Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 2022/104147
PCT/US2021/059254
ADDITIVES FOR REDUCING NON-SPECIFIC INTERACTIONS BETWEEN
FLUORESCENT POLYMER CONJUGATES AND CELLS IN A
BIOLOGICAL SAMPLE
[0001] This application is being filed 12 November 2021, as
a PCT International
Patent application and claims the benefit of priority to U.S. Provisional
Application Serial
No. 63/113,703, filed 13 November 2020, which is incorporated by reference
herein in
its entirety.
BACKGROUND
[0002] Polymer dye conjugates are bright and provide
excellent performance
that can be utilized in single color or multi-color flow cytometry assays. In
general,
polymer dye conjugates exhibit high brightness due to their unique and complex
structure. But that same unique and complex structure also may lead to some
significant
limitations. The instant disclosure addresses these limitations.
SUMMARY
[0003] Because of their nature, polymer dye conjugates can
bind non-
specifically to cells in a biological sample, such as monocytes and
granulocytes in a
peripheral blood sample. Non-specific binding could lead to misinterpretation,
resulting
in false positive inferences. For example, when a polymer dye conjugate comes
in
contact with blood during the analysis of cellular markers, the conjugates may
bind to
cells, such as monocytes and/or granulocytes, non-specifically thereby either
giving a
signal that can be misinterpreted as positive population or pulls out
populations other
than the desired ones.
[0004] The instant disclosure provides a solution to these
and other problems
associated with use of polymer dye conjugates. In some embodiments, the
disclosure
provides a composition for reducing or eliminating non-specific binding of a
dye
conjugate to cells in a biological sample, the composition comprising a dye
conjugate
and a surfactant as described herein.
[0005] In some embodiments, the instant disclosure provides
a method for
reducing or eliminating non-specific binding of at least one dye conjugate to
cells in
biological sample, the method comprising: contacting that at least one dye
conjugate
with at least one zwitterionic surfactant before, during, or after the dye
conjugate is
contacted with the biological sample, the contacting resulting in decreased
non-specific
binding of the at least one dye conjugate in the sample. In some embodiments,
the
instant disclosure provides a method for reducing or eliminating non-specific
binding of
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at least one to cells in a blood sample, the method comprising: contacting the
at least
one dye conjugate with at least one anionic surfactant before, during or after
the dye
conjugate is contacted with a blood sample, the contacting resulting in
decreased non-
specific binding of the at least one dye conjugate in the blood sample. The
compositions
and methods of the disclosure reduce or eliminate non-specific binding of a
polymer dye
conjugate or a non-polymeric dye conjugate to monocytes and/or granulocytes in
a
blood sample.
[0006] A method is provided for reducing or eliminating non-
specific binding of
at least one dye conjugate in a biological sample, such as a blood sample, the
method
comprising: contacting the at least one dye conjugate with at least one
zwitterionic or
anionic surfactant before, during, or after the polymer dye conjugate is
contacted with a
biological sample, the contacting resulting in decreased non-specific binding
of the at
least one polymer dye conjugate in the biological sample.
[0007] In some embodiments, the biological sample may be a
blood sample. In
some embodiments, the cell may be white blood cell(s) and the decreased non-
specific
binding may comprise decreased non-specific binding to a white blood cell in
the blood
sample. In some embodiments, the white blood cell is selected from the group
consisting
of monocytes and granulocytes.
[0008] In some embodiments, the method comprises adding the
surfactant to
the polymer dye conjugate before contacting the polymer dye conjugate with the
biological sample, such as a peripheral blood sample.
[0009] In some embodiments, the method comprises adding the
surfactant to
the blood sample before the contacting with the polymer dye conjugate.
[0010] The surfactant may be a compound of the formula:
R1[CO-X(CH2);]g4N+(R2')(R3')]k-(CH2)f[CH(OH)CH21,-Y-, wherein
R1' is a saturated or unsaturated C5-24 alkyl;
X is NH, NR4', wherein R4' is 01_4 alkyl, 0 or S;
j is an integer from 1 to 10;
g is 0 or 1;
R2' and R3' are independently a Ci_4 alkyl;
k is 0 or 1;
the hydroxyl is optionally substituted by methyl, ethyl, hydroxymethyl, or
hydroxyethyl;
f is an integer from 0 to 4;
his 0 or 1; and
Y is COO, SO3, OPO(0R5')O, or P(0)(0R5')O, wherein R5' is H or C1_4 alkyl, and
when
k=0, the surfactant may be in acidic form, or sodium, or potassium salts
thereof.
