g., avidin or Neutravidin) in order to allow capture of the
ATL-bound cells. In certain embodiments, such capture-moieties are
not antibodies. In some embodiments, such capture moieties are
ligands. In some embodiments, the enrichment device comprises a
surface coated or functionalized with a ligand that is used to
directly enrich cells expressing a specific marker from a mixed
cell population. In some embodiments, the surface of such
enrichment device is functionalized with folate in order to enrich
cells expressing the folate receptor. In some embodiments, the
enrichment device is functionalized with Dolichos Biflorus
Agglutinin (DBA), epidermal growth factor (EGF), EGF peptide (amino
acid sequence YHWYGYTPQNVI), epi peptide (amino acid sequence
QMARIPKRLARH), transforming growth factor-alpha (TGF-alpha),
Urokinase Plasminogen Activator (UPA), FasL, MUC1/sec, catenin,
ICAM-1, plasminogen, or the like. Parameters such as timing,
temperature, and/or buffer composition may be optimized to promote
optimal capture by the enrichment device.
[0053] The enrichment device may include one or more arrays of
obstacles that allow lateral displacement of cells and other
components of fluids. Such arrays of obstacles comprise different
configurations. In one configuration, a row or rows of obstacles
are arranged with a 50% offset from other randomly- or
non-randomly-arranged obstacles creating restricted and expanded
gaps.
[0054] In some embodiments, the obstacles within the array of
obstacles are coated or treated with one or more capture moieties
or ligands. In some embodiments, the enrichment device is capable
of sorting cells based on another variable, e.g. hydrodynamic size,
as well as capturing ATL-bound cells. Enrichment devices include,
but are not limited to centrifuge, microfuge and ultracentrifuge
test tubes. In some embodiments, the enrichment device is a column
or other such receptacle such as a magnetic activated cell sorting
(MACS) column.
[0055] Any of the ATLs herein may be designed to enrich cancer
cells as well as prognose and diagnose cancer conditions. For
example, cancer can be staged or diagnosed by enriching rare cells
(e.g., epithelial cells, circulating cancer stem cells) using ATL's
by any of the means described above. Subsequently, the enriched
cells can be further evaluated or quantified. In one instance,
diagnosis or prognoses comprises comparing the percentage or
quantity of epithelial cells in the blood of a test subject with
that in the blood of a healthy control subject, a panel of healthy
control subjects, known standard levels, or similar information
obtained from the same patient at a different point in time.
[0056] Peptide Library
[0057] In some embodiments, the present disclosure provides a
method for screening a combinatorial peptide library for peptide
ligands capable of binding cell surface markers. Such methods
comprise contacting a sample of cancer cells with a combinatorial
peptide library, wherein the peptides have been pre-labeled with
one or more affinity tags, described herein; purifying out the
cells selectively binding to candidate peptide ligands; and
identifying the candidate ligands bound to the cells. In certain
embodiments, the purifying may comprise the lateral flow of the
cellular sample through a device coated with capture moieties that
selectively bind the ATs so as to permit capture of the ATL bound
cells
EXAMPLES
Example 1
Capture of H1650 Lung Cancer Cells with Biotinylated Anti-EpCam
Antibody
[0058] The example depicted in FIG. 1 illustrates a method and
device for isolating cancer cells. The affinity-tagged ligand (ATL)
in this example comprised biotin as the affinity tag. The ligand
was anti-Epithelial Cell Adhesion Molecule (EpCam) antibody which
recognizes an epithelial cellular marker. For the experiment, H1650
lung cancer cells were incubated with biotinylated goat anti-EpCam
antibody for 1 hour on ice (FIG. 1B). Approximately 1000 cells were
input into each experiment. The left and right images represent
replicate experiments. As a negative control, H1650 lung cancer
cells were incubated with biotinylated goat IgG antibody under the
same or similar conditions (FIG. 1A). Following the incubation, the
cells were washed and run through an array of obstacles [comprising
a restricted gap--Chip 7, please confirm] chip coated with
Neutravidin.TM.. Cells were then fixed and visualized by
fluorescence microscopy.
[0059] The results of this experiment demonstrated that it is
possible to use ATLs to isolate cancer cells. In this example,
between 20 and 30% of the total H1650 lung cancer cell population
were recovered when anti-EpCam antibody was used as the ligand. To
calculate the percentage (%), the # of cells captured was divided
by the total # of cells passed onto the chip, and the resulting
value was multiplied by 100. In contrast, only 7-8% of cells were
recovered when the goat IgG was used.
Example 2
Capture of Cells after Solution Phase Binding of the Cells with an
Affinity-Tagged Ligand
[0060] The examples depicted in FIG. 2 illustrate the capture of
cells labeled with an affinity tag before being subjected to a
microfluidic device. FIG. 2A is a diagram depicting a cell being
contacted with an affinity-tagged ligand in solution (top panel)
and the capture of such cell with a microfluidic device coated with
a capture moiety capable of recognizing the affinity-tag (lower
panel).
[0061] FIG. 2B illustrates the capture of cells bound to EGF
peptide tagged with biotin. In this example, MDA-MB-231 cells (a
breast cell line which expresses low levels of EpCam) were
contacted with biotin-tagged EGF peptide in solution under
conditions favorable for binding. The cells were then subjected to
a chip coated with neutravidin, a capture moiety capable of
recognizing the affinity tag. Following fixation, the captured
cells were visualized by fluorescent microscopy. The results
demonstrate significant capture of cells using this method.
* * * * *