Note: Descriptions are shown in the official language in which they were submitted.
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CAPTURE AGENTS AND RELATED METHODS AND SYSTEMS FOR DETECTING
AND/OR SORTING TARGETS
By Gabriel A. Kwong, Caius G. Radu, Antoni Ribas, Owen Witte, and James R.
Heath
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application
entitled "A unified
Platform for Multiplexed Cell Sorting and Detection of Genes and Proteins"
Serial No.
61/123,478, filed on April 9, 2008 Docket No. CIT-5127-P, herein incorporated
by reference in
its entirety. This application is also a continuation-in-part of the U.S.
Application entitled
"Methods and Systems for Detecting and/or Sorting Targets" Serial No.
11/888,502 filed on
August 1, 2007, Docket Number P017-US, which on its turn claims priority of
provisional
application entitled "A unified Platform for Multiplexed Cell Sorting and
Detection of Genes and
Proteins" Serial No. 60/834,823, filed on August 2, 2006 Docket No. CIT-4707,
and to U.S.
Provisional Application entitled "Digital DEAL: A quantitative and digital
Protein Detection
Immunoassay" serial No. 60/959,665 filed on July 16, 2007 Docket No. CIT-4944,
the
disclosures of which are incorporated herein by reference in their entirety.
This application might
also be related to U.S. Application entitled "Microfluidic Devices, Methods
and Systems for
Detecting Target Molecules" Serial No. 12/174,598 filed on July 16, 2008,
Docket Number
P235-US and of U.S. Application entitled "Arrays, Substrates, Devices, Methods
and Systems
for Detecting Target Molecules" Serial No. 12/174,601 filed on July 16, 2008,
Docket Number
P262-US, the disclosures of both of which are also incorporated herein by
reference in their
entirety.
STATEMENT OF GOVERNMENT GRANT
[0002] The U.S. Government has certain rights in this invention pursuant to
Grant No.
CA119347 awarded by the National Institute of Health.
TECHNICAL FIELD
[0003] The present disclosure relates to detection and/or sorting of one or
more targets, in
particular biomarkers, in a sample, such as a biological sample. More
specifically, it relates to
capture agents and related methods and systems for detecting and/or sorting
targets.
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BACKGROUND
[0004] High sensitivity detection of targets and in particular of biomarkers
has been a
challenge in the field of biological molecule analysis, in particular when
aimed at detection of a
plurality of targets and/or at detection of target of a certain dimension or
present in the sample at
a low concentration. Whether for pathological examination or for fundamental
biology studies,
several methods are commonly used for the detection of various classes of
biomaterials and
biomolecules.
[0005] Some of the techniques most commonly used in the laboratory for
detection of single
biological targets include gel electrophoresis, polyacrylamide gel
electrophoresis (PAGE),
western blots, fluorescent in situ hybridization (FISH), Florescent activated
cell sorting (FACS),
Polymerase chain reaction (PCR), and enzyme linked immunosorbent assay
(ELISA). These
methods have provided the ability to detect one or more biomarkers in
biological samples such
as tissues and are also suitable for diagnostic purposes.
[0006] Subsequent polynucleotide encoding approaches, developed by Applicants,
provided
improvements over previous techniques, and in particular, allowed performance
of a highly
sensitive and selective multiplex detection of targets.
SUMMARY
[0007] Provided herein are polynucleotide encoded capture agents and in
particular modular
capture agents and related arrays methods and systems that allow, in several
embodiments,
selective and sensitive detection of a vast series of targets, through a
flexible and versatile
modular molecular tool.
[0008] In particular, a modular polynucleotide-encoded capture agent herein
described
comprises a target binding component, a scaffold component and an encoding
component
formed by standardized molecular units that can be coupled and decoupled in a
controlled
fashion. Accordingly, in the modular capture agent here described, not only a
same scaffold can
be combined with different target binding structures, but also a same scaffold
can be combined
with a plurality of target binding structures, thus significantly improving
detection, sensitivity
and selectivity achievable by the capture agents.
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[0009] According to a first aspect, a modular polynucleotide-encoded capture
agent
configured for specific binding to a target is described. The modular
polynucleotide-encoded
capture agent comprises at least one binding molecule configured to
specifically bind to the
target, an encoding polynucleotide configured to specifically bind to a
substrate polynucleotide
attached to a substrate, and a scaffold molecule configured to attach the at
least one binding
molecule and the encoding polynucleotide with positionally distinguishable
scaffold binding
domains. In the scaffold molecule, the positionally distinguishable scaffold
binding domains are
arranged to allow, upon binding with the at least one binding molecule and
with the encoding
polynucleotide, presentation of the at least one binding molecule for specific
binding to the target
and of the encoding polynucleotide for specific binding to the substrate
polynucleotide.
[0010] According to a second aspect, a method and a system to detect a target
in a sample are
disclosed, the method and system based on the combined use of a substrate
polynucleotide
attached to a substrate, and a modular polynucleotide-encoded capture agent,
comprising a
scaffold molecule attaching at least one binding molecule and an encoding
polynucleotide. In the
modular polynucleotide-encoded capture agent the at least one binding molecule
is configured to
specifically bind to the target and the encoding polynucleotide is configured
to specifically bind
to the substrate polynucleotide attached to the substrate.
[0011] In the method, the modular polynucleotide-encoded capture agent and/or
the units
composing said modular polynucleotide-encoded capture agent, the sample and
the substrate
polynucleotide are contacted for a time and under conditions to allow binding
of the at least one
binding molecule with the target in a modular polynucleotide-encoded capture
agent-target
complex, and binding of the encoding polynucleotide with the substrate
polynucleotide, thus
providing a modular polynucleotide-encoded capture agent-target complex bound
to the substrate
polynucleotide. In the method, the modular polynucleotide-encoded capture
agent-target
complex bound to the substrate polynucleotide is then detected by way of
detecting techniques
which will be identifiable by a skilled person upon reading of the present
disclosure.
[0012] In the system, a substrate with a substrate polynucleotide attached to
the substrate is
provided, together with at least one binding molecule that is configured to
specifically bind to the
target, an encoding polynucleotide specifically binding to the substrate
polynucleotide attached
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to the substrate, and a scaffold molecule configured to attach the at least
one binding molecule
and the encoding polynucleotide with positionally distinguishable scaffold
binding domains. In
the scaffold molecule, the positionally distinguishable scaffold binding
domains are arranged to
allow, upon binding with the at least one binding molecule and with the
encoding
polynucleotide, presentation of the at least one binding molecule for specific
binding to the target
and of the encoding polynucleotide for specific binding to the substrate
polynucleotide.
[0013] According to a third aspect, a method and system for sorting targets of
a plurality of
targets are disclosed, the method and system based on the combined use of a
plurality of
substrate polynucleotides attached to a substrate and a plurality of modular
polynucleotide-
encoded capture agents. In some embodiments, the targets are cells and the
method and systems
are for sorting a plurality of cells.
[0014] In the method and system, each substrate polynucleotide is sequence-
specific and
positionally distinguishable from another. In the method and system, each
modular
polynucleotide-encoded capture agent is comprised of at least one binding
molecule configured
to specifically bind to a complementary target of the plurality of targets, an
encoding
polynucleotide configured to specifically bind to a substrate polynucleotide
of the plurality of
substrate polynucleotides, and a scaffold molecule configured to bind to the
at least one binding
molecule and the encoding polynucleotide with positionally distinguishable
scaffold binding
domains, the positionally distinguishable scaffold binding domains being
arranged on the
scaffold molecule to allow, upon binding with the at least one binding
molecule and with the
encoding polynucleotide, presentation of the at least one binding molecule for
specific binding to
the target and of the encoding polynucleotide for specific binding to the
substrate polynucleotide.
[0015] In the method, the plurality of modular polynucleotide-encoded capture
agents and/or
the units forming said plurality of modular polynucleotide-encoded capture
agents, are contacted
with the sample for a time and under conditions to allow binding of the at
least one binding
molecule with the targets, thus providing a plurality of modular
polynucleotide-encoded capture
agent-target complexes. In the method, the plurality of polynucleotide-encoded
capture agent-
target complexes is then contacted with the plurality of substrate
polynucleotides for a time and
under conditions to allow binding of the encoding polynucleotides to the
substrate
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polynucleotides attached to the substrate, thus sorting the plurality of
targets in a plurality of
polynucleotide-encoded capture agent-target complexes bound to the substrate.
[0016] In the system, a substrate with the plurality of substrate
polynucleotides attached to the
substrate is comprised, together with a plurality of binding molecules, each
binding molecule
specifically binding to a complementary target of the plurality of targets, a
plurality of encoding
polynucleotides, each encoding polynucleotide specifically binding to each
polynucleotide of the
plurality of substrate polynucleotides attached to the substrate, at least one
a scaffold molecule
configured to bind to the each binding molecule of the plurality of binding
molecules and each
encoding polynucleotide of the plurality of the encoding polynucleotides with
positionally
distinguishable scaffold binding domains, the positionally distinguishable
scaffold binding
domains being arranged on the scaffold molecule to allow, upon binding with
the at least one
binding molecule and with the encoding polynucleotide, presentation of the at
least one binding
molecule for specific binding to the target and of the encoding polynucleotide
for specific
binding to the substrate polynucleotide.
[0017] According to a fourth aspect, a scaffold molecule is described, the
scaffold molecule
comprising a first scaffold binding domain configured to attach at least one
binding molecule and
a second scaffold binding domain configured to attach an encoding
polynucleotide, wherein the
at least one binding molecule is configured to specifically bind to a target
and the encoding
polynucleotide is configured to specifically bind to a substrate
polynucleotide attached to a
substrate. In the scaffold molecule, the first scaffold binding domain and the
second scaffold
binding domain are positionally distinguishable and arranged in the scaffold
molecule to
minimize interaction of the at least one binding molecule and the encoding
polynucleotide, upon
binding of the at least one binding molecule with the first binding domain and
of the encoding
polynucleotide with the second binding domain.
[0018] According to a fifth aspect, a polynucleotide encoded capture agent is
described. The
polynucleotide encoded capture agent is comprised of a binding molecule that
specifically binds
to a target and of an encoding-polynucleotide attached to the binding
molecule. The encoding
polynucleotide is comprised of a sequence that specifically binds to a
substrate polynucleotide.
The substrate polynucleotide is attached to a substrate and is comprised of a
sequence that
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specifically binds to the encoding polynucleotide. In the polynucleotide
encoded capture agent,
the encoding polynucleotide comprises at least one restriction enzyme site
arranged in the
encoding polynucleotide to be presented for cleavage by a corresponding
restriction enzyme. In
several embodiments, the polynucleotide encoded capture agent can be a modular
polynucleotide
encoded capture agent herein described.
[0019] According to a sixth aspect, the substrate of each of the methods,
systems and arrays
disclosed herein is in operable association with a microfluidic component
comprising a
microfluidic feature for carrying a fluid. Accordingly, in the methods herein
described, at least
contacting the encoding-polynucleotide with the substrate polynucleotide can
be performed in
the fluid carried by the microfluidic feature. Additionally, each of the
systems herein disclosed
can further include the microfluidic component comprising the microfluidic
feature.
[0020] In modular polynucleotide-encoded capture agents, and related arrays
methods and
systems, herein described, the target binding structure is decoupled from a
scaffold structure,
thus allowing an improved flexibility and versatility of use if compared with
corresponding
instruments of the art.
[0021] Additionally, modular polynucleotide-encoded capture agents, and
related arrays
methods and systems herein described allow, in several embodiments, to attach
to a single
polynucleotide-encoded capture agent a plurality of binding molecules, in
particular proteins,
each binding a same target. Accordingly, the sensitivity and/or selectivity of
the resulting
modular polynucleotide encoded capture agent can be controlled and, in
particular, improved.
[0022] In particular, modular polynucleotide-encoded capture agents, and
related arrays,
methods and systems herein described allow, in several embodiments, an
improved selectivity
and sensitivity of target detection when compared to certain methods and
systems of the art.
[0023] More particularly, modular polynucleotide-encoded capture agents, and
related arrays,
methods and systems herein described allow, in several embodiments, to detect
and/or sort of
targets such as cells for which only low-affinity ligands exist, and targets
such as cells for which
ligands are present in very low abundance <<O. I % even from a complex
mixture.
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[0024] Modular polynucleotide-encoded capture agents, and related arrays,
methods and
systems herein described also allow, in several embodiments, to perform target
detection using a
robust platform which does not necessarily include antibodies.
[0025] In particular, modular polynucleotide-encoded capture agents, and
related arrays,
methods and systems herein described allow, in several embodiments, to
generate robust and
modular arrays for high efficiency target detection and/or sorting, which, in
view of their
stability are capable to significantly outperform literature approaches that
utilize surface-bound
proteins for target capture and detection.
[0026] Furthermore, modular polynucleotide-encoded capture agents, and related
arrays
methods and systems, in several embodiments allow selective release of the
polynucleotide
encoded capture agent-target complex, and therefore allow for the deployment
of a host of
bioanalytical methods on detected and/or sorted targets, with particular
reference to target cells.
[0027] The modular polynucleotide-encoded capture agents, and related arrays
methods and
systems herein described can be used in connection with performance of several
assays designed
to detect and/or sort targets, which include, but are not limited to,
monoparameter and
multiparameter assays such as genomic and proteomic assays, and other assays
identifiable by a
skilled person. In particular, the modularity of the platform herein described
allows performance
on captured target (and in particular of captured cell) of standardized assays
traditionally
performed on glass substrates, such as immunohistochemistry, FISH, and
additional assays
identifiable by a skilled person upon reading of the present disclosure.
[0028] The modular polynucleotide-encoded capture agents, and related arrays
methods and
systems herein described can be used in various fields including but not
limited to molecular
diagnostics, molecular therapeutics, fundamental biological studies, tissue
engineering, and
biomaterials,
[0029] The details of one or more embodiments of the disclosure are set forth
in the
accompanying drawings and the description and examples below. Other features,
objects, and
advantages will be apparent from the description, examples and drawings, and
from the claims.
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BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The accompanying drawings, which are incorporated into and constitute a
part of this
specification, illustrate one or more embodiments of the present disclosure
and, together with the
detailed description and the examples, serve to explain the principles and
implementations of the
disclosure.
[0031] Figure 1 shows a schematic illustration of a modular polynucleotide
encoded capture
agent, according to embodiments herein disclosed.
[0032] Figure 2 shows a schematic illustration of a modular polynucleotide
encoded capture
agent, according to embodiments herein disclosed.
[0033] Figure 3 shows a schematic illustration of modular polynucleotide
encoded capture
agents, methods and systems according to an embodiment herein disclosed.
[0034] Figure 4 shows a schematic illustration of modular polynucleotide
encoded capture
agents, methods and systems according to an embodiment herein disclosed.
[0035] Figure 5 shows results of cell sorting experiments performed using
modular
polynucleotide-encoded capture agents according to an embodiment herein
described. Panel A
shows a grayscale fluorescence image of an array of ssDNA-p/MHC tetramer
specific for
Tyrosinase TCR contacted with T cells. The image shows detection and cell
sorting of the T cells
on a substrate where substrate DNA complementary to the encoding
polynucleotide of the
ssDNA-p/MHC tetramers are attached. Panel B illustrates cell sorting using two
sets of ssDNA-
p/MHC tetramers each encoded with a ssDNA bindingly distinguishable from the
other. The
arrows indicate binding of each set after contact with target T cells.
[0036] Figure 6 shows results of cell sorting experiments performed using
modular
polynucleotide-encoded capture agents according to an embodiment herein
described. Panel A
shows a grayscale version of a fluorescence image of ssDNA-SA-p/MHC tetramer
arrays
specific for Tyrosinase TRC contacted with a complementary substrate
polynucleotide and T
cells at different frequencies as indicated. Panel B shows a diagram
illustrating a quantification
of the data shown in Panel A.
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[0037] Figure 7shows a representation of the structure of a scaffold Panel A)
and a
corresponding optimized scaffold (Panel B) according to an embodiment herein
described.
[0038] Figure 8 shows results of experiments exemplifying the binding capacity
of an
optimized polynucleotide encoded capture agent according to an embodiment
herein described.
In particular, Panel A shows a denaturing PAGE gel for a SAC protein detected
at various stages
of expression, refolding, and purification. The molecular weight of a SAC
monomer is -12kDa.
Panel B shows a gel mobility shift assay performed to verify the formation of
ssDNA-SAC
conjugates. Panel C shows a formula for determining a molar ratio of
association of biotin to SA
using the molecule 2-(4'-Hydroxyazobenzene) benzoic acid (HABA).
[0039] Figure 9 shows results of experiments exemplifying the capture
efficiency of modular
polynucleotide encoded capture agent according to an embodiment herein
described. Panel A
shows grayscale brightfield images of arrays of ssDNA encoded proteins
including a streptavidin
scaffold and p/MHC binder proteins specific for Human TCR transduced T cells
(left) and
Murine TCR transgenic T cells (right). Each sub-panel of Panel A, shows the
brightfield image
of an array after contact of the ssDNA-encoded protein with a complementary
substrate DNA
and with a specific cell as indicated. Panel B shows grayscale brightfield
images of arrays of
ssDNA-encoded proteins including an optimized streptavidin scaffold and p/MHC
binder
proteins specific for Human TCR transduced T cells (left) and Murine TCR
transgenic T cells
(right) (cells identical to top panel). Each sub-panel of Panel B shows the
brightfield image of an
array after contact of the SAC-ssDNA p/MHC with a complementary substrate DNA
and with
the specific cells as indicated.
[0040] Figure 10 shows a comparison of target capture performed with
conventional protein
arrays and with modular polynucleotide encoded capture agents according to an
embodiment
herein described. Panel A shows grayscale fluorescence images of arrays of
p/MHC comprised
in polynucleotide-encoded proteins further including an optimized SA scaffold
compared with
conventional direct p/MHC protein spotting strategies on various model
substrates as indicated.
Panel B shows a diagram illustrating a quantification of the data showed in
Panel A. Each data
point was derived from three representative spots.
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[0041] Figure 11 shows a comparison of target capture performed with
conventional protein
arrays and with modular polynucleotide encoded capture agent according to an
embodiment
herein described. Panel A shows a grayscale version of fluorescence images of
arrays of p/MHC
protein comprised in an ssDNA-encoded protein further including a SAC
scaffold. Each row
represents a separate experiment performed on a different slide. Panel B shows
a grayscale
version of fluorescence images of derivatized surfaces spotted with p/MHC
protein alone. Each
row represents a separate experiment performed on a different slide.
[0042] Figure 12 shows the results of cell sorting experiments performed using
modular
polynucleotide-encoded capture agents according to an embodiment herein
described. Panel A
shows grayscale fluorescence images of an array of three different substrate
polynucleotides
(indicated as A, B and C) following contact with p/MHC tetramers encoded with
an ss-DNA
complementary to polynucleotide A and with the target provided by Jurkata-Tyr
cells. Panel B
shows grayscale fluorescence images of arrays of different substrate
polynucleotides (indicated
as A, B and C) following contact with p/MHC tetramers specific for Mart-l-
specific TCR
encoded with ss-DNA complementary to polynucleotide A, with p/MHC tetramers
specific for
Tyrosinase-specific TCR encoded with ss-DNA complementary to polynucleotide B
and with the
targets provided by mixed population of Jurkata-MART-1 and Jurkata-Tyr cells
prestained with
lipophilic dyes (green and red respectively, illustrated in the grayscale
version as light gray and
dark gray). Panel C shows grayscale fluorescence images of arrays of
polynucleotide encoded
protein specific for Tyrosinase-specific TCR after contact with Jurkata-Tyr at
different serial
dilutions (50%, 10%, 1%, 0.1%) as indicated. Panel D shows a diagram
illustrating a
quantification of the data shown in Panel C.