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[0011] In some embodiments, the surfactant may be a
zwitterionic surfactant
compound of the formula:
RICO-X(CH2)j]g-N+(R2')(R3')-(CH2)f-[CH(OH)CH21h-Y,
wherein:
R1' is saturated or unsaturated 05-24 alkyl;
X is NH or N R4', wherein R4' is 01-4 alkyl, 0 or S;
j is an integer from 1 to 10;
g is 0 or 1;
R2' and R3' are independently a Ci_4 alkyl;
the hydroxyl is optionally substituted by methyl, ethyl, hydroxymethyl, or
hydroxyethyl;
f is an integer from 1 to 4;
his 0 or 1; and
Y is COO, SO3, OPO(0R90 or P(0)(0R90, wherein R5' is H or a 01_4 alkyl
residue.
[0012] The zwitterionic surfactant may be a compound of the
formula:
R'"-N-f(CH3)2-C1-12C00-:
R'"-CO-NH(CH2),-N'(CH3)2-CH2C00--;
R''-i\l'(CH3)2-CH2CH(OH)CH2S03-; or
Ri'-CO-NH-(C1-12)3-N+(CH3)7-CH2CH(OH)CH2S03-.
[0013] In some embodiments, the zwitterionic surfactant may
be selected from
the group consisting of almondamidopropyl betaine, apricotamidopropyl betaine,
avocadamidopropyl betaine, babassuamidopropyl betaine, behenamidopropyl
betaine,
behenyl betaine, canolamidopropyl betaine, capryl/capramidopropyl betaine,
carnitine,
cetyl betaine, cocamidoethyl betaine, cocamidopropyl betaine, cocamidopropyl
hydroxysultaine, coca betaine, coca hydroxysultaine, coco/oleamidopropyl
betaine,
coco sultaine, decyl betaine, dihydroxyethyl leyl glycinate, dihydroxyethyl
soy
glycinate, dihydroxyethyl stearyl glycinate, dihydroxyethyl tallow glycinate,
dimethicone
propyl PG-betaine, drucamidopropyl hydroxysultaine, hydrogenated tallow
betaine,
isostearamidopropyl betaine, lauramidopropyl betaine, lauryl betaine, lauryl
hydroxysultaine, lauryl sultaine, milk amidopropyl betaine, milkamidopropyl
betaine,
myristamidopropyl betaine, myristyl betaine, oleamidopropyl betaine,
oleamidopropyl
hydroxysultaine, oleyl betaine, olivamidopropyl betaine, palmamidopropyl
betaine,
palmitamidopropyl betaine, palmitoyl carnitine, palm kernel amidopropyl
betaine,
polytetrafluoroethylene acetoxypropyl betaine, ricinoleamidopropyl betaine,
sesamidopropyl betaine, soyamidopropyl betaine, stearamidopropyl betaine,
stearyl
betaine, tallowamidopropyl betaine, tallowamidopropyl hydroxysultaine, tallow
betaine,
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tallow dihydroxyethyl betaine, undecylenamidopropyl betaine, and wheat germ
amidopropyl betaine. In some embodiments, the surfactant is lauryl betaine.
[0014] In some embodiments, the surfactant may be an
anionic surfactant
compound of the formula:
RI00-X(CH2)j]g-(0H2)f[CH(OH)CH2b-Y-, wherein
RI is a saturated or unsaturated 05-24 alkyl;
X is NH, NR4', wherein R4' is 01-4 alkyl, 0, or S;
j is an integer from 1 to 10;
g is 0 or 1;
R2' and R3' are independently a 01-4 alkyl;
the hydroxyl is optionally substituted by methyl, ethyl, hydroxymethyl, or
hydroxyethyl;
f is an integer from 0 to 4;
his 0 or 1; and
Y is COO, SO3, OPO(0R5)0 or P(0)(0R90, wherein R5' is H or 01_4 alkyl and
wherein
the anionic surfactant may be in acidic form, or sodium, or potassium salt
forms thereof.
In some embodiments, f=0. In some embodiments, f=1. In some embodiments, f=3.
In
some embodiments, f=4. In some embodiments, Y is COO or S03. In some
embodiments, R2' and R3' are methyl.
[0015] The anionic surfactant may be a compound according
to the formula
R1'¨CO-N(CH3)-CH2-000-; or
R1'¨CO-N(CH3)-CH2-S03-, and sodium or potassium salts thereof, wherein
R1' is saturated or unsaturated 05-24 alkyl. In some embodiments, R1' may be a
saturated
or unsaturated C7_19 alkyl, or C11-17 alkyl.
[0016] In some embodiments, the anionic surfactant may be
selected from the
group consisting of N-lauroyl sarcosine, sodium lauroylsarcosinate, sodium
palmitoyl
sarcosinate, sodium stearoyl sarcosinate, N-methyl-N-(1-oxotetradecyl)-glycine
sodium
salt, sodium caproyl sarcosinate, sodium capryloyl sarcosinate, N-methyl-N-(1-
oxo-9-
octadecen-1-y1)-glycine, sodium salt, sodium oleoyl sarcosinate, and sodium
linoleoyl
sarcosinate. In some embodiments, the anionic surfactant is N-lauroyl
sarcosine.
[0017] In some embodiments, the polymer dye conjugate
comprises a binding
partner conjugated to a polymer dye having the structure of Formula III:
G1 ___________________ A a ( __________________ G2
-m(III)
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wherein,
each A is independently selected from the group consisting of an aromatic co-
monomer and a heteroaromatic co-monomer;
each optional M is independently selected from the group consisting of an
aromatic co-monomer, a heteroaromatic co-monomer, a bandgap-modifying monomer,
optionally substituted ethylene, and ethynylene;
each optional L is a linker moiety;
each G1 and G2 are independently selected from an unmodified polymer
terminus and a modified polymer terminus;
a, c, and d define the mol % of each unit which each can be evenly or randomly
repeated and where each a is a mol % from 10 to 100%, each c is a mol % from 0
to
90%, and each d is a mol % from 0 to 25%;
each b is independently 0 or 1;
and each m is an integer from 1 to about 10,000.
[0018] In some embodiments A comprises a DHP moiety.
In some
embodiments, A comprises a fluorene moiety. In some embodiments, A comprises a
DHP and a fluorene moiety.
[0019] In some embodiments, the polymer dye conjugate is a
polymer of
Formula
(I)
R1
G2.
a \ b
c /a
¨ In
wherein
each X is independently C or Si;
each Y is independently CR1R2 or SiR1R2,
each R1 is independently an ammonium alkyl salt. an ammonium alkyloxy salt, an
ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt; a
sulfonate
oiigoether salt, a sulforiamido oiigoether, or a water solubilizing moiety:
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..õ...õ.so2
(CH2r
rffj\rr'-r
each R2 is independentiy H, akyi, akenyl, alkynyl, cycloalkyl, haloalkyl,
alkoxy,
(hetero)aryloxy, :aryl, (hetero)arylarnino, a PEG group, an ammonium alkyl
salt, an
ammonium alkyloxy salt, an amrric.)nium oiigoether salt, a sulfonate alkyl
salt, a sultanate
alkoxy salt, a sulfonate oligoether salt, a sulfonarnido oligoether, or a
water solubzing
moiety
=
SO2
(CH2)"
r>rfr =
each R3 is independently selected from the group consisting of H, alkyl,
aikene, alkyne,
cycloalkyl, haloalkyl, aikoxy, (hetero)aryloxy, aryl, (hetero)arylarnino, and
a PEG group;
each Z is independently selected from the group consisting of C, 0, and N;
each 0 is independently selected from the group consisting of a bond, NH,
NR4., and
CH-::; and
each subscript n is independently an integer from 0 to 20;
each M is a unit capable of altering the polymer band gab and are evenly or
randomly
distributed along the polymer main chain; L is a linker; G' and G2 which are
each
independently selected from the group consisting of hydrogen, halogen, alkyne,
optionally substituted aryl, optionally substituted heteroaryi, halogen
substituted aryl,
silyi, diazonium salt, triflate, acetyloxy, azlde, suifonate, phosphate,
boronic acid
substituted aryl, boronic ester substituted aryl, boronic ester, boronic acid,
optionally
substituted dihydrophenanthrene (DHP), optionally substituted fluorene, aryl
or hetroaryl
substituted with one or more pendant chains terminated with a functional group
selected
from amine, carbarnate, carboxylic acid, carboxylate, maleimide, activated
ester. N-
hydroxysuccinimidyl, hydrazine, hydra.zide, hydrazone, azide, alkyne,
aldehyde, thlol,
and protected groups thereof conjugated to a binding partner. in some
embodiments, L
is an aryl or hetroaryl group evenly or randomly distributed along the polymer
main chain
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and substituted with one or more pendant chains terminated with a functional
group
selected from the group consisting of arnine, carbarriate, carboxylic acid,
carboxylate,
maleirnide, activated ester, N-hydroxysuccinirniciyi, hydrazine, hydrazide,
hydrazone,
azide, alkyne, aldehyde, thiol, and protected groups thereof conjugated to a
binding
partner; a, c, and d, define the mol % of each unit within the structure which
each can
be evenly or randomly repeated and where a is a mol % from 10 to 100%, c is a
mol %
from 0 to 90%, and each d is a mol% from 0 to 25%; each b is independently 0
or 1; m
is an integer from 1 to about 10,000; and each n is independently an integer
from 1 to
20.