[0043] Figure 13 shows detection of engineered cells performed using modular
polynucleotide
encoded capture agents according to an embodiment herein described. Panel A
shows a
schematic illustration of the experimental approach for transduced PMBC cells
with F5 MART-1
TCR and related diagrams showing transduction efficiency as determined by flow
cytometry.
Panel B shows a gray scaled brightfield image of transduced PBMCs sorted on a
microarray
containing the cognate capture protein (MART-126-35 /HLA-A2. 1) and the
control capture protein (CMV
pp65495-503 /HLA-A2.1) encoded to A and B respectively. Panel C shows an
overlay of confocal and
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brightfield images, verifying that cell capture illustrate in Panel B was
specific (MART-1 cells shown in
light gray).
[0044] Figure 14 shows diagrams illustrating detection specificity and
sensitivity of modular
polynucleotide-encoded capture agents according to an embodiment herein
described. Panel A
shows diagrams illustrating quantity and specificity of human CD8+ T cell that
are specific for
EBV BMLF1259-267, as determined by flow cytometry. Panel B shows diagrams
illustrating
quantity and specificity of human CD8+ T cell that are specific for CMV
pp65495-503, as
determined by flow cytometry.
[0045] Figure 15 shows detection of endogenous primary cells performed using
modular
polynucleotide encoded capture agents according to an embodiment herein
described. Panel A
shows images of arrays containing polynucleotide encoded p/MHC capture
proteins EBV
BMLF1259-267/HLA-A2.1 and CMV pp65495-503/HLA-A2.1 as indicated, following
contact with
the CD8+ T cells specific for EBV-BMLF-1 of Figure 13 (Patient NRA 13). The
left panel
shows an array localized with p/MHC capture proteins EBV BMLF1259-267/HLA-A2.1
and CMV
pp65495-503/HLA-A2.1 (to A and B cDNA spots respectively) after contact and
capture of patient
NRA 13 cells containing CD8+ T cells specific for EBV-BMLF-1 but not CMV-pp65
(independently verified by flow cytometry in Figure 13). The right two panels
are representative
gray-scaled fluorescence images of the arrays after staining of the cells with
fluorescent EBV
BMLF1259-267/HLA-A2.1 (blue, shown in the figure as white arrows) and CMV
pp65495-503/HLA-
A2.1 p/MHC tetramers (red shown in the grayscale version of the figure as
black arrows). Panel
B shows images of arrays of ss-DNA-SAC-p/MHC specific for EBV or CMV as
indicated,
following contact with a 1:1 mixture of the CD8+ T cells specific for EBV-BMLF-
1 shown in
Figure 13 (Patient NRA 13) and CD8+ T cells specific for CMV-pp65 (Patient
NRA11). The left
panel shows brightfield image immediately after cell capture, with T cells
localizing on spots A
and B. The right two panels are representative fluorescence images of the
arrays after staining of
the cells with fluorescent EBV (blue, shown in the figure as white arrow) and
CMV p/MHC
tetramers (red shown in the grayscale version of the figure as black arrow).
Panel C shows
diagrams illustrating quantity and specificity of mixtures of -0.4%, 0.2% and
0.1% human EBV-
specific T cell populations, as determined by flow cytometry. Panel D shows a
grayscale version
of fluorescence images of arrays of modular polynucleotide-encoded protein
specific for EBV
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following contact with the mixture of Panel C. Populations of EBV-specific T
cells are marked
with light gray arrows and non-specific cells are marked with black arrows
[0046] Figure 16 shows controlled release of targets captured using modular
polynucleotide
encoded capture agents according to an embodiment herein described. Panel A
shows a
schematic illustration of the experimental approach. Panel B shows grayscale
version of
fluorescence images Jurkata-MART-1 (red shown in the grayscale version as dark
gray) and Jurkata-
Tyr cells (green shown in the grayscale version as light gray) (i) captured on
p/MHC array (ii)
after treatment with BamHI (iii) after treatment with EcoRI and (iv) after
treatment with BamHI
and EcoRI.
[0047] Figure 17 shows fluorescently labeled ssDNA-p/MHC tetramers can be
utilized as
staining reagents for flow cytometry and can localize antigen-specific T cells
from suspension by
DNA hybridization. In panel a, Cy3-labeled A'-SAC conjugates were used to
generate
fluorescent tyrosinase368-376 ssDNA-p/MHC tetramers and used to detect Jurkat-
Tyr cells by
flow cytometry. Similar staining profiles were observed between Cy3 NACS p/MHC
tetramer
(lower panels) and conventional tyrosinase368-376(PE) p/MHC tetramers (upper
panels). In
panel b, Jurkata'-Tyr cells were stained with tyrosinase368-376-A' p/MHC
tetramers prior to
exposure to a DNA array. The Jurkata'-Tyr cells were localized to spot A by
DNA hybridization.
[0048] Figure 18 shows a schematic of a branched peptide. The reactive primary
amine is
highlighted by a "star" and the thiol reactive maleimide groups are
highlighted by a dashed box.
[0049] Figure 19 shows the detection of Jurkat-Tyr T cells using tyrosinase368-
376/HLA-A2.1
tetramers encoded to strand A' made from the optimized protein scaffolds SAC
(left panel) and
SAC3 (right panel).
[0050] Figure 20 shows the detection of cell surface receptors using immuno-
PCR. Panel A
shows the flow cytometry analysis of Jurkata-MART-1 and Jurkata-Tyr T cells
stained with
fluorescent MART-1 p/MHC tetramers encoded with ssDNA. Panel B shows flow
cytometry
analysis of CD glioma cells and Jurkat T cells stained with fluorescent anti-
EGFR antibodies
labeled encoded with ssDNA. The bottom panels illustrate the detection of the
cognate cell
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receptor (a-MART-I TCR, panel C; EGFR, panel D) by amplifying the encoded
ssDNA with
PCR.
[0051] Figure 21 shows a schematic illustration of the functional profiling of
TCR triggered
activation of capture antigen-specific cells using DNA-encoded p/MHC tetramers
and DNA-
encoded antibodies.
[0052] Figure 22 shows functional profiling of antigen specific T cells
captured and activated
on a glass substrate. Panel A contain the fluorescent images of individual
spots encoded with
three different cytokines at three different time points. Panel B shows
fluorescent magnifications
of one individual IFN-y cluster.
DETAILED DESCRIPTION
[0053] Polynucleotide encoded capture agents and in particular modular
polynucleotide encoded
capture agents and related arrays methods and systems are herein described,
which can be used
in combination with substrate polynucleotides to detect one or more targets in
a sample,
according to an approach herein also identified as NACS approach or
technology.
[0054] The wording "polynucleotide-encoded capture agent" refers to a
polynucleotide encoded
molecular construct that specifically binds to a target. In particular, a
polynucleotide-encoded
capture agent typically comprises a binding component that specifically binds
to, and is thereby
defined as complementary to, the target, a structural component that supports
the binding
component and an encoding polynucleotide attached to the structural component
that encodes the
molecular structure.
[0055] In a "modular polynucleotide-encoded capture agent" the binding
component, the
structural component and the encoding component of the polynucleotide encoded
capture agent
are formed by standardized molecular units that can be coupled or decoupled to
each other in a
controlled fashion. In particular, in the modular polynucleotide-encoded
capture agents herein
described, the binding component is formed by at least one binding molecule,
that is configured
to specifically bind to, and be thereby defined as complementary to, a target;
the encoding
component is formed by an encoding polynucleotide configured to specifically
bind, and be
thereby defined as complementary to, a substrate polynucleotide attached to a
substrate; and the
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structural component is formed by a scaffold molecule attaching the at least
one binding
molecule and the encoding polynucleotide. In particular, in the modular
polynucleotide-encoded
capture agents, the at least one binding molecule specifically binding to a
target, the scaffold
molecule and an encoding polynucleotide, are attached or to be attached one to
the other
according to the schematic illustration of Figure 1 or Figure 2 as will also
be further described
herein below.
[0056] The term "attach" or "attached" as used herein, refers to connecting or
uniting by a
bond, link, force or tie in order to keep two or more components together,
which encompasses
either direct or indirect attachment such as, embodiments where a first
molecule is directly
bound to a second molecule or material, and embodiments wherein one or more
intermediate
molecules are disposed between the first molecule and the second molecule or
material.
Molecules include but are not limited to polynucleotides, polypeptides, and in
particular proteins
and antibodies, polysaccharides, aptamers and small molecules.
[0057] The term "polynucleotide" as used herein indicates an organic polymer
composed of
two or more monomers including nucleotides, nucleosides or analogs thereof.
The term
"nucleotide" refers to any of several compounds that consist of a ribose or
deoxyribose sugar
joined to a purine or pyrimidine base, and to a phosphate group and that are
the basic structural
units of nucleic acids. The term "nucleoside" refers to a compound (as
guanosine or adenosine)
that consists of a purine or pyrimidine base combined with deoxyribose or
ribose and is found
especially in nucleic acids. The term "nucleotide analog" or "nucleoside
analog" refers
respectively to a nucleotide or nucleoside in which one or more individual
atoms have been
replaced with a different atom or a with a different functional group.
Accordingly, the term
polynucleotide includes nucleic acids of any length DNA RNA analogs and
fragments thereof. A
polynucleotide of three or more nucleotides is also called nucleotidic
oligomers or
oligonucleotide.
[0058] The term "polypeptide" as used herein indicates an organic polymer
composed of two
or more amino acid monomers and/or analogs thereof. The term "polypeptide"
includes amino
acid polymers of any length including full length proteins and peptides, as
well as analogs and
fragments thereof. A polypeptide of three or more amino acids is also called a
protein oligomer
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or oligopeptide. As used herein the term "amino acid", "amino acidic monomer",
or "amino acid
residue" refers to any of the twenty naturally occurring amino acids including
synthetic amino
acids with unnatural side chains and including both D an L optical isomers.
The term "amino
acid analog" refers to an amino acid in which one or more individual atoms
have been replaced,
either with a different atom, isotope, or with a different functional group
but is otherwise
identical to its natural amino acid analog.
[0059] The term "protein" as used herein indicates a polypeptide with a
particular secondary
and tertiary structure that can participate in, but not limited to,
interactions with other
biomolecules including other proteins, such as antibodies, DNA, RNA, lipids,
metabolites,
hormones, chemokines, and small molecules.
[0060] The term "antibody" as used herein refers to a protein that is produced
by activated B
cells after stimulation by an antigen and binds specifically to the antigen
promoting an immune
response in biological systems and that typically consists of four subunits
including two heavy
chains and two light chains. The term antibody includes natural and synthetic
antibodies,
including but not limited to monoclonal antibodies, polyclonal antibodies or
fragments thereof.
Exemplary antibodies include IgA, IgD, IgGl, IgG2, IgG3, IgM and the like.
Exemplary
fragments include Fab Fv, Fab' F(ab')2 and the like. A monoclonal antibody is
an antibody that
specifically binds to and is thereby defined as complementary to a single
particular spatial and
polar organization of another biomolecule which is termed an "epitope". A
polyclonal antibody
refers to a mixture of monoclonal antibodies with each monoclonal antibody
binding to a
different antigenic epitope. Antibodies can be prepared by techniques that are
well known in the
art, such as immunization of a host and collection of sera (polyclonal) or by
preparing continuous
hybridoma cell lines and collecting the secreted protein (monoclonal).
[0061] The term "polysaccharide" as used here indicates polymers formed by
monosaccharides units joined together by glycosidic bonds. Polysaccharides
include very large,
often branched, macromolecules, including polymers of any length, from a mono-
or di-
saccharide polymer to polymers including hundreds or thousands of
monosaccharides and that
can have a molecular weight from about 1000 Da to about 20KDa. Exemplary
polysaccharides
comprise glycogen, cellulose, starch, and chitin.
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[0062] The term "aptamers" as used here indicates oligonucleic acid or peptide
molecules that
bind a specific target. In particular, nucleic acid aptamers comprise nucleic
acid species that have
been engineered through repeated rounds of in vitro selection or equivalently,
SELEX
(systematic evolution of ligands by exponential enrichment) to bind to various
molecular targets
such as small molecules, proteins, nucleic acids, and even cells, tissues and
organisms. Aptamers
are useful in biotechnological and therapeutic applications as they offer
molecular recognition
properties that rival that of the antibodies. Peptide aptamers are proteins
that are designed to
interfere with other protein interactions inside cells. They consist of a
variable peptide loop
attached at both ends to a protein scaffold. This double structural constraint
greatly increases the
binding affinity of the peptide aptamer to levels comparable to an antibody's
(nanomolar range).
[0063] The term "small molecule" as used herein indicates an organic compound
that is not a
polymer and has a dimension from about 10 Da to 2000-3000 Da. Small molecules
comprise
molecule that are biologically active and molecules that do not have a
biological activity.
Exemplary small molecules comprise 4-[(4-methylpiperazin-1-yl)methyl]-N-[4-
methyl-3-[(4-
pyridin-3-ylpyrimidin-2-yl)amino]-phenyl]-benzamide (Gleevec(g),
Sulfosuccinimidyl-6-
(biotinamido) hexanoate, and Succinimidyl 6-hydrazinonicotinate acetone
hydrazone.
[0064] In the modular polynucleotide capture agents herein described, the at
least one binding
molecule specifically binds to a target, and the encoding polynucleotide
specifically binds to a
substrate polynucleotide attached to a substrate.
[0065] The wording "specific", "specifically", or specificity" as used herein
with reference to
the binding or attachment of a molecule to another refers to the recognition,
contact and
formation of a stable complex between the molecule and the another, together
with substantially
less to no recognition, contact and formation of a stable complex between each
of the molecule
and the another with other molecules. Exemplary specific bindings are antibody-
antigen
interaction, cellular receptor-ligand interactions, polynucleotide
hybridization, enzyme substrate
interactions etc. The term "specific" as used herein with reference to a
molecular component of a
complex, refers to the unique association of that component to the specific
complex which the
component is part of. The term "specific" as used herein with reference to a
sequence of a
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polynucleotide refers to the unique association of the sequence with a single
polynucleotide
which is the complementary sequence.
[0066] The wording "substrate polynucleotide" as used herein refers to a
polynucleotide that is
attached to a substrate so to maintain the ability to bind to its
complementary polynucleotide. A
substrate polynucleotide can be, in particular, comprised of a sequence that
specifically binds and
is thereby defined as complementary with an encoding-polynucleotide of a
polynucleotide
encoded protein.
[0067] The term "substrate" as used herein indicates an underlying support or
substratum.
Exemplary substrates include solid substrates, such as glass plates,
microtiter well plates,
magnetic beads, silicon wafers and additional substrates identifiable by a
skilled person upon
reading of the present disclosure.
[0068] In some embodiments, the encoding polynucleotide attached to the
scaffold component is
specific for the binding component. Those embodiments can be used to perform
assays that
exploit the binding component-target specific interaction to detect proteins,
cytokines,
chemokines, small molecules, DNA, RNA, lipids, etc., whenever a target is
known, and sensitive
detection of that target is required. In several embodiments, the binding
component and the
structural component are formed by a protein.
[0069] The term "detect" or "detection" as used herein indicates the
determination of the
existence, presence or fact of a target or signal in a limited portion of
space, including but not
limited to a sample, a reaction mixture, a molecular complex and a substrate.
A detection is
"quantitative" when it refers, relates to, or involves the measurement of
quantity or amount of
the target or signal (also referred as quantitation), which includes but is
not limited to any
analysis designed to determine the amounts or proportions of the target or
signal. A detection is
"qualitative" when it refers, relates to, or involves identification of a
quality or kind of the target
or signal in terms of relative abundance to another target or signal, which is
not quantified.
[0070] The term "target" as used herein indicates an analyte of interest. The
term "analyte"
refers to a substance, compound or component whose presence or absence in a
sample has to be
detected. Analytes include but are not limited to biomolecules and in
particular biomarkers. The
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term "biomolecule" as used herein indicates a substance compound or component
associated to a
biological environment including but not limited to sugars, aminoacids,
peptides proteins,
oligonucleotides, polynucleotides, polypeptides, organic molecules, haptens,
epitopes, biological
cells, parts of biological cells, vitamins, hormones and the like. The term
"biomarker" indicates a
biomolecule that is associated with a specific state of a biological
environment including but not
limited to a phase of cellular cycle, health and disease state. The presence,
absence, reduction,
upregulation of the biomarker is associated with and is indicative of a
particular state.
[0071] The term "sample" as used herein indicates a limited quantity of
something that is
indicative of a larger quantity of that something, including but not limited
to fluids from a
biological environment, specimen, cultures, tissues, commercial recombinant
proteins, synthetic
compounds or portions thereof.
[0072] In several embodiments, the modular polynucleotide-encoded molecule
comprises at
least one binding molecule, a scaffold molecule and an encoding
polynucleotide.
[0073] The term "binding molecule" or "affinity agent" as used herein
indicates a molecule that
is able to specifically bind to a target under appropriate conditions. The
binding molecule or
affinity agent is, in particular, able to specifically bind to at least one
target under appropriate
conditions, which includes for example binding to the target in a solution
(e.g. biologically
derived, or synthetic), on a cell surface, on artificial surfaces (e.g.
derivatized beads,
nanoparticles) or in other mixtures or surfaces identifiable by a skilled
person. Examples of
binding molecules include any molecule that exhibits a binding affinity for a
predetermined
target, including but not limited to protein binders such as antibodies,
lectins, Fc receptors, MHC
protein A/G, peptide aptamers etc, non-protein binders such as polynucleotides
(e.g. RNA c/o
DNA) nucleic acid aptamers, peptides, small molecules, and drugs such as
imatinib mesylate
(Gleevac(g), bungarotoxins, inhibitor for nicotinic acetylcholine receptors
etc,. In the modular
polynucleotide encoded capture agent, the affinity agent is attached to the
scaffold where the
binding can be performed directly or indirectly, e.g. through an intermediate
molecule (e.g. a
biotin molecule), through any covalent attachment scheme (readily identified
by a person in the
field) or through non-covalent schemes such as electrostatic, Fc-protein A/G
interaction, biotin
etc.
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[0074] Typically, a binding molecule used in the modular polynucleotide-
encoded protein herein
described, exhibits a binding affinity and binding selectivity to a target to
be detected that can be
measured with technologies known in the art, such as surface Plasmon resonance
(SPR), enzyme
linked immunosorbant assays (ELISAs), and additional techniques identifiable
by a skilled
person. The choice of a binding molecule for a modular polynucleotide capture
agent is made by
a skilled person in view of the target to be detected and the experimental
design. For example,
when the target is a cell, the binding molecule exhibits a binding affinity
and selectivity to
biomolecules, and in particular biomarkers, that are presented at the surface
of specific cell
types. Exemplary surface molecules include but are not limited to membrane
proteins, receptors,
glycoproteins, ion channels or major histocompatibility complexes and other
molecules
identifiable by a skilled person upon reading of the present disclosure.
[0075] A skilled person would also be able to identify an appropriate binding
molecule for a
certain target based on determination of the binding affinity and selectivity
exhibited by
candidate binding molecules towards the target and in view of the number of
binding molecules
that are attachable to the scaffold (see below).
[0076] In several embodiments, upon selection of the appropriate scaffold, it
is possible to
modulate the number of binding molecules to be attached to the scaffold to
achieve a desired
binding affinity and/or selectivity by controlling the valency of the capture
agent for the target
(i.e. the number of chemical bonds formed by a capture agent with the target).
[0077] For example, a binding molecule with low affinity (e.g., Kd > 10-6) for
a certain target
(e.g., a cell) will most likely require an increased number of molecules
attached to the scaffold to
ensure specific binding of the modular polynucleotide-encoded capture agent
that comprises
such a binding molecule.