[0020] The binding partner may be a molecule or complex of
molecules capable
of specifically binding to target analyte. The binding partner may be a
protein, an affinity
ligand, an antibody, or an antibody fragment. In some embodiments, the binding
partner
may be selected from the group consisting of a monoclonal antibody, a
polyclonal
antibody, an immunoglobulin, an immunologically active portion of an
immunoglobulin,
a single chain antibody, Fab fragment, Fab' fragment, and F(a1:02 fragments,
and scFv
fragment.
[0021] A composition is provided comprising a polymer dye
conjugate; an
aqueous buffer; and a zwitterionic or anionic surfactant. The composition may
comprise
the zwitterionic or anionic surfactant at a concentration below the critical
micellar
concentration (CMC). In some embodiments, the surfactant may be at a
concentration
of 0.05 to 0.25% (w/v), 0.06 to 0.20% (w/v), or 0.08 to 0.16% (w/v). The
aqueous buffer
may comprise an additional additive selected from the group consisting of a
protein
stabilizer, a preservative, and an additional surfactant. The composition may
exhibit,
following exposure to a blood sample and flow cytometry analysis, decreased
non-
specific binding of polymer dye conjugate to white blood cells in a sample.
The
decreased non-specific binding may be compared to the same composition without
the
zwitterionic or anionic surfactant. The white blood cells may be selected from
the group
consisting of monocytes and granulocytes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1A shows a plot of fluorescence intensity as a
function of wavelength
for fluorene (FF), dihydrophenanthrene (DD) and fluorene-DHP (DF) polymer
dyes.
[0023] FIG. 1B shows a graph of absorption spectra of
fluorene (Fl-Fl) polymer
and dihydrophenanthrene (DHP-DHP) polymer. The DHP-DHP polymer (black curve)
exhibits lambda max (Amax) at 390 and 410 rim, whereas the Fl. Fl (grey curve)
polymer
shows lambda max (Amax) at about 400 nrn.
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[0024] FIG. 2 shows flow cytometry dot plots of unstained
blood cells (upper
panel); blood cells stained with polymer dye and no surfactant (lower left
panel); and
blood cells stained with a composition comprising polymer dye and a surfactant
(lower
right). The fluorescent polymer dye SN v605 without antibody was used to stain
a blood
sample and analyzed in a flow cytometer. Polymer dye without surfactant
exhibited non-
specific binding to the monocytes/granulocytes (lower left). Polymer dye with
Ervi PlGEN
BE exhibited a substantial decrease in the non-specific binding to
monocytes/granulocytes (lower right).
[0025] FIG. 3 shows dot plots for blood cells without
polymer dye conjugate
(upper panel) and blood cells stained with SN 605-CD20 conjugate with (lower
left panel)
and without surfactant (lower right panel). The percentage of non-specifically
bound
granulocytes was reduced (check the "P2" gate in the dot plot) with the usage
of
surfactant. Also, the functional aspect of the conjugate did not change (check
the "P1"
gate in the dot plot), since the percentage of the positive population is
similar in both the
cases.
[0026] FIG. 4 shows a bar graph of Median Fluorescence
Intensity (MdFl) values
of monocytes in presence and absence of surfactant for two lots of polymer dye
conjugates (SN v605-CD20) compared to unstained monocytes (autofluorescence).
In
the presence of surfactant, the non-specific interaction on monocytes was
substantially
reduced for both Lot-1 and Lot-2 5N605 0D20 conjugates.
[0027] FIG. 5 shows a bar graph of MdFI values of
granulocytes in presence
and absence of surfactant for two lots of polymer dye conjugates (SN v605-
CD20)
compared to unstained granulocytes (autofluorescence). In the presence of
surfactant,
the non-specific interaction on granulocytes was reduced for both Lot-1 and
Lot-2 SN605
CD20 conjugates.
[0028] FIG. 6 shows dot plots of blood cells without
polymer dye conjugate
(upper left) and stained with SN v786-CD103 conjugate with Empigen BB
surfactant
(lower left) and without surfactant (upper right). The dot plot compares one
of the
claimed polymers and BV786-CD103, tandem fluorochrome (lower right) (available
from
Becton Dickinson). The percentage of non-specifically bound granulocytes and
monocytes were reduced (check the "P1" and "P2" gate respectively in the dot
plot) in
the presence of surfactant.
[0029] FIG. 7 shows a bar graph of MdFI values of monocytes
in presence and
absence of surfactant for two lots of polymer dye conjugates (SN v786-CD103)
compared to unstained monocytes (autofluorescence). In the presence of
surfactant,
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non-specific binding of polymer conjugates to monocytes was substantially
reduced for
both lots of the polymer dye conjugates.
[0030] FIG. 8 shows a bar graph of MdFI values of
granulocytes in presence
and absence of surfactant for two lots of polymer conjugates compared to
unstained
granulocytes (autofluorescence). In the presence of surfactant; non-specific
binding of
polymer conjugates to granulocytes was substantially reduced for both lots of
the
polymer dye conjugates,
[0031] FIG. 9 shows dot plots of blood cells stained with
SN v605-CD20
conjugate with and without surfactants (upper panel), where the lower left
panel is
nonionic surfactant Tween-20 and the lower right panel is nonionic surfactant
Pluronic
F-68. The percentage of non-specifically bound monocytes was not reduced
(check the
"non-specific monocytes" gate in the dot plot) with the usage of nonionic
surfactants
Tween-20 and Pluronic F-68.
[0032] FIG. 10 shows a dot plot of blood cells without dye
conjugate (upper left
panel); blood cells stained with SN v605-CD20 conjugate with BSA (upper right
panel),
oxidized BSA (lower left panel), and BSA-Cy5-ox (lower right panel). The
percentage of
non-specifically bound monocytes and granulocytes was not substantially
reduced
(check the "P1" gate in the dot plot) with the usage of protein blockers.
[0033] FIG. 11 shows three graphs each showing the effect
of surfactant
concentration on negative monocytes (MFI) for Donor 1 (D1) and Donor 2 (D2)
blood
samples for unstained and stained samples for SN v428 CD19 (FIG. 11, upper
panel),
SN v428 CD22 (FIG. 11, lower panel), and SN v428 CD25 (FIG. 11, middle panel)
specificities. SN conjugates in the presence of 0.06 to 0.20 % Empigen BB
exhibited
lower non-specific monocyte interactions than in absence of surfactant.
[0034] FIG. 11 (cont.) shows three graphs each showing the
effect of surfactant
concentration on negative monocytes (MFI) for Donor 1 (D1) and Donor 2 (D2)
blood
samples for unstained and stained samples for SN v428 CD19 (FIG. 11, upper
panel),
SN v428 CD22 (FIG. 11, lower panel), and SN v428 CD25 (FIG. 11, middle panel),
with
data shown as negative monocyte MFI in % of no Empigen BB samples. Samples
stained with BD polymer dye conjugates in the presence of 0.06 to 0.20 %
Empigen
BB exhibited lower percent of non-specific monocyte interactions than in
absence of
surfactant.
[0035] FIG. 12 (nine graphs) shows the effect of surfactant
concentration on
negative granulocytes (upper three panels), positive lymphocytes (middle
panels), and
positive lymphocytes in % of no Empigen BB samples (lower three panels).
Unstained
and stained blood samples from donor 1 (D1) and donor 2 (D2) are shown.
Somewhat
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lower negative interaction to granulocytes was exhibited in presence of 0.06
to 0.20%
surfactant, compared to absence of surfactant for each of CD19 BD, 0D25 BD,
and
CD22 BD polymer dye conjugates (upper three panels). Positive lymphocyte data
were
similar or slightly higher in presence of surfactant, compared to absence of
surfactant
for each of CD19 BD, CD25 BD, and CD22 BD polymer dye conjugates (middle three
and lower three panels).