[0078] Accordingly, in several embodiments, the binding affinity of the
capture agents for a
certain target can be controlled and, in particular, increased by providing
multiple copies of a
same or different binding molecule. In particular, multiple copies of a same
or different binding
molecule, if located appropriately on the scaffold, can lend the binding
molecule/scaffold
construct a significantly higher binding affinity to the cell type of
interest, than can the affinity
agents by themselves.
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[0079] In particular, multiple ligand capture agents can be assembled by
combining various
binding molecules (such as the ones listed above) to the same scaffold. This
may be
advantageous in certain situations when it is desirable to probe multiple
elements that are present
within a target sample (for example, multiple cell surface markers, endogenous
and exogenous
peptide/MHC from the same antigen presenting cell, etc).
[0080] In several embodiments, the binding molecule is a protein, such as
antibodies, lectins,
Fc receptors, MHC', protein A/G, and additional proteins identifiable by a
skilled person.
[0081] In particular, in some embodiments, the binding molecule is an
antibody. In particular,
in those embodiments, the antibody can be produced that specifically bind to a
desired target.
The target can be a biomarker to be detected in a mixture or on a cell surface
(see e.g. CD4,
CD8, and CD3). In the latter cases, the antibodies can also be used as binding
molecules of
modular polynucleotide encoded capture agent used to detect and/or sort cell
targets (see
Example 13).
[0082] In some embodiments, the protein can be an MHC complex. The term "MHC"
or
"p/MHC" as used herein indicates peptide major histocompatibility complex
molecules
(p/MHC), and, more particularly, heterotrimer protein binders that are the
cognate binders to T
cell receptors found on T cells. In particular, p/MHC complexes are hetero-
trimeric proteins
found on the cell surface of antigen presenting cells and comprise MHC class
I, MHC class II
and MHC class III proteins. MHC class I proteins contain a peptide, an a chain
and (32-micro-
globulin. APCs expressing MHC class I proteins present antigen fragments to
cytotoxic T-cells,
stabilized by the surface molecule CD8. MHC class II including heterodimeric
peptide-binding
proteins and proteins that modulate antigen loading onto MHC class II proteins
in the lysosomal
compartment such as MHC II DM, MHC II DQ, MHC II DR, and MHC II DP. On antigen-
presenting cells, MHC class II proteins contain a & (3 chains and they present
antigen fragments
to T-helper cells by binding to the TCR and the CD4 receptor on the T-helper
cells. MHC class
III comprise other immune components, such as complement components (e.g., C2,
C4, factor B)
and some that encode cytokines (e.g., TNF-a) and also hsp.
[0083] A typical receptor/target for MHC is the T cell receptor found on T
cells that can be
specific for various antigens (e.g. MART-1, Cytomegalo virus, Tyrosinase etc)
as presented in
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the MHC complexes. The appropriate MHC for a specific target can be identified
by a skilled
person based on the target of interest in view of the binding specificity MHC
candidate
molecules.
[0084] In some embodiments, MHC monomers provide the binding molecule of the
modular
polynucleotide-encoded capture agent herein described. In those embodiments,
the targets are
preferably provided by molecular biomarker or other molecules in a state or
form when they are
not comprised within a cell to be detected. In some embodiments, MHC dimers
and trimers can
be utilized to detect antigen-specific T cells in solution via flow cytometry.
In some
embodiments, MHC dimers and trimers with higher affinity interactions (e.g.
TCR-p/MHC), are
expected to detect antigen specific T cells also in a complex environment, as
exemplified in
Example 5 and Figure 9a (lower left panel) where the Jurkat cells expressing a
TCR against
tyrosinase antigen were sorted with a suboptimal capture agent (see also the
mix of dimers and
trimers, of Figure 8) In some embodiments, MHC tetramers are used in
connection with a
polynucleotide encoded scaffold and can be advantageously used in applications
directed to cell
detection and/or sorting. (e.g. see Examples 9 and 10 and Figures 5, 6, 9, 10,
11, 12, and 13-14,
15,16,19, 22.)
[0085] In particular, in some embodiments, a tetramer of an antigen-presenting
MHC provides
the binding molecule of a modular polynucleotide encoded capture agent herein
described. MHC
tetramers exhibit a substantially higher affinity for a T-cell of interest
than MHC monomers or
dimers do, and are now well-established reagents for the detection of antigen-
specific T cells by
flow cytometry. When coupled with a scaffold in the modular capture agents
herein disclosed an
MHC tetramer allows detection and/or sorting of target with an increased
sensitivity and
specificity when compared with some methods known in the art.
[0086] In the following disclosure, including figures and examples, reference
will often be made
to MHC whenever discussing properties and use of a binding molecule in
connection with the
modular polynucleotide-encoded molecules herein disclosed. A skilled person
would be able to
adapt the description to making and using of proteins and other molecules
other than MHC. In
particular, the skilled person will appreciate that, when using binding
proteins other than p/MHC
molecules, the main determinant of the choice is the target to be detected.
For example, if it is
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desirable to detect a small molecule (e.g. caffeine), and an aptamer exists
that is specific for
caffeine, then the skilled person will choose the aptamer as the binding
portion and adapt it to the
scaffold construct. (see Example 13)
[0087] The term "scaffold" or "scaffold molecule" as used herein indicates a
molecular
structure of a capture agent that serves to assemble an affinity agent (e.g.,
MHC) to an encoding
polynucleotide (e.g., ssDNA tags). This structure can be derived from proteins
(such as
Streptavidin or SA), other biopolymers (such as polynucleotides, like RNA and
DNA, peptide
nucleic acid, etc.), or other polymers which can bind to the affinity agent
and the encoding
polynucleotide in distinct and separate portions of the polymer.
[0088] In modular polynucleotide encoded capture agents here described, the
scaffold molecule
is configured to bind the at least one binding molecule and an encoding
polynucleotide, with
scaffold binding domains.
[0089] The term "domain" as used herein with indicates a region that is marked
by a distinctive
structural and functional feature. In particular, a scaffold binding domain is
a region of the
scaffold that is configured for binding with another molecule. Accordingly, a
scaffold binding
domain in the sense of the present disclosure includes a functional group for
binding the another
molecule and a scaffold binding region on the scaffold that is occupied by the
another molecule
bound to the scaffold. Once the functional group has been identified, the
relevant scaffold
binding region can be determined with techniques suitable to identify the size
and in particular
the largest diameter of the another molecule of choice to be attached. The
average largest
diameter for a protein according to the present disclosure in several
embodiments is between
about 101 and about 501 depending on the protein of choice, between about 3 A
and about 10
A for a small molecule, and is between about 10 A and about 20 A for a
polynucleotide.
Techniques suitable to identify dimensions of a molecule include but are not
limited to X-ray
crystallography for molecules that can be crystallized (see e.g., Refs. 39-41)
and techniques to
determine persistence length for molecules such as polymers that cannot be
crystallized (see e.g.,
Refs. 42-43). Those techniques for detecting a molecule dimensions are
identifiable by a skilled
person upon reading of the present disclosure.
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[0090] In the modular polynucleotide encoded capture agent herein described,
the scaffold
binding domains are positionally distinguishable among each other, and
therefore, do not
overlap.
[0091] The wording "positionally distinguishable" as used herein refers to
molecules or domains
thereof, indicates molecules or domains thereof that are distinguishable based
on the point or
area occupied by the molecules or domains. Accordingly, positionally
distinguishable scaffold
binding domains are binding domains that occupy different points or areas on
scaffold and are
thereby positionally distinguishable.
[0092] In particular, in modular polynucleotide encoded capture agents here
described, the
scaffold molecule comprises a first scaffold binding domain that is configured
to attach at least
one binding molecule and a second scaffold binding domain that is configured
to attach the
encoding polynucleotide.
[0093] In the modular polynucleotide encoded capture agents, the first
scaffold binding
domains and the second scaffold binding domains can be selected by identifying
positionally
distinguishable functional groups and related scaffold binding regions that
are configured, to
allow, upon attachment, that the attached molecule is presented on the
scaffold.
[0094] The term "present" as used herein with reference a molecule or portion
thereof, (e.g., a
functional group or a restriction site) that has a chemical reactivity and is
comprised in a
structure, indicates a configuration of the molecule or functional group in
the structure wherein
the molecule or portion thereof maintains a detectable level of such chemical
reactivity.
Accordingly, a molecule or a functional group presented on a scaffold is a
molecule or portion
thereof comprised in that scaffold in a configuration that allows performing,
and detecting, under
the appropriate conditions, the one or more chemical reactions that chemically
and/or
biologically characterize the molecule or portion thereof at issue.
[0095] Therefore in modular polynucleotide encoded capture agents of the
present disclosure,
upon attachment of the binding molecule and the encoding polynucleotide with
the scaffold, the
binding molecule is presented for binding to the target and the encoding
polynucleotide is
presented for binding to a substrate polynucleotide.
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[0096] In modular polynucleotide encoded capture agents here described,
presentation of the
binding molecule and encoding polynucleotide on the scaffold is achieved by
selecting a scaffold
with appropriate first and second scaffold binding domains.
[0097] Functional groups for binding a binding molecule, that can be included
in a first scaffold
binding domain, depend on the chemical nature of the binding molecule and are
identifiable by
the skilled person upon reading of the present disclosure. For example,
functional groups for
binding a binding molecule include but are not limited to BirA Ligase (enzyme
that attaches
biotin group to predefined peptide sequences), other enzymes such as
formylglycine-generating
enzyme (site-specific introduction of aldehyde groups into recombinant
proteins described for
example in Ref. 44).
[0098] Functional groups for binding a polynucleotide, that can be included in
a second scaffold
binding domain, are also identifiable by the skilled person upon reading of
the present disclosure.
Exemplary functional groups presented on the scaffold for binding a
polynucleotide include
functional groups such as sulfulhydryl (e.g. in a cysteine residue), primary
amines and other
functional groups that attach derivatized DNA via conventional conjugation
strategies, that
would be identifiable by the skilled reader.
[0099] Those functional groups can either be endogenous groups on the scaffold
(e.g. native
lysine residues on a scaffold protein), or introduced by methods such as gene
cloning (e.g.
proteins), synthetic techniques (polymers, small molecules), and other
methods. The number of
copies of polynucleotides or binding molecules that can attach to the scaffold
will be directly
proportional to the number of functional groups available on the scaffold.
[00100] The specific first and second functional groups and related scaffold
binding domain are
selected in view of the experimental design. Usually, the scaffold is selected
so that the
functional groups of the first and second scaffold binding regions allow
attachment of the
binding molecule and the encoding polynucleotide using orthogonal chemistries.
A set of
attachment chemistries is orthogonal if, when performing any particular
chemistry, the functional
groups that participate and/or undergo a chemical reaction in that particular
chemistry do not
react with any other chemistry within the orthogonal set. Exemplary orthogonal
chemistries
include cysteine-maleimide coupling, amine-NHS coupling, and streptavidin-
biotin binding,
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when a scaffold is a protein, and controlled oxidization of OH functional
groups in different
scaffold binding regions with Na104 when the scaffold is a polysaccharide.
[00101] In some embodiments, in addition to containing distinct scaffold
binding domains to
accommodate the affinity agent and encoding DNA, the scaffold is also selected
to be compatible
with the environment of the target of interest (e.g. it should be soluble in
aqueous solutions if the
target is cell surface markers).
[00102] In several embodiments, the scaffold consists of a macromolecular
scaffold that is
customized, via multi-ligand interactions, for the high affinity binding to
specific cell types, and
then for the spatially directed, multiplexed sorting of those different cell
types.
[00103] In particular, in some embodiments, the scaffold is provided by a non-
naturally
occurring molecule that is expressed with modular design characteristics. In
those embodiments,
the protein scaffold is designed so that multiple and controlled numbers of
copies of specific
binding molecules and encoding polynucleotides may be attached to the scaffold
at specific
scaffold polynucleotide binding domains.
[00104] In some embodiments, the scaffold can be configured to enable or ease
attachment of
multiple copies of single-stranded encoding polynucleotide (e.g. DNA
oligomers) in multiple
second scaffold binding domains. In those embodiments, the second scaffold
binding domain can
be selected to allow hybridization with an encoding polynucleotide to be used
to spatially direct
the scaffold to particular spots on a surface that are coated with the
substrate polynucleotides.
[00105] A scaffold, thus configured, can be useful, in embodiments where the
modular
polynucleotide-encoded capture agents is used for the spatially selective
sorting of specific cell
types. For example, multiple scaffolds, each containing a different set of
affinity agents, and
uniquely labeled with bindingly distinguishable ssDNA oligomers, can be
harnessed in parallel
to spatially separate a mixture of many cell types into its individual
components as it will be
apparent to a skilled person in view of the present disclosure. For example,
in some
embodiments, it is feasible to use modular capture agents with biotinylated-
antibodies along with
p/MHC proteins as the affinity reagents, where each is encoded to bindingly
distinguishable
ssDNA oligomers. The antibodies can be used to sort cells according to cell
surface markers like
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CD4, CD8, CD3, etc., while the p/MHC proteins will sort cells according to
antigen-specificity
as determined by the TCRs.
[00106] In some embodiments, a desired configuration of a scaffold and, in
particular, a
scaffold protein, can be achieved through modification of candidate scaffolds
that are modified
with techniques known to the skilled person such as traditional cloning
techniques or other
techniques identifiable by a skilled person.
[00107] In some embodiments, the scaffold can be optimized for a specific
capture agent. In
particular, in a specific capture agent an optimized scaffold has well defined
scaffold binding
regions for independently coupling a binding molecule and an encoding-
polynucleotide, so that
upon binding the binding molecule and the encoding polynucleotide, possible
interferences
between the polynucleotide and the assembly of the binding molecule are
minimized. This is
usually achieved for a capture agent having a desired binding affinity for the
target and the
substrate polynucleotide, by minimizing structural overlapping between the
binding molecule(s)
and the encoding polynucleotide attached to the scaffold while maintaining a
desired binding
affinity of the capture agent for the target and the substrate polynucleotide.
[00108] Reference is made to Figures 1 and 2 showing different configurations
of the modular
capture agents according to the present disclosure. In particular, in the
illustration of Figures 1
and 2 a scaffold domain, a binding molecule domain (protein binder domain) and
a
polynucleotide domain (DNA domain) are schematically illustrated. As already
mentioned, the
term "domain" as used herein with indicates a region that is marked by a
distinctive structural
and functional feature. Accordingly, the term "domain" as referred to the
scaffold molecule, the
binding molecule and the encoding polynucleotide, indicates a special region
defined by
conformational changes of the molecule (scaffold molecule, binding molecule,
polynucleotide)
bound in a capture agent at a certain temperature. The domain of a certain
molecule can be
determined with any techniques suitable to identify the dimension and in
particular the tri-
dimensional structure a molecule, and include X-ray crystallography, size
exclusion
chromatography, mass spectrometry, gel electrophoresis and other techniques
identifiable by a
skilled person.
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[00109] In an optimized scaffold for a certain capture agent, the scaffold
binding domains are
selected to minimize the overlapping between the binding molecule domains and
the
polynucleotide domains on the scaffold, that provide the desired binding
affinity to the capture
agent.
[00110] In several embodiments, an optimized scaffold presents the binding
molecules and
encoding polynucleotides associated to a desired binding affinity of the
resulting capture agent
on all the available positionally distinguishable scaffold binding domains on
the scaffold. On the
other hand, in several embodiments, a non-optimized scaffold the number of
attached binding
molecule and encoding-polynucleotides does not match the total number of sites
available on the
scaffold. For example, if the scaffold has 4 sites to attach binder proteins
and 3 sites to attach
DNA, most likely the non-optimized scaffold will be able to contain <4 binder
protein and <3
DNA per scaffold, regardless of how large the molar excess is. This presence
(or absence) of the
binding molecule and/or polynucleotide per scaffold can be measured (see for
example Fi ure
8). The capture agent can also be tested with traditional assays like ELISA,
flow cytometry, SPR,
etc. to measure the efficacy of the capture agent.
[00111] In a specific capture agent, the scaffold can also be modified to
optimize the scaffold
function as an integration point for two moieties (i.e. the binding molecule,
and the encoding-
polynucleotide), while minimizing any possible interactions between the
scaffold domains that
bind those moieties which would result in a reduced functional efficacy of the
attached moiety.
The reduced functional efficacy can be due to steric hindrance (between
overlapping regions of
the binding molecule and encoding-polynucleotide), irreversible modification
of the attachment
regions on the scaffold due to the nature of the coupling chemistry employed,
etc. A comparison
between T cell capture efficiency with an unoptimized scaffold (native SA) and
an optimized
scaffold (cysteine-SA) is illustrated in Examples 4-6 and Figures 7-9).
[00112] Accordingly a certain scaffold molecule binding a certain binding
molecule and
encoding polynucleotide can be optimized for those binding molecule and
encoding
polynucleotide by identifying the scaffold binding regions binding the binding
molecule and
encoding polynucleotide and modify the remaining portion of the scaffold
molecule to arrange
the scaffold binding domains on the scaffold to minimize interactions between
the binding
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molecule and the encoding polynucleotide. In this way, it is possible to
derive optimized variants
of a certain scaffold molecule.
[00113] In some embodiments the scaffold is a protein. In particular in some
embodiments,
protein scaffolds are provided which already contain functional groups that
allow specific
binding for the binding molecule. For example, streptavidin is a good scaffold
protein because
of its natural affinity for biotin, giving it specificity for biotinylated
p/MHC molecules. Another
example would be protein A/G. These proteins have a natural affinity for the
Fc region of
antibodies, in which the latter would be employed as the binding protein. This
is advantageous
over protein scaffolds in which no inherent specificity exists, in which case
it is necessary to
introduce two chemically orthogonal handles for coupling the binding protein
and the encoding-
polynucleotide. Since most proteins lie within a narrow range of size and
sequence length (i.e.
properties such as solubility, number of available sites for modification), it
is expected that any
protein can be used as a scaffold molecule. In particular, proteins which are
stable in the
conditions used for bioconjugation are in particularly expected to be suitable
as scaffolds.
[00114] In several embodiments, the scaffold protein is formed by a
streptavidin (SA or Sa).
Streptavidin is a tetrameric protein from the bacterium Streptomyces avidinii
having sequence
HMGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGT
ALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGH
DTFTKVKPSAAS (SEQ ID NO: 1). SA has extraordinary affinity and four unique
binding sites,
arranged tetrahedrally for its natural ligand biotin (Kd - 10-1s mol/L). The
molar binding capacity
of Streptavidin for biotin is 4:1 biotin:SA. While SA does not have to
specifically bind to the
binding molecule (e.g. via biotin interaction), embodiments where alternative
coupling methods
are used to bind the binding unit to SA do not take advantage of the 4 fold
valency and strong
interaction of SA for biotin. Specifically in several embodiments where SA is
the scaffold, the C
terminus portion is chosen as the site for attachment of the encoding
polynucleotide in view of
the location of the binding pocket for biotin on the N-terminus portion of the
protein.
[00115] Accordingly, in several embodiments where the scaffold protein is
streptavidin,
binding molecules (e.g. MHC molecules) can be biotinylated, to enable the
tetrameric assembly
with the protein-ligand pair SA. In some embodiments, binding molecules can
also be coupled to
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SA via covalent linkages (such as amide coupling), and therefore not
necessarily through the
biotin-SA interaction. The skilled person will be able to identify the most
appropriate binding
based on the experimental design of choice. In several embodiments of the
present disclosure,
SA is used as standard scaffold used to assemble p/MHC monomers into
tetramers.