[0036] FIG. 13 shows dot plots of SS/FL9 staining patterns
for SN v428 CD19
Lot # D19-094 polymer dye conjugate at 0.5 pg/test without surfactant, with
0.06%,
0.12%, and 0.2% Empigen BB surfactant, and CD19 BV-421 conjugate (Becton
Dickinson) at its commercial dose on donor 1 (upper panels) and donor 2 (lower
panels)
blood samples.
[0037] FIG. 14 shows dot plots of SS/FL9 staining patterns
for SN v428 CD25
Lot # D19-107 polymer dye conjugate at 0.5 pg/test without surfactant, with
0.06%,
0.12%, and 0.2% surfactant and CD25 BV-421 conjugate (Becton Dickinson) at its
commercial dose on donor 1 (upper panels) and donor 2 (lower panels) blood
samples.
[0038] FIG. 15 shows dot plots for SN v428 CD22 Lot # D19-
109 polymer dye
conjugate at 0.5 pg/test without surfactant, with 0.06%, 0.12%, and 0.2%
surfactant and
CO22 BV-421 conjugate (Becton Dickinson) at its commercial dose on donor 1
(upper
panels) and donor 2 (lower panels) blood samples.
[0039] FIG. 16 shows dot plots with the percentage of dead
cells at up to 0.2%
surfactant. CD19-SNv428 D19-094 without EMP1GEN BB (negative control) and
with
0.06%, 0.12%, and 0.2% EMP1GEN BB , was tested on donor 1 and donor 4 whole
blood samples, stained with 7-ADD, to evaluate the percentage of dead cells in
each
condition. VVhole blood samples that had been preserved for greater than 24
hours were
added as positive control of 7-AAD staining (left panel, 12% dead cells). The
percentage
of dead cells was not substantially increased by the presence of EMP1GEN BB
at up
to 0.2%, when compared to samples without surfactant.
[0040] FIG. 17A shows a dot plot of a peripheral blood
sample without single
color conjugate, evident as there is no population in the CD20+ gate.
[0041] FIG. 17B shows a positive control dot plot of a
peripheral blood sample
in the presence of CD2O-SN v605 single-color conjugate in a buffer composition
containing BSA, sodium azide, PluronicTM F-68 (PF-68) and Empigen BB as
additives.
When compared to negative control dot plot (FIG. 17C), the % population in the
gate
"Mons Non-specific binding" and "Grans Non-specific binding are each
considerably
reduced, indicating the effectiveness of Empigen BB in eliminating or
reducing non-
specific binding to monocytes and granulocytes.
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[0042] FIG. 17C shows a negative control dot plot of a
peripheral blood sample
in the presence of CD2O-SN v605 single-color conjugate in a buffer composition
containing only BSA, PF-68 and sodium azide as additives.
[0043] FIG. 17D shows a test dot plot of a peripheral blood
sample in presence
of CD2O-SN v605 single-color conjugate in a buffer composition containing BSA,
sodium
azide, PF-68 and NLS (0.16% w/v) as additives.
[0044] FIG. 17E shows a test dot plot of a peripheral blood
sample in presence
of CD20-SN v605 single-color conjugate in a buffer composition containing BSA,
sodium
azide, PF-68 and NLS (0.08% w/v) as additives.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0045] Reference will now be made in detail to certain
embodiments of the
disclosed subject matter, examples of which are illustrated in part in the
accompanying
drawings and Examples. While the disclosed subject matter will be described in
conjunction with the enumerated claims, it will be understood that the
exemplified
subject matter is not intended to limit the claims to the disclosed subject
matter.
General Disclosure
[0046] The disclosure generally relates to compositions,
and methods for
detecting analytes in a sample using compositions comprising at least one
surfactant
and at least one polymer dye conjugated to binding partners (e.g.,
antibodies), for
example a fluorescent polymer dye conjugated to binding partner. More
specifically, the
disclosure relates to a method for reducing or eliminating non-specific
binding of at least
one polymer dye conjugate in a biological sample, such as a blood sample, the
method
comprising: contacting the at least one polymer dye conjugate with at least
one
zwitterionic or anionic surfactant before, during, or after the polymer dye
conjugate is
contacted with a biological sample, such as a blood sample, the contacting
resulting in
decreased non-specific binding of the at least one polymer dye conjugate to
cells, such
as white blood cells in the blood sample. The surfactant can be added to the
blood
sample before the contacting. The surfactant can be added to the polymer dye
conjugate
prior to contacting with a biological sample.