[00116] In embodiments where the scaffold is SA, a modified SA can be used as
well as
molecules derived therefrom (see in particular SA-phycobiliprotein (PE or APC)
conjugates). In
some embodiments, a scaffold can be used that is a recombinant mutant of SA
for fluorescent
p/MHC tetramer preparations. In some of those embodiments, SA variants can be
used, such as
for example a variant that incorporates a cysteine residue at the carboxy-
terminus [Ref 25, 26,
27], in a site removed from the biotin binding pocket. In those embodiments,
the conjugation of
cysteine-reactive maleimide derivatives can be restricted to the C-terminus
because cysteine
residues are absent in native SA.
[00117] More particularly, in some embodiments, an optimized Streptavidin,
named
streptavidin-cysteine (SAC), can be used that contains several exogenous amino
acids at the c-
terminus. These residues contain a cysteine amino acid, from which derivatized
DNA (or any
other maleimide-derivatized molecule) can be coupled to. The SAC scaffold has
the sequence
HMGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGT
ALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGH
DTFTKVGGSGCP (SEQ ID NO: 2)
[00118] In the present disclosure, reference is often made to SA and SAC
scaffold. A skilled
person will be able to adapt the description to scaffolds and optimized
scaffold other than SA and
SAC, making also reference to the guidance provided by the examples section.
In particular
additional scaffold proteins include but are not limited to Protein A/G,
branched peptides, small
molecules such as NHSester PEG-malemide and optimized variants thereof.
[00119] Additional scaffolds and optimized scaffolds, given a predetermined
target and pre-
selected binding molecule can be derived using the following approach. (a)
selecting a first
coupling chemistry to attach the preselected binding molecule to the scaffold,
and a second
coupling chemistry to attach the polynucleotide to the scaffold (e.g. NHS-
amine and thiol-
maleimide chemistry). (b) selecting a candidate scaffold structure,
considering the valency and
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polarity of the resulting capture agent (e.g. the use of branched peptides for
increasing valency,
the use of glycine-serine-glycine-serine extensions for creating space between
the binder
proteins). (c) performing coupling of the polynucleotide and of the binding
molecule with
candidate scaffold structure thus providing a candidate capture agent. (d)
testing the candidate
capture agent for specific binding with the predetermined target empirically
and optionally (e)
iterate some or all the steps if necessary or desired to increase binding
affinity and/or specificity
of the capture agent to the predetermined target. If optimization is desired
the step of selecting a
candidate scaffold can be performed or comprise a step of modifying the
scaffold to reduce, and
in particular, minimize overlapping between the preselected binding molecule
and the
polynucleotide.
[00120] The term "encoding polynucleotide" as used herein indicates a
polynucleotide that is
attached to the scaffold of a modular capture agent herein described and is
complementary to a
substrate polynucleotide attached to a substrate. In several embodiments, an
encoding
polynucleotide encoding a modular capture agent specific for a first target is
bindingly
distinguishable from an encoding polynucleotide encoding a capture agent
specific for a second
target, in particular when the first target is different from the second
target.
[00121] The wording "bindingly distinguishable" as used herein with reference
to molecules,
indicates molecules that are distinguishable based on their ability to
specifically bind to, and are
thereby defined as complementary to, a specific molecule. Accordingly, a first
molecule is
bindingly distinguishable from a second molecule if the first molecule
specifically binds and is
thereby defined as complementary to a third molecule and the second molecule
specifically binds
and is thereby defined as complementary to a fourth molecule, with the fourth
molecule distinct
from the third molecule. Accordingly, a first and second encoding
polynucleotides are bindingly
distinguishable, if the first encoding polynucleotide specifically binds (and
is thereby defined as
complementary) to a first substrate polynucleotide and the second encoding
polynucleotide
specifically binds (and is thereby defined as complementary to) a second
substrate
polynucleotide, with the first substrate polynucleotide distinct from the
second substrate
polynucleotide.
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[00122] In several embodiments of the arrays substrates, methods and systems
herein
described, each substrate polynucleotide and encoding polynucleotide is
bindingly
distinguishable from another. In some embodiments of the methods and systems
herein
disclosed, each substrate polynucleotide of a substrate is sequence specific
and positionally
distinguishable from another.
[00123] As already mentioned, the wording "positionally distinguishable" as
used herein refers
to with reference to molecules, indicates molecules that are distinguishable
based on the point or
area occupied by the molecules. Accordingly, positionally distinguishable
substrate
polynucleotides are substrate polynucleotide that occupy different points or
areas on the substrate
and are thereby positionally distinguishable.
[00124] In several embodiments, the encoding polynucleotide can include one
ore more
restriction sites for one or more restriction enzymes. The wording
"restriction site" indicates
specific sequences of nucleotides that are recognized by restriction enzymes.
The wording
"restriction enzyme" indicates any enzyme that cuts double-stranded or single
stranded DNA at
specific recognition nucleotide sequences known as restriction sites.
Exemplary restriction sites
that can be comprised in an encoding polynucleotide herein described include 6
base restriction
sites such as the ones for EcoRl BamHI, Ndel and other enzyme identifiable by
a skilled person.
Additional exemplary restriction site include but are not limited to Aval,
BglII, Dral, EcoRV and
further restriction site published in Ref. 45, herein incorporated by
reference in its entirety.
[00125] In several embodiments, the scaffold is itself interchangeable between
bindingly
distinguishable capture agents and a single scaffold can be used in the
construction of different
modular capture agents. Additionally the scaffold can be subject to rounds of
improvement and
optimization. In several embodiments, the scaffold and at least one binding
molecule are also
bindingly distinguishable.
[00126] In some embodiments, the scaffold can be configured to provide a polar
capture
agent, with no radial symmetry as shown in the schematic illustration of
Figure 1 (top panel), a
symmetrical capture agent as shown in the schematic illustration of Figure 1
(bottom panel) and
Figure 2 (top right panel), or pseudo-polar capture agent as shown in the
schematic illustration
of Figure 2 (top left panel).
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[00127] In particular, polar capture agents are capture agents where
overlapping of the binding
molecule domain, scaffold domain and encoding-polynucleotide is minimized.
Pseudo polar
capture agents are molecules in which a minority of binding molecule domains
and encoding-
polynucleotide domains overlap between each other and with the scaffold
domain. In a
symmetric capture agent, all the binding molecules and encoding-polynucleotide
domains
overlap to a certain extent. In each of the polar capture agent, pseudopolar
capture agent and
symmetric capture agent, the scaffold can be optimized to minimize overlapping
within the
specific capture agent between the binding molecules domain(s), scaffold
molecule domain and
the polynucleotide domain(s). Such minimization is maximized for the polar
capture agents.
[00128] Accordingly, in several embodiments, a fully-assembled, polar,
polynucleotide
encoded capture agent is expected to have a higher avidity with respect to a
non-polar capture
agent, since in a polar capture agent the binding molecule is free to interact
with the targets of
interest, and the encoding polynucleotide is free to interact with the cDNA
printed on the
substrate. Applicants have demonstrated that an approximation to this "about-
face" construct
results in higher cell surface marker staining as accessed by flow cytometry
(see Figure 2, lower
panels)..
[00129] In particular, Applicants produced SAC-DNA constructs such that 1 DNA
strand
(fluorescently labeled) were attached per SAC. This moiety is pseudo-polar
because the MHC
capture proteins (the binding domain) are radially distributed across the
scaffold, while the DNA
domain is singular (hence polar, when compared to the rest of the construct)
(see below). This
construct binds better to cell surface receptors when compared directly with
radial symmetric
constructs (178 vs. 153 mean intensities) (see Figure 2 lower panels).
[00130] In several embodiments, a single scaffold with its associated encoding
polynucleotide
can be repeatedly used to generate a library of binding structures, by pairing
a library of binding
structures with the single scaffold. Those embodiments are exemplified in
Examples 5, 8, 9 and
and in Figures 5, 6, 9,12A,B, 13B, 15AB, 16, 19, 22 where the same scaffold
(SAC-A') was
used as NACS capture agents with specificity against MART-1, OVA, Pmel,
tyrosinase, and
CMV. In those embodiments the modularity and interchange-ability of the
system. .
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[00131] In some embodiments, polynucleotide encoded capture agents herein
described and in
particular, modular polynucleotide encoded capture agents here described
comprise a variable
length linker to be used to couple the binding molecule to the scaffold. The
term "linker" as
used herein indicates a molecule comprised in the modular polynucleotide
encoded capture
agents to couple or connect the scaffold with the binding molecule. The linker
can be derived
from any chemical molecule which can be reacted with the scaffold and the
binding molecule of
the capture agent that comprises it. Exemplary linkers include but are not
limited to peptides (e.g.
a 10mer), nucleic acids, polymers (polyethylene glycol), and carbon chains.
The length of the
linker can be controlled by conventional chemical methods, which should be
apparent from a
reader with technical expertise. The linker can be attached with conventional
bioconjugation
strategies which should also be apparent to the skilled reader,
[00132] In those embodiments, the inclusion of a linker is expected to
increase the degrees of
freedom of the binding protein for proper interaction with a target of
interest. This is expected to
increase the strength of the interaction when the target is fixed/confined in
a particular domain
where proper spatial orientation is crucial for high affinity interactions
(for example, target cell
surface proteins confined to the cell membrane).
[00133] In some embodiments, the linker can also function as a spacer between
the binding
molecule and the encoding polynucleotide that is comprised so that the binding
molecule domain
and the polynucleotide domain do not interfere with each other. Exemplary
linkers include
molecules of dimensions between 50 Da and 5000Da.
[00134] In some embodiments, further discussed below a linker can be a
conditional linker that
changes conformation in view of a controlled stimulus.
[00135] In some embodiments, the affinity of capture agent for the target can
be increased by
coupling multiple binding units to a single scaffold. This increase in valency
of the capture
agent improves the avidity of the assembled complex because each neighbor
participates in a
binding event, so the net effect is an increase in the overall association of
the target with the
polynucleotide encoded capture agent as exemplified in Example 5 and Figures
7, 8 and 9. This
is particularly relevant for binding molecules such as MHC that are
characterized by a poor
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affinity for target cells as monomers but that increase the affinity when
bundled together in
multimers.
[00136] In embodiments where the binding molecule is formed by a low affinity
binding
protein, increased valency of the capture agent for the target can be required
for effectively
binding to targets. Variation in affinity associated with a modification of
the molecular
conformation can be exploited to temporally control, the interaction of
polynucleotide encoded
molecule to the respective complementary targets. For example, some cell
surface receptors
function to control cellular activity upon binding to complementary ligands
(molecules to which
the receptors are specific for). The activity can be reversed or restored when
the ligands are no
longer bound to the receptors. Thus, by exploiting the decoupled and
multivalent nature of the
binding molecules to the scaffold, it is possible to control the binding by
using conditional
linkers-linkers which undergo some conformational change in response to
exogenous external
stimuli, resulting in a reduction of the valency-hence of the binding affinity
of the capture agent
-and subsequent inability of the polynucleotide-encoded capture agent to
remain bound to the
target. Examples of conditional linkers are peptides or nucleic acids which
incorporate UV
labile bases which break upon exposure to UV light. An example of this in
practice would be to
use p/MHC proteins coupled to SAC-DNA scaffold via a UV labile peptide linker.
The capture
agent upon binding to T cell receptors will activate the target cell. The
polynucleotide-encoded
p/MHC capture agent can then be removed after a desired amount of time by
exposure to UV
light.
[00137] In several embodiments, polynucleotide encoded capture agents herein
described
include multiple binding molecules bindingly distinguishable between each
other. In those
embodiments, multi-ligand encoded capture agents can be used to interrogate
multiple targets
simultaneously. This would be most advantageous when the targets are assembled
within a
domain, like on the surface of a cell, because the increase in avidity (as
indicated above) would
equally apply to this system, where the avidity of the complex would be
greater than the affinity
each individual binder protein. An example of multi-ligand encoded capture
agents would
include p/MHC complexes where each protein would consist of a distinct peptide
sequence.
Other combinations are possible like Ab Ab, Ab peptide, Ab aptamer, peptide
aptamer, and
additional molecules identifiable by a skilled person upon reading of the
present disclosure.
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[00138] The modular polynucleotide encoded capture agent herein described can
be
manufactured by binding the units in view of the specific capture and the
experimental design
according to procedures that are identifiable by a skilled person. For
example, in embodiments
wherein a binding molecule specifically binds the scaffold, the encoding
polynucleotide (e.g.
DNA) can be first coupled to the scaffold (e.g. SAC) before assembling the
binding molecule
(e.g. MHC). In other embodiments, where the binding molecule does not bind
specifically the
scaffold the binding unit needs to be covalently attached. This can either
occur before or after
attachment of the polynucleotide to the scaffold. Additional procedures to
assemble a modular
capture agent herein described with other scaffolds and binding molecule will
be easily
identifiable to a skilled reader. Those procedure would suitable to assemble
the capture agents
herein disclosed regardless which order the reagent is assembled, as long as
that the specificity of
the binding protein unit needs to be encoded uniquely by the polynucleotide
sequence.
[00139] In some embodiments, where the scaffold is streptavidin, a modular
polynucleotide-
encoded protein can be prepared by enzymatically mixing biotinylated MHC
molecules with
commercial preparations of streptavidin (SA), usually in a 4:1 proportion, the
streptavidin
usually conjugated to a fluorescent dye molecule. Variations on this procedure
all focused on
improving the MHC tetramers for flow-cytometry based cell sorting will be
identifiable by a
skilled person.
[00140] For example, in some embodiments, the binding protein can be attached
via protein-
ligand interaction (streptavidin biotin), protein-protein interaction (Fc
domain and protein A/G),
or bioconjugation strategies (amine coupling, sulihydryl coupling, etc.). In
some embodiments
where the binding molecule is MHC, MHC can be biotinylated at the C terminus.
In some
embodiments where the scaffold is SAC, SAC is modified with DNA at the C
terminus. In some
embodiments, the entire unit is assembled by pooling biotin-MHC with SAC-DNA,
in which
SAC binds specifically to 4 MHC protein molecules via biotin
[00141] Conjugation of an encoding polynucleotide with the scaffold protein of
the modular
polynucleotide-encoded capture aget herein disclosed can be produced with
common
bioconjugation methods, such as chemical cross-linking which include
techniques relying on the
presence of primary amines in the protein to be bound (usually found on Lysine
residues). In
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particular, polynucleotide-encoded-protein can be produced by the covalent
conjugation strategy
such as the ones described in PCT application W02008/016680 incorporated
herein by reference
in its entirety. In particular, chemical conjugation is used to generate
covalent linkages between
scaffold protein and polynucleotide, these include NHS-amine coupling, thiol-
thiol coupling,
thiol-maleimide coupling, hydrazide-aldehyde coupling, etc. These
bioconjugation strategies
should be evident by any person with technical expertise in the area.
[00142] The number of encoding polynucleotides to be conjugated with a
particular
polynucleotide-encoded capture agent can be varied. In particular, the number
of polynucleotides
attached to the protein component can be modulated to minimize the size and
therefore the steric
hindrance of the pending moieties while still maintaining binding specificity.
The optimization
can be performed by way of procedures exemplified in PCT application
W02008/016680
incorporated herein by reference in its entirety, (see in particular Figure 3
and Example 3) In
those embodiments an optimization of the capture agent can be carried forth
chemically (i.e.
varying stoichiometric amounts of reactive small molecule with capture agent
(e.g. Antibody)).
[00143] In some embodiments, the number of encoding polynucleotides to be
attached to each
protein can be any from 1 to 6 or even more than 6. In some embodiments, such
as cell sorting,
attaching 1 to 4 encoding polynucleotides per scaffold provides the further
advantage of
minimizing the steric effects of labeling and therefore allowing a labeling of
a polynucleotide-
encoded capture agent with a plurality of encoding polynucleotides for high
affinity
hybridization with the complementary substrate polynucleotide.
[00144] The length of the polynucleotide forming the pending moieties can also
be controlled
to optimize binding of the polynucleotide-encoded capture agent to the
substrate. In particular,
the length of the encoding polynucleotides can be optimized for
orthogonalization purposes. In
those embodiments, the encoding region contains a 20mer recognition sequence.
These were
generated in silico according to procedures exemplified in PCT application
W02008/016680
incorporated herein by reference in its entirety. In particular the sequences
containing restriction
sites mentioned in the examples were generated by appending the 6base cutting
site to the
sequences originally generated according to procedures exemplified in PCT
application
W02008/016680 incorporated herein by reference in its entirety.
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[00145] The substrate polynucleotides can be produced by techniques known in
the field. For
example, first the polynucleotides can be chemically synthesized. The
polynucleotides can then
be pin spotted according the paradigm outlined by Pat Brown at Stanford [Ref.
46]. The substrate
polynucleotides so produced can be then attached to a substrate according to
techniques
identifiable by a skilled person upon reading of the present disclosure.
Particularly, suitable
polynucleotides for the production of substrate polynucleotides include at
least 75mers long on
polylysine substrates.
[00146] In some embodiments, the encoding polynucleotides and/or the substrate
polynucleotides are orthogonalized to minimize the non-specific binding
between encoding-
polynucleotide and substrate polynucleotide. Accordingly, orthogonalized
polynucleotides
include polynucleotides whose sequence is computationally generated to
minimize incomplete
base pairing, metastable states and/or other secondary structures to minimize
non specific
interactions between polynucleotides and non linear secondary interactions in
the polynucleotide
usually associated with random generation of the relevant sequences.
[00147] The term "orthogonalization" as used herein refers to the process by
which a set of
polynucleotides are generated computationally, in which incomplete base
pairing, metastable
states and other secondary structures are minimized, such that a
polynucleotide only binds to its
complementary strand and none other. Exemplary orthogonalization techniques
used in this
disclosure include orthogonalization performed according to the paradigm
outlined by Dirks et
al. [Ref. 47] herein incorporated by reference in its entirety.
[00148] In particular, in some embodiments, the encoding-polynucleotides and
the
corresponding complementary substrate polynucleotides are orthogonalized
polynucleotides such
as polynucleotides A, B, and C described in detail in Example 6 and in Figures
5, 6, 9-13, 15,
17, 19, 22, and polynucleotides AEcoRI, BBamHI described in Example 7 and in
Figure 16.
[00149] Additional orthogonalized polynucleotides can be further identified by
way of
methods and procedures, such as in silico orthogonalization (i.e. computerized
orthogonalization) of polynucleotides according to procedures that would be
apparent to a skilled
person upon reading of the present disclosure.
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[00150] The modular polynucleotide-encoded capture agents herein described
using MHC as a
binding molecule can be manufactured using a procedure extensively described
in [Ref. 33]
herein incorporated by reference in its entirety. According to this procedure
a library of p/MHC
protein molecules can be generated by first synthesizing a sacrificial peptide
that is modifiable
through a controlled stimulus. For example, in several embodiments this
sacrificial peptide
contains a non-natural amino acid containing a nitro-phenyl side chain. This
functional group is
UV labile; hence in the presence of UV, the sacrificial peptide is cleaved
into two smaller
peptides. Thus it is feasible to generate p/MHC complexes presenting the
sacrificial peptide,
expose the entire complex to UV light, but perform the latter in a solution
containing molar
excess of an exchange peptide. Upon UV exposure, the sacrificial peptide will
be cleaved in
two, and will be displaced for the full length exchange peptide, for which the
MHC will have
higher affinity. By employing a library of peptides, it will be possible to
generate large p/MHC
libraries in one UV exchange step.
[00151] The methods and systems herein disclosed can be used for performing
assays for the
detection of targets, including mono-parameter assays, and multiparameter
assays, all of which
can be performed as multiplex assays.
[00152] The term "monoparameter assay" as used herein refers to an analysis
performed to
determine the presence, absence, or quantity of one target. The term
"multiparameter assay"
refers to an analysis performed to determine the presence, absence, or
quantity of a plurality of
targets. The term "multiplex" or "multiplexed" assays refers to an assay in
which multiple assays
reactions, e.g., simultaneous assays of multiple analytes, are carried out in
a single reaction
chamber and/or analyzed in a single separation and detection format.
[00153] In some embodiments, the methods and systems herein disclosed can
advantageously
used to perform diagnostic assays, wherein the target(s) to be detected are
predetermined
biomarkers associated with a predetermined disease. Those embodiments are
particularly
advantageous in a diagnostic approach where different classes of biomaterials
and biomolecules
are each measured from a different region of a typically heterogeneous tissue
sample, thus
introducing unavoidable sources of noise that are hard to quantitate.
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[00154] In some embodiments of the methods and systems herein disclosed, the
polynucleotide-encoded capture agent and substrate polynucleotide are used in
combination as
schematically illustrated in Figures 3, 4, 21.
[00155] In the embodiment shown in Figures 3, 4, 21 the polynucleotide-encoded
capture
agent herein disclosed form a protein array that can be contacted with a
sample to detect a target
in the sample. The embodiment of Figures 3, 4, 21 is particularly advantageous
for detecting
and/or sorting protein-targets.
[00156] In additional embodiments, particularly suitable for detecting and/or
sorting cells
targets, some or all of the modular polynucleotide-encoded capture agents are
contacted with the
sample before contacting the modular polynucleotide-encoded-antibodies with
the
complementary substrate polynucleotide. In those additional embodiments, the
antibodies and
the one or more corresponding targets can bind in absence of the substrate,
e.g., in a solution
phase, where both molecules have a complete orientational freedom and the
access of the target
to the binding site of the affinity agent is not impaired by the substrate.
Additionally, sensitivity
and specificity of the performed assay is improved as well as the
detectability of the modular-
polynucleotide-encoded target complex bound to the substrate, when compared to
corresponding
methods and system of the art. Exemplary embodiments showing some of the above
advantages
are illustrated in Example 12.and in Figure 17.
[00157] In some embodiments, multiple cell types can be sorted on an array by
employing a
library of protein binders in which the scaffold they are coupled to are
encoded with distinct
polynucleotides, such that each different protein binder specificity is
encoded with a distinct
DNA sequence. Examples of this approach in practice are illustrated in
Examples 8-11 and
Figures 5b, 12b, 15b, 16.
[00158] In some embodiments of methods and systems herein disclosed the
modular
polynucleotide-encoded target complex bound to the substrate is eventually
detected from the
substrate.
[00159] In some embodiments, detection of the complex is performed by
providing a labeled
molecule, which includes any molecule that can specifically bind a modular
polynucleotide-
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encoded-protein target complex to be detected (e.g. an antibody, aptamers,
peptides etc) and a
label that provides a labeling signal, the label compound attached to the
molecule. The labeled
molecule is contacted with the polynucleotide-encoded capture agent-target
complex and the
labeling signal from the label compound bound to the polynucleotide-encoded
capture agent-
target complex on the substrate can then be detected, according to procedure
identifiable by a
skilled upon reading of the present disclosure and, in particular, of the
Examples section.
[00160] In embodiments wherein one or more targets and/or a plurality of
targets is detected
described below in more details, the labeled molecule can be formed of a
plurality of labeled
molecules. Each labeled molecules comprises a molecule that specifically binds
one target of the
one or more targets/plurality of targets and a label compound attached to the
molecule, the label
compound providing a labeling signal, each labeled molecule detectably
distinguishable from
another.
[00161] The wording "detectably distinguishable" as used herein with reference
to labeled
molecule indicates molecules that are distinguishable on the basis of the
labeling signal provided
by the label compound attached to the molecule. Exemplary label compounds that
can be use to
provide detectably distinguishable labeled molecules, include but are not
limited to radioactive
isotopes, fluorophores, chemiluminescent dyes, chromophores, enzymes, enzymes
substrates,
enzyme cofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metal
sols, ligands (such as
biotin, avidin, streptavidin or haptens) and additional compounds identifiable
by a skilled person
upon reading of the present disclosure.
[00162] In some embodiments, the plurality of labeled molecules is contacted
with the plurality
of modular polynucleotide-encoded capture agent-target complexes for a time
and under
condition to allow binding of the plurality of polynucleotide-encoded capture
agent-target
complexes with the plurality of labeled molecules. The labeling signal is then
detected from the
plurality of labeled molecules bound to the plurality of modular
polynucleotide-encoded capture
agent-target complexes on the substrate.
[00163] In some embodiments, the detection method can be carried either via
fluorescent based
readouts, in which the labeled antibody is labeled with flurophore which
includes but not
exhaustively small molecular dyes, protein chromophores, quantum dots, and
gold nanoparticles
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In particular, in some embodiments, in any of the methods and systems herein
disclosed,
detection can be carried out on gold nanoparticle-labeled secondary detection
systems in which a
common photographic development solution can amplify the gold nanoparticles as
further
described below. Also, if the readout comes from dark field scattering of gold
particles, single
molecule digital proteomics is enabled. Additional techniques are identifiable
by a skilled person
upon reading of the present disclosure and will not be further discussed in
details.
[00164] The terms "label" and "labeled molecule" as used herein as a component
of a complex
or molecule refer to a molecule capable of detection, including but not
limited to radioactive
isotopes, fluorophores, chemiluminescent dyes, chromophores, enzymes, enzyme
substrates,
enzyme cofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metal
sols, ligands (such as
biotin, avidin, streptavidin or haptens) and the like. The term "fluorophore"
refers to a substance
or a portion thereof which is capable of exhibiting fluorescence in a
detectable image. As a
consequence the wording and "labeling signal" as used herein indicates the
signal emitted from
the label that allows detection of the label, including but not limited to
radioactivity,
fluorescence, chemolumiescence, production of a compound in outcome of an
enzymatic
reaction and the likes. In particular gold nanoparticles can be used in a
sandwich style detection
assay, in which the detection complex is linked to a gold nanoparticle. This
is most relevant in
detecting small molecules like proteins, peptides, etc, as detecting cells can
be simply carried out
using traditional microscopy techniques.
[00165] In some embodiments, one specific target is detected. In those
embodiments
contacting the modular polynucleotide-encoded capture agent with the target
can be performed
before or after contacting the polynucleotide-encoded capture agent with the
substrate. In
particular, the units forming the modular capture agents can be contacted in a
single reaction
mixture. Such an approach, however, will require specificity of the binding
between scaffold and
binding molecule as well as of scaffold and encoding polynucleotide (which is
usually already
specific).
[00166] The embodiments wherein contacting the modular polynucleotide encoded
capture
agent with the target is performed before contacting the modular
polynucleotide-encoded protein
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with the substrate are particularly suitable to sort or detect cells. This
approach is exemplified in
Example 12 and in Figure 17.
[00167] The embodiments wherein contacting the modular polynucleotide-encoded
capture
agents with the target is performed after contacting the modular
polynucleotide-encoded capture
agents with the substrate are particularly suitable to sort or detect proteins
with high sensitivity.
[00168] Exemplary embodiments of methods and systems herein disclosed wherein
contacting
the polynucleotide-encoded capture agent with the target is performed after
contacting the
polynucleotide-encoded capture agent with the substrate are exemplified in
Examples 3, 7, 8, 9,
10, 11 and illustrated in Figures 5-6, 9-13, 15-16, 19, 22. In those
embodiments, competition for
the same specific substrate polynucleotide between a polynucleotide-encoded-
proteins bound to
the target and polynucleotide-encoded-proteins not bound to the target can be
eliminated and the
sensitivity of the assay consequently increased. Further, in those embodiments
the concentration
of polynucleotides on the substrate can be optimized so that higher
concentration of
polynucleotide-encoded capture agents can be bound to the substrate, which
will in turn result in
higher concentrations of correctly assembled complex, which in turn increase
the overall
detection sensitivity, by virtue of equilibrium thermodynamics law that govern
each binding.
[00169] Monoparameter assays that can be performed with the methods and
systems
exemplified in Figures 5a, 6, 9-11, 12Ac, 13b, 15ad, 17b, 19, 22 and in
Examples 1, 7-10,
include but are not limited to, any assays for the detection of single markers
in serum, single
protein detection in biological samples, cell sorting according to one surface
marker and further
assays identifiable by a skilled person upon reading of the present
disclosure.
[00170] In particular, monoparameter assays can be performed to detect in a
sample CD8 cell,
CD4 cells or antigen-specific T-cells (i.e. cells that are distinguished from
one another by their
T-cell receptors (TCRs), which give them their antigen specificity)
[00171] In some embodiments, detection of a plurality of targets is performed,
according to a
strategy schematically illustrated in Examples 3, 8, 10 and 11 and in Figures
3-6, 12b, 15b, 16,
22.
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[00172] In some embodiments, a protein array composed of a plurality of
bindingly
distinguishable and positionally distinguishable modular polynucleotide-
encoded capture agents
can be produced. Those embodiments are particularly advantageous for sorting
and/or detecting
different protein-targets with a high sensitivity.
[00173] In additional embodiments, the plurality of modular polynucleotide-
encoded capture
agent is contacted with a sample for detection of the related target before
contacting the substrate
polynucleotides. In those embodiments, the methods and systems herein
disclosed can be used to
perform multiplexed multiparameter assays wherein due to the improved
sensitivity and
selectivity associated with binding of a binding protein and target in absence
of a substrate and in
view of the reduced biofouling and protein denaturation, a large number of
biomarkers can be
efficiently detected in a quantitative and/or qualitative fashion.
[00174] Multiparameter assays that can be performed with the methods and
systems
exemplified in Examples 3, 8-11 and illustrated in Figures 3-6, 12b, 15b, 16,
22 include but are
not limited to any proteomic analysis, tissue analysis, serum diagnostics,
biomarker, serum
profiling, multiparameter cell sorting, single cell studies, and additional
assays identifiable by a
person skilled in the art upon reading of the present disclosure.
[00175] In some of those embodiments, multiparameter assays can be performed
to detect in a
sample CD8 cell, CD4 cells and antigen-specific T-cells in a multiplexed
detection approach.
[00176] Embodiments of the methods and systems wherein the plurality of
targets is composed
of different types of cells are particularly advantageous over corresponding
methods and systems
of the art such as panning in which cells interact with surface marker-
specific antibodies printed
onto an underlying substrate [Ref. 48]. In particular, the efficiency of cell
capture on the
substrate is improved with respect to prior art methods and systems, due to
the fact that with
panning the tertiary structure of capture antibodies are detrimentally and
irreversibly damaged by
absorption/covalent attachment to common derivatized substrates. This results
in surfaces which
are less reactive when compared with NACS as exemplified in Example 7 and
illustrated in
Figures 10 and 11.
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[00177] In several embodiments the modular polynucleotide-encoded capture
agents herein
described are used in a cell sorting approach.
[00178] Assays to sort targets performable with the methods and systems
exemplified in
Examples 1-11 and illustrated in Figures 3-13, 15-17, 19, 21, 22, include any
assay that requires
detection of a particular target (including but not limited to cell targets,
protein-target or gene
targets) in a mixture, which will be identifiable by a skilled person upon
reading of the present
disclosure.
[00179] In particular, methods and systems herein described allow multiplexed
sorting of
specific cell types from at least a 1: 1000 dilution within a complex mixture
of cell types. Such
sorting of rare cells is demonstrated even for the case when the cell-specific
affinity agents
exhibit a relatively weak binding affinity.
[00180] In some embodiments, the polynucleotides (e.g. DNA oligos) employed
for encoding
the capture agent are designed to include distinct restriction enzyme sites
which complementary
restriction endonucleases can cleave (herein also releasable polynucleotide-
capture agent).
[00181] In particular, the restriction sites are included so that different
restriction enzymes
recognize different DNA sequences on bindingly distinguishable capture agents.
Thus by using a
plurality of distinct restriction enzymes, the adhesion of distinct
populations of capture agents
and, as a consequence, of distinct population of target, and in particular
cells can be
independently controlled by the addition of the complementary restriction
enzyme specific for
the sequence employed to sort that cell type. The released cells can be
expanded further in vivo
by cell culturing for enrichment, or can be genomically or proteomically
analyzed (e.g. PCR,
western blots, etc.) with monoparameter or multiparameter assays as described
in the present
disclosure.
[00182] The controlled release of any captured target with the polynucleotide
encoded-capture
agent will allow further analysis of the target by other conventional
bioanalytical techniques, like
PCR, mass spec, western blots, etc, and other techniques that will be
identifiable by the skilled
reader.
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[00183] Accordingly in some embodiments releasable polynucleotide encoded
capture agents
here described can be used in connection with a method wherein a target and in
particular a
plurality of targets is provided, the polynucleotide encoded capture agents
are contacted with the
target and a substrate attaching substrate polynucleotides for a time and
under condition to allow
formation of polynucleotide-encoded capture agent-target complexes on the
substrate. A
restriction enzyme for a restriction site of a releasable polynucleotide
encoded capture agent is
then contacted with the releasable polynucleotide-encoded capture agent-target
complexes to
allow cleavage of the complementary restriction site, thus allowing selective
release of the
releasable polynucleotide-encoded capture agent-target complexes comprising
the restriction site
complementary to the restriction enzyme. In some embodiments, the release
performed with the
method herein described can be selectively controlled to release different
releasable
polynucleotide-encoded capture agent-target complexes in a controlled fashion
(e.g., at different
times).
[00184] In some embodiments, the releasable polynucleotide-encoded capture
agent-are
modular polynucleotide-encoded capture agent herein described. In particular,
in some
embodiments, the releasable modular polynucleotide-encoded capture agent
according to this
method further comprise the linker molecule to allow controlled release of the
modular
polynucleotide encoded capture agent herein described thus allowing additional
analysis of the
target in absence of the capture agent.
[00185] In several embodiments, also target captured in an array, and in
particular cells
captured on an array will be amenable to further analysis. Specifically,
immuno-PCR can be
employed to profile the cell surface receptors. In addition or in the
alternative, it is possible to
use a set of polynucleotide-encoded capture agent such as the ones described
in W02008/016680
herein incorporated by reference in its entirety, to perform such analysis,
which can be carried
out according to procedures exemplified in Example 16 and Figures 20-22. In
particular, in the
example of Figures 20-22 antibodies against target surface biomarkers are
labeled with unique
DNA sequences. These polynucleotide-encoded antibodies are then used to stain
the cells
captured on the array. The DNA tags on the encoded antibodies can then be
analyzed, and the
presence or absence of the target cell biomarkers will be correlated to the
presence or absence of
the DNA tags associated with the cell biomarker. The DNA tags can be detected
with
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conventional techniques like PCR, sequencing, microarrays, and additional
techniques
identifiable by a skilled reader.
[00186] Accordingly, in some embodiments, polynucleotide-encoded capture
agents and the
modular polynucleotide-encoded capture agents here described can be used in
combination in a
method to detect and in particular analyze a target wherein at least one of
the modular
polynucleotide-encoded capture agents and polynucleotide-encoded capture
agents is contacted
with a target and/or with a substrate polynucleotide attached on a substrate
to allow formation of
modular polynucleotide-encoded capture agent-target complexes and/or
polynucleotide-encoded
capture agent-target complexes. Additional polynucleotide-encoded capture
agents and/or
modular polynucleotide-encoded capture agents can further be contacted with
those complexes
to allow binding with the target and/or with additional targets presented on
the targets to form
additional polynucleotide-encoded capture agents-complexes. Complexes of the
additional
polynucleotide-encoded capture agents and/or modular polynucleotide-encoded
capture agents
with the additional targets can therefore be detected. Additional variants,
based on the use of
releasable polynucleotide capture agents and/or of polynucleotide-encoded
capture agents
comprising a linker, and in particular a conditional linker, will be apparent
to a skilled person.
[00187] In additional embodiments, the substrate of any of the methods and
systems herein
disclosed can be associated with a microfluidic component so to allow
performance of
microfluidic based assays. Microfluidic-based assays offer advantages such as
reduced sample
and reagent volumes, and shortened assay times [Ref. 49]. For example, under
certain
operational conditions, the surface binding assay kinetics are primarily
determined by the analyte
(protein) concentration and the analyte/antigen binding affinity, rather than
by diffusion [Ref.
50].
[00188] The term "microfluidic" as used herein refers to a component or system
that has
microfluidic features e.g. channels and/or chambers that are generally
fabricated on the micron
or sub-micron scale. For example, the typical channels or chambers have at
least one cross-
sectional dimension in the range of about 0.1 microns to about 1500 microns,
more typically in
the range of about 0.2 microns to about 1000 microns, still more typically in
the range of about
0.4 microns to about 500 microns. Individual microfluidic features typically
hold very small
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quantities of fluid, e.g. from about 10 nanoliters to about 5 milliliters,
more typically from about
100 nanoliters to about 2 milliliters, still more typically from about 200
nanoliters to about 500
microliters, or yet more typically from about 500 nanoliters to about 200
microliters.
[00189] The microfluidic components can be included in an integrated device.
As used herein,
"integrated device" refers to a device having two (or more) components
physically and operably
joined together. The components may be (fully or partially) fabricated
separate from each other
and joined after their (full or partial) fabrication, or the integrated device
may be fabricated
including the distinct components in the integrated device. An integrated
microfluidic array
device includes an array component joined to a microfluidic component, wherein
the
microfluidic component and the array component are in operable association
with each other
such that an array substrate of the array component is in fluid communication
with a microfluidic
feature of the microfluidic component. A microfluidic component is a component
that includes a
microfluidic feature and is adapted to being in operable association with an
array component. An
array component is a component that includes a substrate and is adapted to
being in operable
association with a microfluidic component.
[00190] The microfluidic systems can also be provided in a modular form.
"Modular"
describes a system or device having multiple standardized components for use
together, wherein
one of multiple different examples of a type of component may be substituted
for another of the
same type of component to alter the function or capabilities of the system or
device; in such a
system or device, each of the standardized components being a "module".
[00191] In microfluidic embodiments of the methods and systems herein
disclosed,
measurements of large panels of protein biomarkers within extremely small
sample volumes and
a very reduced background/biofouling are possible.
[00192] In the microfluidic embodiments of the methods and systems herein
disclosed, the
sensitivity of the assay can also be increased.
[00193] Additionally, the microfluidic methods and systems herein disclosed
allow
performance of both (i) mono step assays (wherein the polynucleotide-encoded
capture agent the
target(s) and labeled antibodies are contacted in a single step) and (ii)
multi-steps assays
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(wherein the substrate is sequentially exposed to modular polynucleotide-
encoded capture agent,
target(s), and then secondary antibody) in a reduced amount of time, with
samples reduced in
size and with a higher sensitivity when compared with corresponding
microfluidic methods and
system of the art and with other non-microfluidic methods and systems for
molecule detection
[00194] An additional advantage associated with microfluidic methods and
systems herein
disclosed includes the possibility of performing in a microfluidic environment
any assay that
involves substrate-supported antibodies, which would not have survived
microfluidic chip
assembly with the use of previous techniques.
[00195] Further advantages associated with the methods and systems herein
disclosed are: the
possibility of performing sensitive measurements using low cost reagents, such
as glass, and
plastic; and of using the substrate in combination with additional components
for sample
pretreatment and purification.
[00196] The methods and systems herein disclosed allow the multiplexed
multiparameter
detection, sorting and of biomarkers of interest and related diagnostic
analysis. Exemplary
illustration of applications of the methods and systems herein disclosed for
diagnostic analysis
are described in Examples 9-10 and shown in Figures 13-15, 21-22, and any
additional assay
identifiable by a skilled person upon reading of the present disclosure.
[00197] In particular, in the exemplary diagnostic assays of Figures 13-15,
antigen-specific T
cell populations were directed detected with NACS from human PBMCs. Detection
of these T
cells is important for diagnostic purpose because they are involved in the
immune response
against cancer and viral pathogens. Examples of therapeutic assays are instead
provided in
Figure 13. Here human PBMCs are transduced with a TCR specific for a cancer
associated
antigen. These T cells are detected on a NACS array prior to subsequent
infusion into a patient.
[00198] The systems herein disclosed can be provided in the form of arrays or
kits of parts. An
array sometimes referred to as a "microarray" includes any one, two or three
dimensional
arrangement of addressable regions bearing a particular molecule associated to
that region.
Usually the characteristic feature size is micrometers. Examples 3-11, and
Figures 3-6, 9-13, 15-
17, 19, 21-22 provide exemplary microarrays.
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[00199] In a kit of parts, the modular polynucleotide-encoded capture agents
and/or any of the
relevant components are independently comprised in the kit together with a
substrate. In
particular, the modular polynucleotide-encoded capture agent can be included
in one or more
compositions, and each modular polynucleotide-encoded capture agent can be
comprised in a
composition together with a suitable vehicle carrier or auxiliary agent.
[00200] The substrate provided in the system can have substrate polynucleotide
attached
thereto. In some embodiments, the substrate polynucleotides can be further
provided as an
additional component of the kit. Additional components can include labeled
polynucleotides,
labeled antibodies, labels, microfluidic chip, reference standards, and
additional components
identifiable by a skilled person upon reading of the present disclosure. In
particular, the
components of the kit can be provided, with suitable instructions and other
necessary reagents, in
order to perform the methods here disclosed. The kit will normally contain the
compositions in
separate containers. Instructions, for example written or audio instructions,
on paper or electronic
support such as tapes or CD-ROMs, for carrying out the assay, will usually be
included in the kit.
The kit can also contain, depending on the particular method used, other
packaged reagents and
materials (i.e. wash buffers and the like).
[00201] Further details concerning the identification of the suitable carrier
agent or auxiliary
agent of the compositions, and generally manufacturing and packaging of the
kit, can be
identified by the person skilled in the art upon reading of the present
disclosure.
EXAMPLE S
[00202] The methods and system herein disclosed are further illustrated in the
following
examples, which are provided by way of illustration and are not intended to be
limiting.
Example 1: Production of polynucleotide encoded SAC scaffold protein
[00203] Expression of SaC was expression was performed according to previously
published
protocols [Ref. 36]. Briefly the streptavidin-cysteine (SaC) gene cloned into
the pET-3a plasmid
was a generous gift from Dr. Takeshi Sano (Harvard Medical School). Following
colony
selection and overnight start culture, B121(DE3)-pLysE containing the plasmid
was grown at
37 C with shaking in LB medium and selection antibiotics ampicilin (50 ltg/ml)
for pET-3a-SaC
and chlorophenicol (25 ltg/ml) for pLysE. Induction of the cells occurred when
A600 reached 0.6
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at which b-D-thiogalactopyranoside (IPTG) was added to a final concentration
of 0.4 MM.
Following induction the cells were kept spinning at 37 C for 4 hours. The
culture was then
centrifuged at 1600g for 10 min and washed with 100mM NaCl, 1 mM EDTA, 10 mM
Tris-HCI,
pH 8Ø The cells were then lysed with lysis buffer (2 mM EDTA, 30 mM Tris-HC
1, 0.1% Triton
X-100, pH 8.0).
[00204] To reduce the viscosity of the solution, the lysate was then treated
with DNase and
RNase (10 ltg/ml each, with J2mM MgSO4) for 20 min at room temperature. The
insoluble
inclusion bodies were then separated from the lysate by centrifugation at
39,000g for 15 min
after which the precipitate was washed again with lysis buffer. The
precipitate was dissolved in 6
M guanidine-HCI, pH 1.5 to the original culture volume. To remove cellular
biotin, 100 mL of
dissolved precipitate solution was dialyzed in 1L of 6M guanidine-HC1, pH 1.5.
The dialysate
was then transferred to 0.2 M NaAcetate, pH 6.0 to remove guanidine and in the
process refold
SaC. It is critical here to perform the dialysis slowly, by removing the stir
bar. The dialysate was
then spun at 3000g for 10 min to remove precipitated material and filtered
through a 0.2 !-Lm
tllter (amicron). Refolded SaC was lastly dialyzed against 50mM Bicarbonate,
500 mM NaCl,
pH 11.
[00205] SaC were purified as follows. Refolded volumes of SaC were mixed 1:1
with binding
buffer (50 mM Sodium Bicarbonate, 500 mM NaCl, lOmM b-Me, pH 11). A gravity
column
packed with 1.5 ml of iminobiotin agarose resin (Pierce) was washed with 10 ml
of binding
buffer. The refolded mixture was then applied to the column and the eluted
fractions were
collected and reapplied to the column again, to maximize SaC recovery. After
washing the
column with 20 ml binding buffer, SaC was eluted with pH 4 elution buffer (50
MM Sodium
Acetate, lOmM --me). Fractions containing SaC, as monitored by OD280, were
collected,
buffer exchanged to PBS containing 10 mM --me, and concentrated to 1 mg/ml
final
concentration using 10K mwco filters (Millipore)
[00206] SaC Oligonucleotide Conjugation was performed as follows. Prior to
use, stock SaC
was buffer exchanged to Tris buffered Saline (TBS) containing 5mM TCEP using
desalting
columns (Pierce). TCEP is a nonthiol containing reductant that maintains the
reduced form of
SaC during conjugation. MHPH (3-N-Maleimido-6-hydraziniumpyridine
hydrochloride,
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Solulink) in DMF was added to SaC at a molar excess of 300: 1. In parallel,
SFB in DMF
(succinimidyl 4-formylbenzoate, Solulink) was added in a 40: 1 molar excess to
5' aminated
oligos (IDT).
[00207] The mixtures were allowed to react at room temperature for 3-4 hours.
Excess MHPH
and SFB were removed and samples were buffer exchanged to citrate buffer (50mM
sodium
citrate, 150 mM NaCl, pH 6.0) using desalting spin columns (Pierce). The SFB-
labeled oligos
were then combined in a 20:1 molar excess with the derivatized SaC and allowed
to react for 2-3
hours at room temperature before transferring to overnight incubation at 4 C.
Unreacted oligos
were removed using a Pharmacia Superdex 200 gel filtration column at 0.5
mllmin isocratic flow
of PBS. Fractions containing the SaC-oligo conjugates were concentrated using
10K mwco
concentration filters (Millipore). The synthesis of SaC-oligo constructs were
verified by non-
reducing 8% Tris-HCI SDS-PAGE.
Example 2: Microarray Fabrication
[00208] DNA microarrays were printed via standard methods by the microarray
facility at the
Institute for Systems Biology (ISB--Seattle, WA) onto amine-coated glass
slides. Typical spot
size was 600 f.lm (SMPXB15 pin, Arrayit). All DNA strands were purchased
Integrated DNA
technologies, and all complements were 5' aminated. Sequences for all six 3D-
mers and their
respective designations are given in Table 1 and Table 2 below.
Table 1. Sequence Designation
Name Sequence SEQ ID NO
A 5'-AAA AAA AAA AAT CCT GGA GCT AAG TCC GTA 3
B 5'- AAA AAA AAA AGC CTC ATT GAA TCA TGC CTA 4
C 5' -AAA AAA AAA AGC ACT CGT CTA CTA TCG CTA 5
Table 2. Complement
Name Sequence SEQ ID NO
A' 5' - NHz- AAA AAA AAA ATA CGG ACT TAG CTC CAG GAT 6
B' 5' -NHz- AAA AAA AAA ATA GGC ATG ATTCAA TGA GGC' 7
C' 5' -NHz- AAA AAA AAA ATA GCG ATA GTA GAC GAG TGC 8
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Example 3: Detection and Sorting Methods and Systems using NACS
[00209] Cell sorting of antigen-specific CD8+ T-cells using antigen presenting
MHC molecules
organized on a ssDNA-oligomer-labeled SaC scaffold was performed as
schematically illustrated
in Figure 3 and Figure 4. In particular, the assays schematically illustrated
in Figures 3 and 4
are directed to cell sorting, by which different cell types can be sorted on a
glass substrate and
detected by conventional microscopy techniques. The modular DNA-SAC-MHC
construct is
first hybridized to the array, after which a complex cell sample containing
the target T cell of
interest is applied and sorted on the array.
[00210] According to a first series of experiments, the protein SaC was
expressed, and ssDNA
oligomers are coupled to the cysteine residues using thiol coupling according
to procedure
exemplified in Example 1 (see Figure 3).
[00211] A biotinylated antigen-presenting MHC was coupled to the SaC at the
biotin/SaC
binding sites, by combining molar excess of p/MHC monomers to SaC-oligo and
incubating at
37 C for 20 minutes. (see Figure 3).
[00212] The SaC/MHC/ssDNA scaffold was then combined with a solution
containing the
CD8+ cells of interest, and then the entire SaC/MHC/ssDNA/CD8+ assembly is
localized to a
particular spot on a surface that has been prespotted with complementary
ssDNA' oligomers
according to procedure exemplified in Example 2 (see Figure 3).
[00213] According to a second series of experiments, self-assembled ssDNA-
p/MHC tetramer
arrays for multiplexed sorting of antigen-specific cells were prepared.
(Figure 4). ssDNA-tagged
p/MHC tetramers were produced by coupling ssDNA site-specifically to SAC prior
to exposure
to molar excess of biotinylated p/MHC monomers (Figure 4 . p/MHC tetramer
arrays are
formed by pooling ssDNA-p/MHC tetramers of select specificity and
hybridization to a
complementary printed ssDNA microarray (figure 4). T cells expressing the
cognate TCR were
detected by binding to the surface confined tetramer.
[00214] To perform both series of experiments, two T cell lines Tyrosinase-TCR
transgenic
Jurkats and Mart-I-TCR transgenic Jurkats, genetically engineered to express
TCRs specific for
melanoma antigens, were used together with corresponding HLA-A2 restricted
class I major
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histocompatibility complexes (MHC) monomers with tyrosinase 369-377 (YMDGTMSQV
-
SEQ ID NO: 9) and Marti 26-35 (ELAGIGILTV - SEQ ID NO:10) peptides.
[00215] Prior to sorting experiments, the microarray slides were blocked to
prevent non-
specific cell absorption with 1 mg/ml PEG-NHS ester (Sunbio) dissolved in PBS
for 2 hours at
room temperature. The slides were then dipped 5 times in 0.5x PBS to remove
excess PEG,
immediately followed by another 5 dips in dH2O to remove salts. The slides
were blown dry with
a nitrogen gun. The slides can be stored in a dessicator for up to 2 weeks
with no degradation in
efficacy.
[00216] Solid state sorting was carried out by first combining molar excess of
p/MHC
monomers to SaC-oligo and incubating at 37 C for 20 minutes. After
tetramerization, 200 ul of
DMEM supplemented with 10% FBS was added and the solution pipetted on top of
the DNA
microarray.
[00217] The tetramers were allowed to localize to the complementary spots for
1 hour at 37 C.
The slide was then rinsed with 3% FBS in PBS. After rinsing, 106 T cells in
100 III of fresh
DMEM were added to the slide on top of the array. The slide was then
transferred to a 37 C
incubator for 20-30 min to allow the cells to interact with the p1MHC array.
After incubation, the
slide was slide gently with 3% FBS in PBS to dislodge unbound cells and
visualized via
brightfield and fluorescence microscopy. Similarly solution phase capture was
carried out by
combining 106 cells with 0.5 g of assembled NACS tetramer, allowing the
capture agent to bind
to the cells in solution phase. After 20 min incubation at 37 C, the cells
were spun at 500g for 5
min and the excess tetramers removed by aspiration. The cells were resuspended
in 100 / L
DMEM, 10% FBS, and directly applied to a pre-blocked microarray. The slide was
further
incubated for 20 min at 37 C. Subsequent washing and imaging steps are
identical to the solid
state sorting.
[00218] The results of the above procedure performed to obtain a single
parameter and
multiplexed cell sorting are illustrated in Figure 5. In particular, the
results of Figure 5 show the
usefulness of NACS for single parameter (Figure SA) and multiplexed (Figure
SB) sorting of
antigen-specific CD8+ T-cells. A skilled person would appreciate from the
details of Figure 5
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the specificity of DNA hybridization as well as functionality of spatially-
localized p/MHC
proteins that allow target sorting and in particular cell sorting, with
minimal inter-spot noise.
[00219] To evaluate the sensitivity of detection/sorting performed through the
above
procedure, a further series of the experiments was carried out where the above
procedure was
performed to selectively detect and sort antigen-specific CD8+ T-cells diluted
in a mixture of
other cells at levels of 10%, 1%, and 0.1 % with ssDNA-SA-p/MHC tetramer
arrays.
[00220] The results illustrated in Figure 6 show the usefulness of NACS
approach to
selectively detect and/or sort antigen-specific cells in a mixture of other
cells. In particular, the
micrograph of Figure 6A illustrates cell sorting efficiency, in which the
target T cell populations
are spiked in at the percentages indicated at the top left of each subpanel,
while the histogram of
Figure 6B is a quantitative representation of the such experiments. The
shorter bars represent a
CD8+ T-cell of different antigen-specificity than the object of the sorting
experiment, and so
represent the level of background signal.
Example 4: Optimized scaffolds
[00221] The structure of streptavidin is shown Figure 7A, the structure of
optimized streptavidin
SAC is shown in Figure 7B.
[00222] Here is the crystal structure of the optimized streptavidin-cysteine
protein construct.
Streptavidin is made of four identical subunits (in various shades of gray).
The biotin binding
domain for the protein is illustrated in the figure where the biotin ligand is
bound (white). The
cysteine residues are illustrated as red "balls" at the carboxy termini. This
is the site of
attachment of derivatized DNA. Notice that the two regions are separated by a
distance of about
20-30Angstroms. There is a third element in the crystal structure, illustrated
by lysl2l (gray).
This can also serve as the site of attachment of derivatized DNA (via amine-
NHS coupling)
instead of attachment to the cysteine residues. The problem with this site for
attachment is that it
is too close in proximity to the biotin pocket (i.e. the domains overlap, 7
Angstroms), so this
illustrates an unoptimized scaffold. The diameter of ssDNA is about 10
Angstroms and dsDNA
is about 20 Angstroms. Thus if the DNA were attached to the lysine residue, it
would physical
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overlap the biotin binding region (I OA > 7 A) preventing specific interaction
with biotinylated
binder proteins (e.g. biotinylated p/MHC).
[00223] A synthetic approach in scaffold design allows precise control over
the various
parameters of an optimized scaffold. For example, in the case where peptides
are used as the
scaffold, several parameters involved in scaffold optimization can be
controlled, including the
linker length separating the binding protein and encoding-polynucleotide
attachment regions,
polarity, and valency.
Example 5: Optimized and non-optimzed ssDNA-encoded p/MHC tetramers
[00224] Applicants expressed SAC, coupled the protein with 5'-maleimide ssDNA,
and
verified the formation of conjugates with mobility shift assays.
[00225] In particular, production of ssDNA-SAC conjugates was performed as
follows. The
expression of SAC was performed according to previously published protocols
from a pET 3a
plasmid [Ref. 36]. Prior to conjugation, stock SAC was buffer exchanged to PBS
containing
5mM Tris(2-Carboxyethyl) phosphine Hydrochloride (TCEP) using zeba desalting
columns
(Pierce). MHPH (3-N-Maleimido-6-hydraziniumpyridine hydrochloride, Solulink)
in DMF was
added to SAC at a molar excess of 300:1. In parallel, SFB in DMF (succinimidyl
4-
formylbenzoate, Solulink) was added in a 40:1 molar excess to 5'aminated
oligos. The mixtures
were reacted at room temperature (RT) for 3-4 hours before buffer exchanged to
citrate (50mM
sodium citrate, 150 mM NaCl, pH 6.0) using zeba columns. The SFB-labeled
oligos were
combined in a 20:1 molar excess with the derivatized SAC and incubated
overnight at RT.
Unreacted oligos were removed using a Pharmacia Superdex 200 gel filtration
column at 0.5
ml/min isocratic flow of PBS. Fractions containing the SAC-oligo conjugates
were concentrated
using 10K mwco concentration filters (Millipore). In parallel, ssDNA was
coupled to native SA
for direct comparison.
[00226] The results illustrated in Figure 8, indicate that an engineered
variant of streptavidin
expressing C-terminal cysteine residues has superior biotin binding capacity
compared to native
streptavidin post conjugation with ssDNA. In particular, the results of Figure
8 shows that the
molecular weight of a SAC monomer is -12kDa (Fiture 8A), individual bands
representing
SAC-oligo conjugates differing by one DNA strand can be resolved and that
lower order SAC-
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oligo conjugates (1-2 oligos per protein) run "lighter" when compared to
unmodified SAC
because of the difference in charge/mass density of nucleic acids (Figure 8B).
Higher order
SAC-oligo conjugates corresponding to 3-4 DNA strands per SA were favored
(Figure 8B .
[00227] To test biotin binding capacity, SAC-oligo conjugates were probed with
2-(4'-
Hydroxyazobenzene) benzoic acid (HABA) [Ref. 28], a molecular mimic of biotin
with distinct
optical density coefficients dependent on whether biotin is bound to SA or
not. A biotin:SA
molar ratio of association significantly below 4 in the assay would indicate a
reduction in biotin
binding capacity. Conjugates derived from native SA were greater than one full
unit below the
expected value (2.86 versus 4.0), while conjugates formed with SAC maintained
near optimal
(3.7) binding capacity (Figure 8C).
[00228] In particular, as shown by Figure 8 native SA-oligo conjugates bound -
2.9 moles of
biotin per mole of SA, a significant decline when compared to the 4:1 ratio of
unmodified SAC.
SAC-oligo conjugates maintained near optimum binding capacity (3.7:1).
[00229] These conjugates were then tested across 4 different monoclonal T cell
populations (2
human TCR-transduced cell lines and 2 murine TCR-transgenic splenocyte cell
suspensions).
ssDNA-tagged SAC constructs had markedly higher cell capture efficiencies
compared with
p/MHC tetramers prepared with native SA Fi ure 9). In particular, Figure 9A
show the results
of cell capture experiments performed using native streptavidin where each sub-
panel within
Figure 9A shows the results of a separate experiment using a different cell
type. In Figure 9B,
we are testing cell capture efficiency using the same cells, but with the
optimized SAC scaffold.
A skilled person would appreciate that while the cell capture efficiency of
the experiments of
Figure 9A is highly varied, cell capture efficiency of the experiments of
Figure 9B is uniform
amongst all the cell types. All subsequent NACS tetramers SA constructs were
prepared with the
SAC variant.
[00230] Here the DNA encoding domain, when attached to the un-optimized
scaffold (SA)
reduces biotin binding capacity (binding protein domain) resulting in marked
reduced T cell
capture efficiencies. The optimized scaffold (SAC) minimizes the interaction
between the two
distinct modalities, resulting in higher efficiency T cell sorting.
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[00231] The results illustrated in this example further show an increased
affinity of the binding
molecule by increasing the number of binding molecules comprised in a single
capture agent.
p/MHC proteins as each individual monomer is characterized by a poor affinity
(disassociation
constant = Kd - 10-5 , [Ref 2]). However when four p/MHC proteins are bundled
together to
form a p/MHC tetramer the avidity of the complex is greatly increased (Kd - 10-
9, reference 1).
This increase in avidity due to multi-valency should likewise extend to any
family of protein
binders, aptamers, peptides, small molecules. The effect of valency on the
avidity of the capture
agent complex is clearly seen in Figures 8-9. Here capture agents formed with -
3 p/MHC
proteins were of sufficient avidity to capture a subset of T cell populations
while fully formed
tetramers (4 p/MHC) were able to capture and bind to all 4 T cell populations.
[00232] A skilled person will understand that the principle illustrated in of
this series of
experiments can be adapted to construe other optimized scaffolds, since a
valency a capture
agent can be calculated along the same line of reasoning. In particular, a
skilled person will
understand that independently on the specific binding molecule used a poly
valent scaffold will
always be more avid when compared to a monomer.
Example 6: Microarray Fabrication with polynucleotides including restriction
sites
[00233] All DNA strands were purchased from IDT. DNA microarrays were printed
by the
microarray facility at the Institute for Systems Biology (ISB--Seattle, WA) on
amine-coated
glass slides (GAPS II, Coming) in identical triplicate 12x12 arrays containing
alternative rows of
A, B and C spots, or AEaoRI and BBamx1 with a SMPXB15 pin (Arrayit). Sequences
for all strands
are reported in the following Table 3.
Table 3. Orthogonal DNA sequences for spatial encoding of p[MHC tetramers
Name Sequence SEQ ID NO
A 5' - AAA AAA AAA AAA AAT CCT GGA GCT AAG TCC 11
GTA AAA AAA AAA AAT CCT GGA GCT AAG TCC
GTA AAA AAA AAA AAA A
A' 5'- NHZ- AAA AAA AAA ATA CGG ACT TAG CTC CAG 12
GAT
B 5'- AAA AAA AAA AAA AGC CTC ATT GAA TCA TGC 13
CTA AAA AAA AAA AGC CTC ATT GAA TCA TGC
CTA AAA AAA AAA AAA A
B' 5'- NHZ- AAA AAA AAA ATA GGC ATG ATT CAA TGA 14
GGC
C 5' - AAA AAA AAA AAA AGC ACT CGT CTA CTA TCG 15
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Table 3. Orthogonal DNA sequences for spatial encoding of p[MHC tetramers
Name Sequence SEQ ID NO
CTA AAA AAA AAA AGC ACT CGTCTA CTA TCG CTA
AAA AAA AAA AAA A
C' 5' - NHZ- AAA AAA AAA ATA GCG ATA GTA GAC 16
GAG TGC
AEcoRI 5' - AAA AAA AAA AAA GAG CTA AGT CCG TAG 17
AAT TCA AAA AAA AAA GAG CTA AGTCCG TAG
AAT TCA AAA AAA AAA AAA
AEcoRI' 5' - NH2 - AAA AAA AAA AGA ATT CTA CGG ACT 18
TAG CTC CAG GAT
BBamHI 5' - AAA AAA AAA AAA TTG AAT CAT GCC TAG GAT 19
CCA AAA AAA AAA TTG AAT CATGCC TAG GAT
CCA AAA AAA AAA AAA
BBamHI' 5'- NH2 - AAA AAA AAA AGG ATC CTA GGC ATG ATT 20
CAA TGA GGC
[00234] All sequences of Table 3 to be conjugated to SAC (A', B', C', AEcoRI',
and BBamHI')
were designed with a polyA linker followed by a 20mer hybridization region.
The 5' amine is
required for the attachment of the hetero-bifunctional maleimide derivative
MHPH. Sequences
printed on glass substrates (A, B, C, AEcoRI, and BBamHI) were designed with
two hybridization
regions separated by polyAs. This was designed to facilitate electrostatic
adsorption to amine
glass substrates.
[00235] Sequences AEcoRI and BBamH were designed to include the restriction
site indicated to
allow release, and in particular selective release of the target according to
procedures such as the
ones exemplified in Example 11 below.
Example 7: Performance of p/MHC arrays produced via DNA immobilization and
direct
spotting in comparison with conventional protein arrays
[00236] Applicants directly compared the performance of NACS on the arrays
provided in
Example 6, with conventional direct spotting strategies on various model
substrates. The
substrates were selected to represent the spectrum of surface chemistries
typically used to
immobilize proteins (covalent, electrostatic, hydrophobic, and hydrophilic
adsorption). Serial
dilutions of fluorescent MART-1 SA-PE tetramers (melanoma peptide epitope MART-
126.35
loaded onto HLA-A2.1 MHC molecules) were spotted on the substrates according
to
manufacturer's instructions.
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[00237] T cells were prepared according to the following procedure. cDNA from
the alpha and
beta chains of a TCR specific for tyrosinase368_376 was used. The TCRTYTO
alpha and beta chains
were cloned into a lentiviral vector where both transgenes were linked by a 2A
self-cleaving
sequence as described [Ref. 37]. Concentrated supernatant from this lentiviral
vector was used to
infect Jurkat cells to generate Jurkata-Tyr cells. A MSGV1-F5AfT2AB
retroviral vector
expressing the F5 MART-1 TCR was used. In particular, the MSGV1-F5Aff2AB
retroviral
supernatant was used to infect Jurkat cells to generate the Jurkat '-MART_A
cell line. To generate
primary human T lymphocytes cultures expressing the F5 MART-1 TCR, PBMCs
obtained from
leukapheresis were activated for 48 hours with 50 ng/ml of OKT3 (muromonab
anti-human CD3
antibody, Ortho-Biotech, Bridgewater, NJ) and 300 U/ml of IL-2 (adesleukin,
Novartis,
Emeryville, CA). MSGV1-F5AfT2AB retrovirus supernatant was applied to
retronectin-coated
wells (Takara Bio Inc., Japan).
[00238] Then activated PBMC in RPMI plus 5% human AB serum supplemented by 300
IU of
IL-2 were added to these wells and incubated at 37 C overnight at 5% CO2. On
the following
day, PBMC were transferred to a second set of pre-coated retronectin
retroviral vector tissue
culture plates and incubated at 37 C overnight at 5% CO2. Cells were
subsequently washed and
re-suspended in culture media described above. Frozen leukapheresis fractions
from patients
NRA11 and NRA 13 (UCLA IRB#03-12-023 ) were thawed and incubated overnight in
RPMI
supplemented with 10% human AB serum and 1% penicillin, streptomycin, and
amphotericin
(Omega Scientific) prior to CD8+ enrichment (anti-CD8 microbeads, Miltenyi
Biotech) using an
AutoMACS machine according to the manufacturer's instructions. Following
separation, the
cells were kept at in RPMI-humanAB media containing 30 U IL2/mL.
[00239] Jurkata-MART-1 T cells (the human T leukemia cell line Jurkat
transduced with the F5
MART-1 TCR [Ref. 29] specific for peptide epitope MART-126.35) were then
applied to the array
of Example 6 and the other protein arrays using procedures such as the one
exemplified in
Example 3. The representative images of the arrays collected and quantified
are illustrated in
Figure 10.
[00240] In particular. the results illustrated in Figure 10A (images
collected) and Figure 10B
(images quantified) show little to no T cell capture (electrostatic,
hydrophilic) or significant
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noise (hydrophobic) on the majority of the surfaces investigated compared to
NACS arrays
immobilized with identical concentrations of p/MHC tetramers. In particular,
the illustration of
Figure 10A shows that while cell binding was consistent and uniform for the
optimized scaffold
using SAC, cell binding was observed only on one surface (covalent) but cell
capture was highly
variable. The illustration of Figure 10B shows a quantification of the results
of Figure 10A. A
skilled person will appreciate from the detail of Figure 10 that to achieve
equivalent cell capture
densities, conventional arrays required >5 times more protein material than
the SAC scaffold
(see in particular quantification of Figure 10B)
[00241] In a further series of experiments, the robustness of T cell binding
performed with
NACS was tested in comparison with T cell binding performed with direct
spotting. In particular,
p/MHC tetramers were spotted unto a derivatized surfaced (epoxy functional
group) that was
found to capture T cells (compared to the other surfaces in 00164 that did not
bind cells).
p/MHC tetramers were spotted according to manufacturer's instructions and
directly compared
with a NACS array assembled with the same amount of localized p/MHC protein.
[00242] The results, illustrated in Figure 11 consistency and robustness of
the SAC cell
capture platform (Figure 11A), and corresponding inconsistency of conventional
approaches as
evidence by the large inter-spot cell capture heterogeneity (Figure 11B)
[00243] In particular, T cell binding on a surface (covalent) directly spotted
was observed but
cell capture was highly variable as evidenced by both intra-spot and inter-
spot heterogeneity and
the cross experimental variation illustrated (see Figure 11B).
[00244] On the contrary, the consistency and robustness of T cell
immobilization with NACS
is evident from the image of Figure 11A when compared directly with spotted
arrays, which
suffers from significant levels of inter-spot, intra-spot, and inter-
experimental heterogeneity (see
Figure 11B) .
[00245] Moreover, to achieve equivalent T cell capture densities, NACS p/MHC
arrays
required >5 times less material than covalent immobilization. (p/MHC monomer
at half max
K112 = 1.1 ng for NACS and 5.7 ng for covalent immobilization).
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[00246] The performance and reproducibility of NACS p/MHC arrays is markedly
improved
and represents an integral step towards expanding array-based T cell detection
schemes for
broader applications. This likely has a few causes. First, surface-tethered
ssDNA-p/MHC
tetramers may enjoy greater orientational freedom at the surface/solution
interface compared
with adsorbed proteins which are required to conform to the surface. This
effect may increase
the density of functional protein and consequently reduce the amount of
material required for
array production. Second, the hydration state of the environment during the
production and
subsequent storage of protein arrays is an important factor for array
reproducibility [Ref. 9, 12,
17]. This effect is minimized with NACS because DNA chips can be printed and
stored dry for
extended periods of time and ssDNA-tagged p/MHC tetramer arrays are self-
assembled in
solution immediately prior to an experiment.
Example 8: NACS detection specificity and detection sensitivity
[00247] To evaluate the specificity of p/MHC array assembly and T cell
sorting, a DNA
microarray was printed with the complementary strand (A) along with two
additional distinct
sequences (designated B and C) according to the procedure exemplified in
Example 6.
[00248] NACS ssDNA-p/MHC tetramers (human HLA-A*0201 MHC molecules loaded with
melanoma antigen peptide epitope tyrosinase368_376 with pendant DNA sequence
A') were
hybridized to the DNA microarray so prepared.
[00249] A homogeneous population of Jurkata-Tyr cells (Jurkat cells transduced
with a TCR
specific for tyrosinase368-376) [Ref. 30] was then hybridized to the array
according to procedure
exemplified in Example 3.
[00250] The results, illustrated in Figure 12 show that Jurkata-Tyr T cells
localized to the
complementary spots (A) containing the hybridized cognate p/MHC but not to
spots printed with
the non-complementary sequences B and C (Figure 12A The mean binding capacity
calculated
from three spots (-600 m) was 1486 62 Jurkata-Tyr T cells.
[00251] To illustrate the multiplexing capability of NACS, MART-1 and
tyrosinase ssDNA-
p/MHC tetramers encoded to DNA sequences A and B respectively were combined
and
assembled simultaneously to a three element DNA microarray (strands A, B, and
C). A 1:1
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mixed population of Jurkat"-MART-1 and Jurkata-Ty' cells prestained with
lipophilic dyes (green and
red respectively illustrated in the grayscale version as light gray and dark
gray) was applied to
the array and localized into alternating columns (Figure 12B). Minimal cross-
reactivity was
observed. The average density of spots A and B was a factor of two less than
homogeneous
sorting (661 19 T cells/spot) (Figure 12B). To determine the limit of
detection, target
populations of Jurkata-Tyr cells were spiked in at 10%, 1 % and 0.1 % into
wild type (w.t.) Jurkat
cells and sorted (Figure 12C). The T cell capture density per spot per species
for each mixture
was enumerated and averaged (Figure 12D).
[00252] The number of non-specific w.t. Jurkat cells that adhered to the array
was constant
throughout all dilutions while the number of Jurkata-Tyr T cells captured per
spot decreased
linearly in relation to the fractional composition of Jurkata-Ty' cells with a
detection limit that was
1 in 1000 cells - a limit that corresponds well to the total number of cells
that can be captured
per spot. Thus, the sensitivity of this approach is strictly a geometric
constraint since antigen-
specific T cells that settle on inert areas cannot sample and bind to their
cognate p/MHC
tetramer. The sensitivity can be improved by increasing the size of the
capture region (i.e.
increase spot diameter and/or incorporate spot redundancy) or by reducing
inert regions (i.e.
increase printing density).
[00253] In addition, gentle agitation or microfluidics integration could
potentially allow the
entire T-cell population to sample the entire protein array during cell
sorting. The geometric
layout of the array determines the number of antigen-specificities that can be
interrogated
simultaneously. In the current instance, 600 m spots are printed in 12 by 12
grids (-4 in2),
enabling the potential identification of 144 distinct antigen-specificities
from 106 T cells (typical
number of cells required to cover 1 in2 region).
Example 9: NACS sorting and detection of TCR engineered primary human T cells
[00254] TCR engineering of peripheral blood mononuclear cells (PBMCs) is an
emerging
clinical approach to rapidly generate large numbers of tumor antigen-specific
T cells for adoptive
transfer cell therapy in patients with melanoma and other cancers [Ref. 31,
32]. In this approach
T cells are collected from a patient and transduced with a TCR specific
against a target cancer
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antigen followed by autologous infusion. Demonstrating the feasibility of
detecting TCR
engineered primary human lymphocytes has importance for the clinical
application of NACS.
[00255] Accordingly, in a first series of experiments, human PBMCs containing
CD8+ cells
were obtained from a patient via leukapheresis, expanded and transduced with a
retrovirus vector
containing the F5 MART-1 TCR.
[00256] The results, illustrated in Figure 13A show a transduction efficiency
greater than 90%
as determined by flow cytometry.
[00257] These cells were subsequently sorted on a NACS array with MART-1
(positive control)
and Cytomegalovirus (CMV pp65495-503/HLA-A2.1, negative control) p/MHC
tetramers.
[00258] In particular, the HLA-A*0201 restricted MHC class I monomers loaded
with
tyrosinase369-377 (YMDGTMSQV) (SEQ ID NO: 10) and MART-126-35 (ELAGIGILTV)
(SEQ ID
NO: 11) were produced in house according to previous published protocols (38).
A2.1-restricted
EBV BMLF1259-267 (GLCTLVAML) (SEQ ID NO:21), CMV pp6549s-5o3 (NLVPMVATV) (SEQ
ID NO:22), murine H-2Kb/-OVA257-264 (SIINFEKL) (SEQ ID NO:23), and murine H-
2Db/-
gp10025-33 (KVPRNQDWL) (SEQ ID NO:24) as well as all fluorescent HLA-A*0201
tetramers
were purchased from Beckman Coulter. Lipophilic cell membrane staining dyes
DiO, DiD, and
DiL were purchased from Invitrogen.
[00259] Prior to experiments, microarray slides were blocked to prevent non-
specific cell
binding with 1 mg/ml PEG-NHS ester (Sunbio) in PBS for 2 hours at RT. Four-
fold molar
excess of p/MHC monomers were combined with ssDNA-SAC at 37 C for 20 min.
ssDNA-
p/MHC tetramers were hybridized to DNA arrays for 1 hour at 37 C in 200 l
media and rinsed
with 3% FBS in PBS. T cells (106 /100 l media) were incubated on the array at
37 C for 30
min. The arrays were rinsed with 3% FBS in PBS and cell capture visualized via
brightfield
(Nikon Eclipse TE2000) and/or confocal microscopy (Nikon E800). Post T cell
capture p/MHC
tetramer staining was done by incubating the array with 200 l of media
containing fluorescent
p/MHC tetramer along with fluorescent cDNA (Cy5-A' and/or Cy3-B'). The arrays
were rinsed
with 3% FBS in PBS prior to imaging. For selective T cell release experiments,
three identical
arrays were used to immobilize cells. Treatment with EcoRl, BamHI, or DNase
was in RPMI
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media for 1-2 hours at 37 C. DNase was purchased from Sigma, all other enzymes
from
NEbiolabs.
[00260] For p/MHC comparative studies, SuperEpoxy and SuperProtein
(representing covalent
and hydrophobic surfaces respectively) were purchased from Arrayit (Sunnyvale,
CA). Amine
GAPS II slides (electrostatic) were purchased from Coming. Polycarboxylate
hydrogel
(hydrophilic) slides were purchased from XanTec (Germany). Fluorescent MART-1
tetramers
were printed according to manufacturer's instructions for each slide. Cell
sorting images were
quantified with ImageJ (NIH) and fitted to the Hill Function (NACS n=2,
R2=0.95, Covalent
n=2. 1, R2 =0.97) with Origin (OriginLab, MA).
[00261] The transduced T cells were immobilized to the MART-1 regions only.
The antigen-
specificity of the captured cells was doubly validated by staining with
fluorescent MART-1 and
CMV p/MHC tetramers (red and blue respectively) after the cells were
immobilized on the
arrays.
[00262] The results illustrated in Figure 13B and Figure 13C show that no
cells were stained
specific for CMV (see dotted circles in Figure 13B) and that the cell capture
was specific (see
overly of confocal and brightfield images of Figure 13C)
Example 10: NACS sorting and detection of endogenous primary human T cells
[00263] NACS detection of primary human T cells isolated from peripheral blood
was
performed. This is because, in general detection of primary human T cells
isolated from
peripheral blood is generally more demanding than cultured cell lines because
a single
population of antigen-specific T cells is present within a large background of
differing blood
cells and of T cells expressing monoclonal and polyclonal TCRs of diverse
specificities. In
addition, these T cells would be expressing endogenous levels of TCR.
Applicants explored
whether the same attributes of NACS that were found in the above examples
would apply
equally to endogenous primary human T cells.
[00264] Frozen leukapheresis samples from patients NRA 11 and NRA13 were CD8+
enriched
and prior to NACS sorting.
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[00265] Quantity and specificity of EBV specific and CMV specific T cells in
patient NRA11
and NRA13 were stained with fluorescent EBV and CMV p/MHC tetramers and
analyzed by
flow cytometry
[00266] The results, illustrated in Figure 14, show that lymphocytes isolated
from NRA13
contained significant levels of EBV specific T cells (4.9%) with minimal CMV
specific T
cells.(see Figure 14A) while Lymphocytes isolated from NRA11 contained high
levels of CMV
specific T cells (9%) with a low population of EBV-specific cells (0.12%) (see
Figure 14B).
[00267] Following this determination, the leukaphersis from patient NRA 11 and
NRA 13 were
analyzed with NACS technology in one-target detection and a multiplexed
detection
experiments.
[00268] For the one target detection experiments, frozen leukapheresis samples
from patient
NRA13 were CD8+ enriched and applied to a CMV and Epstein-barr virus (EBV
BMLF1259_
267/HLA-A2.1) p/MHC array provided by the procedure of Example 6.
[00269] For multiplexed detection, a 1:1 mixture of EBV-specific and CMV-
specific CD8+ T
cells was produced by combining NRA13 lymphocytes with CMV-specific T cells
from patient
NRA11 and the mixture applied to a CMV and Epstein-barr virus (EBV
BMLF1259.267/HLA-
A2. 1) p/MHC array provided by the procedure of Example 6.
[00270] The results illustrated in Figure 15 show that in both experiments T
cells were
selectively and quantitatively detected. In particular, in the detection
performed with
leukepheresis of patient NRA13 only T cells were captured within the EBV
regions only (see
Figure 15A). T cells detected from a 1:1 mixture of NRA11 and NRA 13 (left
panel) were
verified to be specific for EBV and CMV. In particular, following NACS cell
sorting and
fluorescent p/MHC tetramer staining, the populations were complementary
stained for the
appropriate antigen-specificity (Figure 15B).
[00271] T cells from patient NRA 13 were serially diluted to create mixtures
of cells that
contained EBV-specific T cells (-0.4%, 0.2%, and 0.1% by FACS (figure 14C).
The three
mixtures of EBV specific T cells were detected on a array encoded with EBV/HLA-
A2.1
tetramers, represented in Figure 15D
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[00272] The results, illustrated in Figures 15C and 15D show that isolated
hits were resolved
in frequencies as low as -0.1 % (Figure 15D, dark grey arrows). The number of
unstained cells
within the capture regions (black arrow) was constant throughout all dilutions
(- 1-2 cells/spot)
and likely represents the level of background from non-specific interactions.
It should be noted
that while we incorporated fluorescent p/MHC tetramer staining after T cell
immobilization for
illustrative purposes, the specificity of the captured cells could be
determined solely from the
registry of the array.
Example 11: Controlled and Selective release throuth restriction endonucleases
of T cells
Immobilized using NACS approach
[00273] Antigen-specific T cells immobilized onto glass are immediately
available for
secondary assays, since many such as immunohistochemistry (IHC), fluorescent
in situ
hybridization (FISH) and cytokine secretion assays [Ref. 5, 7] are
traditionally performed or are
compatible with cells localized to a substrate. However, several other
relevant assays, such as
those designed to assess T cell phenotype or functional status like
genomic/mRNA analysis or
simply, further culture for phenotypic enrichment would require a method for
releasing the
captured cells. Any release scheme should ideally be selective for given cell
types. For NACS,
Applicants explored whether the DNA tethers could be selectively cleaved by
exploiting the
sequence specificity of restriction endonucleases.
[00274] Applicants integrated unique restriction sites to each DNA sequence
employed for cell
sorting (see Example 6 above), and found that the adhesion of different
populations of antigen-
specific T cells could, in fact, be independently controlled (Figure 16b).
[00275] In particular oligonucleotides A and B of Example 6 were modified by
incorporating 6
bp restriction sites specific for endonucleases EcoRl and BamHI respectively
thus obtaining
oligonucleotides AECOR1 and BBanx1 following the approach schematically
illustrated in Fi ure
16A.
[00276] DNA microarrays were therefore printed with orthogonal sequences
containing EcoRl
and BamHI restriction sites. Jurkata-MART-1 and Jurkata-Tyr cells prepared as
exemplified in
Example 7 and prestained with lipophilic dyes (red and green respectively)
were then sorted on
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an array printed with DNA sequences AEaoR1 and BBamx1 according to a procedure
exemplified in
Example 6.
[00277] The results illustrated in Figure 16B show sorting of AEaoR1 and
BBamH1 as well as
correct release of a selected target following treatment with the
corresponding restriction
enzyme.
[00278] In particular, the cell were first immobilized on the array as
illustrated in Figure 16Bi
where the red dye is shown as dark gray and the green dye is shown as light
gray.
[00279] After T cell immobilization, the array was treated with Ban HI which
cleaved the BBamx1
spots and selectively released the bound Jurkata-T" cells (Figure 16B ii).
Conversely, on a
separate but identical array, Jurkata-M`e'RT-1 cells were released after
treatment with EcoRl (Figure
16Biii . A second round of enzymatic treatment with the complementary
endonuclease (EcoRI
to state (ii) or BamHI to state (iii)) removed the remaining adherent cells
(Figure 16Biv).
Alternatively all captured cells (i) could be released non-selectively in a
single step with the
addition of DNase (data not shown).
Example 12: Procedure for performing NACS Detection of Targets captured in
solution
[00280] Target population of cells can be captured according to a procedure
where the encoded
capture agent is contacted with a target before binding the substrate.
[00281] An exemplary series of experiments performed according to this
approach is illustrated
in Figure 17. The biomarkers of interest in a biological sample can bind to
polynucleotide-
encoded capture agents in solution. After binding, the entire sample can be
applied to a substrate
printed with the cDNA. The biological sample, upon application to the
substrate, with localize to
spatially distinct locations mediated by DNA hybridization. The cargo bound to
the
polynucleotide-encoded capture agents can then be identified using convention
fluorescent and
other visualization techniques identifiable by the skilled reader.
[00282] In the experiments of Figure 17, encoded-polynucleotide capture agents
can be used
to bind to biological targets on cell surfaces in solution. This is
demonstrated in panel A, in
which fluorescent polynucleotide-encoded tyrosinase/HLA-A2.1 p/MHC tetramers
were used to
stain Jurkato'-T cells and analyzed with flow cytometry. This was directly
compared with
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fluorescent tyrosinase/HLA-A2.1 p/MHC tetramers. Both reagents stained Jurkat
'-Tyr with
equal intensities. Jurkata-Tyr cells prestained with polynucleotide-encoded
tyrosinase/HLA-
A2.1 p/MHC tetramers were captured and detected on an array mediated by DNA
hybridization
(panel B). Please provide description of the results with reference to the
figure.
Example 13: Additional optimized scaffolds Capture agents comprising a protein
binding
molecule
[00283] Additional optimized scaffolds can be provided by Protein A, Protein
G, and Protein
A/G which is a family of bacterial recombinants that bind to the Fc domain of
all subclasses IgG,
and in limited extent to IgA, IgE, IgM and IgD. In particular, these proteins
can serve as an un-
optimized scaffold in the embodiment where DNA is attached randomly to free
lysine residues
on the surface of the protein via NHS-amide coupling chemistry. The binding
proteins are IgG
antibodies against a desired biomarker (e.g. CD3 found on T cells). Molar
excess of IgG
antibodies can be incubated with ssDNA-Protein A/G and employed to sort and
capture
biological targets of interest (e.g. anti-CD3 -proteinA/G-ssDNA conjugates can
be used to sort
out T cells from a complex mixture of cells).
[00284] Illustrated in Figure 18, is a hypothetical figure of a branched
peptide. Here the length
of the peptide is 9 amino acids, stretching from the N-terminal alanine to C-
terminal tyrosine
residues. There is a branch point at the lysine residue in position 4 from the
C-terminus to
provide a two-fold valency to this scaffold. The reactive maleimide groups at
the N-terminus is
the chemical handle to attach two binder proteins with free thiols, and the
encoding-
polynucleotide can be attached to the lysine residue next to the c-terminal
tyrosine residue via
NHS-amide coupling. Because the attachment sites for the binder proteins and
the encoding-
polynucleotide are separated with this branched-peptide approach, the
resulting capture agent is
fully polar and optimized. Exemplary binder molecules can be RGD peptides
containing
cysteine residues (Anaspec, #29897). The targets to detect include cells which
express integrin
receptors (ATCC, #CCL-92).
[00285] Another optimized protein scaffold is the protein SAC3 which is
represented by the
primary amino acid sequence:
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HMGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGT
ALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGH
DTFTKVGGSGCGGSGCGGSGCP (SEQ ID NO: 25)
[00286] This alternate optimized streptavidin scaffold contains 3 cysteine
residues at the c-
terminus for site specific attachment of the encoding-polynucleotide. By
combining with molar
excess of biotinylated p/MHC (tyrosinase/HLA-A2. 1) with ssDNA-SAC3, the
resulting capture
agent can be used to target T cells from a complex mixture. In Figure 19, the
efficiency of T
cell capture (Jurkat-TS'r ) of the optimized scaffold SAC3 (right panel) is
directly compared with
the optimize scaffold SAC. The cell capture efficiency is identical.
[00287] Any form of optimization scheme of SA will preserve the 4:1 ratio
binding with biotin.
Applicants have provided more than one SA optimized and different optimized SA
work
differently for certain target. The scaffold is eventually selected in view of
the ability to bind the
target. Branched peptides, such as the one exemplified in Figure 18 can be
used as a multimeric
scaffold alternative to SA bind, we can have monomeric or dimeric or n meric
as well depends
on the experimental design.
Example 14: Capture agents comprising a protein binding molecule
[00288] Alternative binder proteins and scaffolds can be employed. Integrins
are heterodimeric
cell surface receptors that bind to extracellular matrix proteins generally
consisting of the peptide
motif arginine-glycine-aspartate. Biotionylated peptides containing the RGD
motif can be
purchased from Anaspec (#62347). These peptide binding molecules can be
assembled with an
optimized scaffold like SAC-A and used to sort cell populations contain target
cells expressing
integrins, including 3T3 fibroblasts (ATCC, #CCL-92).
Example 15: Capture agents comprising a linker
[00289] The linker serves to connect the binding protein to the scaffold. This
can be composed
of a peptide sequence. For example, the linker GGGLNDIFEAQKIEWHE (SEQ ID NO:
26)
can be appended to the C-terminus of a HLA-A2.1 MHC molecule. A biotin
molecule can be
attached to the resulting linker with the enzyme BirA ligase. This
biotinylated p/MHC construct
can be used in conjunction with an optimized A-SAC scaffold to sort antigen
specific cells on a
cDNA printed substrate.
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[00290] Another linker can be composed of the peptide LCTPSRGSLFTGR (SEQ ID
NO: 27)
which can also be appended to the c-terminus of binding proteins like MHC
molecules. The
glycine residue can be modified to an aldehyde group with the enzyme
formylglycine generating
enzyme (FGE). Scaffolds containing hydrazide (R-NH-NH2) (e.g. reacting SANH
(Succinimidyl 6-hydrazinonicotinate acetone hydrazone, Solulink) with the
scaffold SA) can be
used to attach the aldehyde-MHC binder protein.
[00291] An example of a linker can be the poly-uracil RNA sequence U U U U UU
U U U U (SEQ
ID NO: 28). This linker can be cleaved in the presence of RNase A, an
endoribonuclease that
cleaves the 3' end of unpaired (i.e. single stranded) C and U residues.
Example 16: ImmunoPCR to profile surface markers of Cells capture with NACS
[00292] T cells that are captured on an array can be analyzed further. One
analytical assay is
immunoPCR [Ref. 51]. Here antibodies labeled with distinct DNA tags (DEAL
conjugates) are
used to bind to a target of interest, which in this example, is a biomarker
expressed on the cell
surface. After binding, the DNA tag on the antibody can be amplified and
detected using PCR.
While this has been shown to work with protein from solutions, Applicants show
that this
concept is feasible when the biomarker is confined to a cell surface. A cell
line expressing
EGFR (CD cells) and a cell line with null expression of EGFR (Jurkat cells) as
shown by Fi ure
20 (top right panel, validated by flow cytometry) are both stained with a-EGFR-
ssDNA DEAL
conjugate. After staining the tag on the EGFR antibodies were amplified with
PCR and Q-PCR
(lower panels). The presence of the tags was detected within 14.6 cycles (CD
cells) and 23.2
cycles (Jurkat cells). This corresponds to a signal to noise of - 400:1.
[00293] In parallel, p/MHC tetramers encoded with DNA were used to detect the
presence of a
TCR on T cells. Two cell lines, one expressing a TCR specific for the MART-1
antigen (Jurkata'-
MART1) and another cell line expressing a TCR specific for Tyrosinase antigen
(Jurkata-Tyr, both
cell lines were verified by staining with fluorescent p/MHC tetramers and
analyzed by flow
cytometry, Figure 20, top left panel) were stained with MART-1/HLA-A2.1 p/MHC
tetramers
encoded with DNA. The DNA tag was amplified by PCR and detected with gel
electrophoresis.
Jurkata'-Mart-A cells were detected within 15 cycles, while the Jurkata-Tyr
cells that did not express
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the cognate TCR appeared within the gel at around 10 cycles. Thus the presence
of the MART-1
specific TCR was detected specifically.
Example 17: Dynamic functional profiling of T cells using NACS and DEAL
coniuuates:
[00294] Applicants proceeded by integrating an ELISPOT-type sandwich assay
with p/MHC
NACS to detect cytokines produced by captured murine TCR transgenic
splenocytes "on-the-
spot" (Figures 21, 22). Three murine anti-cytokine antibodies (IL-2, IFN-y and
TNF-a) were
encoded with DNA strands A', B', and C' respectively. H-2Kb-OVA257-264 ssDNA-
p/MHC
tetramers were encoded to all three strands. The ssDNA-p/MHC tetramers and
antibody
conjugates were pooled and assembled to a microarray printed with the
complementary strands
A, B and C. Murine OT1 lymphocytes (derived from TCR transgenic mice in which
most
splenocytes are specific for the model antigen OVA257-264), were then seeded
on the array.
Following incubation periods of 2, 5, or 18 hours, pooled cytokine detection
antibodies were
added and the slide imaged by confocal microscopy (Fig. 22A . The inflammatory
cytokine
IFN-y was detected at time points 5 and 18, manifest as discrete diffusive
clusters (-50-100 m
in diameter at 5 hrs) that increased in average diameter temporally,
attributable to molecular
diffusion and sustained secretion. Examination of the local vicinity of each
burst showed that
underlying each fluorescent cluster was a single cell while neighboring cells
appeared to be non-
responders (Fig. 22B), suggestive that each IFN-y burst was derived from a
single cell. The
number of IFN-y clusters remained constant at -3 between hours 5 and 18,
indicating no increase
in the number of activated T cells between those hours. No significant levels
of murine IL-2 and
TNF-a were detected at these time points.
[00295] In the preceding examples, the Applicants have described a method for
generating
robust and modular p/MHC arrays for high efficiency target detection and
sorting, exemplified
with particular reference to T cells. The inclusion of a larger set of
orthogonal DNA sequences
[Ref. 19, 24] will enable the modular assembly of higher order p/MHC arrays
for T cell
screening experiments (e.g. one working set of DNA sequences can be used
interchangeably to
generate any combination of p/MHC arrays). This would find immediate utility
in the field of
TCR peptide epitope discovery where recently, novel antigen peptides were
discovered via high-
throughput CD8+ screening experiments utilizing multi-color flow cytometry in
mice and
humans [Ref. 33, 34] (as many as 2,000 distinct p/MHC tetramers were prepared
and tested).
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[00296] NACS arrays are expected to streamline such experiments. Although
certain
traditional methods of producing single p/MHC monomers are time and labor
intensive, recent
reports using conditional peptide exchange technology enables the relatively
straightforward
construction of 1000 element p/MHC libraries rapidly [Ref. 34-36]. The
integration of NACS
with these peptide exchange technologies is a realistic option. NACS arrays
outperform
conventional spotted arrays assessed in key criteria such as repeatability and
homogeneity. The
versatility of employing DNA sequences for cell sorting is exploited to enable
the programmed,
selective release of target populations of immobilized T cells with
restriction endonucleases for
downstream analysis. Because of the performance, facile and modular assembly
of p/MHC
tetramer arrays, NACS holds promise as a versatile platform for multiplexed T
cell detection.
[00297] Applicants have also demonstrated a number of advantages of the NACS
platform. It
significantly outperforms certain literature approaches that utilize surface-
bound p/MHC
tetramers to capture cells. It is a simple and inexpensive to implement since
cell sorting is
performed on glass substrates prepared via traditional DNA printing
technologies. In addition,
sorted cells may be selectively released, which should permit for the
deployment of a host of
bioanalytical methods on NACS sorted cells.
[00298] Applicants expect that NACS will find uses beyond multiplexed sorting
of T cells
based on TCR specificity. The principal components of the platform used in the
preceding
examples -streptavidin-cysteine core and orthogonal single stranded DNA
sequences-were
rationally developed to enable oriented coupling and spatial addressing. Thus
this platform is
amenable to any family of binding proteins or small molecule binders labeled
with biotin. The
increase in avidity of p/MHC tetramers over monomers as a consequence of the
valency of SA
should likewise extend to other capture agents, making it feasible to generate
cellular arrays with
probes ranging from high to moderate affinities like antibodies, aptamers or
peptides.
[00299] In particular, the NACS approach can be used in therapeutic and/or
diagnostic
applications involving MHC complexes. The MHC complex consists of a fragment
of an antigen
(a peptide) lying within the groove of a major histocompatibility complex
(MHC) molecule. A
portion of the TCR has affinity for the MHC, and a variable portion has an
affinity for the
peptide antigen. This peptide may be a very short fragment <<10 amino acids
long), and so the
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affinity between the TCR and the MHC/antigen may be weak. Nevertheless, for
many
fundamental and clinical purposes, sorting T-cells by their antigen
specificity is critically
important. Such sorting can be used to determine how the immune system is
responding to some
disease, such as an infection or a cancer. It is also a key part of various
immunotherapies that
find applications in the treatment of certain cancers, such as melanoma. In
that case, T-cells from
a patient are collected, they are genetically modified so that they become
antigen-specific T-cells
that are encoded to identify and kill certain cancer cells.
[00300] Once modified, those T-cells are put back into the patient. All of the
steps in this
therapy involve sorting T-cells, but the last step - identifying which T-cells
have evolved into
antigen specific T-cells, involves sorting of the T-cells by their antigen
specificity. High affinity
reagents that can sort the various antigen specific T-cells from a complex
mixture are central to
such therapies.
[00301] Other examples exist in which it is desirable to sort cells by various
membrane
proteins, but those membrane proteins do not provide a good 'handle' for
antibody-based sorting
methods.
[00302] An example is cancer cells that express a genetic mutant of the EGFR
protein called
EGFR-VIII. EGFR-VIII is an onco-protein - meaning that it is an important
genetic mutation that
can lead to cancer. However, it is also a membrane protein in which most of
the extracellular
portion of that protein has been cleaved. The remaining portion represents a
small 'handle', and
so antibodies to EGFR-VIII exhibit only a weak affinity for that protein. The
multiplexed sorting
of cells within a tumor by various cancer-related proteins such as EGFR, EGFR-
VIII, etc., can
provide key diagnostic information about the cancer, which in turn can be
utilized to direct
therapies or combination therapies.
[00303] In summary, in several embodiments provided herein are polynucleotide-
encoded
capture agents for target detection and in particular modular polynucleotide-
capture agents
comprising a target binding component, a scaffold component and an encoding
component
formed by standardized molecular units that can be coupled and decoupled in a
controlled
fashion, and related compositions methods and systems.
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[00304] The examples set forth above are provided to give those of ordinary
skill in the art a
complete disclosure and description of how to make and use the embodiments of
the, capture
agents, devices, systems and methods of the disclosure, and are not intended
to limit the scope of
what the inventors regard as their disclosure. Modifications of the above-
described modes for
carrying out the disclosure that are obvious to persons of skill in the art
are intended to be within
the scope of the following claims. All patents and publications mentioned in
the specification are
indicative of the levels of skill of those skilled in the art to which the
disclosure pertains. All
references cited in this disclosure are incorporated by reference to the same
extent as if each
reference had been incorporated by reference in its entirety individually.
[00305] The entire disclosure of each document cited (including patents,
patent applications,
journal articles, abstracts, laboratory manuals, books, or other disclosures)
in the Background,
Detailed Description, and Examples is hereby incorporated herein by reference.
Further, the hard
copy of the sequence listing submitted herewith and the corresponding computer
readable form
are both incorporated herein by reference in their entireties.
[00306] It is to be understood that the disclosures are not limited to
particular compositions or
biological systems, which can, of course, vary. It is also to be understood
that the terminology
used herein is for the purpose of describing particular embodiments only, and
is not intended to
be limiting. As used in this specification and the appended claims, the
singular forms "a," "an,"
and "the" include plural referents unless the content clearly dictates
otherwise. The term
"plurality" includes two or more referents unless the content clearly dictates
otherwise. Unless
defined otherwise, all technical and scientific terms used herein have the
same meaning as
commonly understood by one of ordinary skill in the art to which the
disclosure pertains.
[00307] Although specific methods and materials are described in the present
disclosure, any
methods and materials similar or equivalent to those described herein can be
used in the practice
for testing of the specific examples described herein.
[00308] A number of embodiments of the disclosure have been described.
Nevertheless, it will
be understood that various modifications may be made without departing from
the spirit and
scope of the present disclosure. Accordingly, other embodiments are within the
scope of the
following claims.
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