Definitions
[0047] The abbreviations used herein have their
conventional meaning within
the chemical and biological arts.
[0048] The singular forms "a", "an" and "the" are intended
to include the plural
forms as well, unless the context clearly indicates otherwise.
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[0049] The term "and/or" refers to and encompasses any and
all possible
combinations of one or more of the associated listed items. Unless otherwise
specified,
the term phrase "room temperature" refers to 18 to 27 C.
[0050] Unless otherwise specified, the term "percent", or
"ck" refers to weight
percent.
[0051] All patents, patent applications and publications
referred to herein are
incorporated by reference in their entirety.
[0052] The term "Analyte" refers to a molecule, compound,
or other component
in a sample. Analytes may include but are not limited to peptides, proteins,
polynucleotides, organic molecules, sugars and other carbohydrates, and
lipids.
[0053] The term "Binding partner" refers to a molecule
capable of specifically
binding an analyte. A binding partner can be any of a number of different
types of
molecules, including an antibody or antigen-binding fragment thereof, or other
protein,
peptide, polysaccharide, lipid, a nucleic acid or nucleic-acid analog, such as
an
oligonucleotide, aptamer, or PNA (peptide nucleic acids).
[0054] The term "CD" refers to Cluster of differentiation.
[0055] The term "Compensation" in flow cytometry is a
mathematical process of
correcting for fluorescence spillover (spectral overlap of multiparameter flow
cytometric
data). For example, compensation may be performed by removing the signal of
any
given fluorochrome from all detectors except the one devoted to measuring that
dye.
Since fluorochromes may have wide-ranging spectrum, they can overlap, causing
the
undesirable confusion during data analysis.
[0056] The term "Labeled binding partner' refers to a
binding partner that is
conjugated to a dye.The term "Reactant solution" refers to solution comprising
the
labeled binding partner. In some embodiments, besides the labeled binding
partner, a
reactant solution may further comprise stabilizers, salt, buffer, surfactants,
and/or other
reagents.
[0057] The term "linker" or "linkage" refers to a linking
moiety that connects two
groups and has a backbone of 100 atoms or less in length. A linker or linkage
may be a
covalent bond that connects two groups or a chain of between 1 and 100 atoms
in length,
for example a chain of 1,2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more
carbon atoms in
length, where the linker may be linear, branched, cyclic or a single atom. In
some
embodiments, the linker is a branching linker that refers to a linking moiety
that connects
three or more groups. In certain cases, one, two, three, four or five or more
carbon atoms
of a linker backbone may be optionally substituted with a sulfur, nitrogen or
oxygen
heteroatom. In some embodiments, the linker backbone includes a linking
functional
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group, such as an ether, thioether, amino, amide, sulfonamide, carbamate,
thiocarbamate, urea, thiourea, ester, thioester or imine. The bonds between
backbone
atoms may be saturated or unsaturated, and in some cases not more than one,
two, or
three unsaturated bonds are present in a linker backbone. The linker may
include one
or more substituent groups, for example with an alkyl, aryl or alkenyl group.
A linker may
include, without limitations, polyethylene glycol, ethers, thioethers,
tertiary amines,
alkyls, which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1-
methylethyl
(iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like.
The linker
backbone may include a cyclic group, for example, an aryl, a heterocycle or a
cycloalkyl
group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are
included in
the backbone. A linker may be cleavable or non-cleavable.
[0058] A linker moiety can be attached to "A", as taught in
US Published
Application No. 2020/0190253A1, which is incorporated herein by reference in
its
entirety, or to "L", as taught in US Published Application No.2019/0144601,
which is
incorporated here by reference in its entirety. A linker moiety can comprise a
sulfonamide, disulfonamide, a selenomide, a sulfinamide, a sultam, a
disulfinamide, an
amide, a seleninamide, a phosphonamide, a phosphinamide, a phosphonamidate, or
a
secondary amine.
[0059] As described therein, and as each pertains to a
linker moiety, the term
"sulfonamide," refers to a moiety ¨S(0)2NR-; the term "disulfonamide," refers
to a moiety
¨S(0)2NRS(0)2-; the term "selenonamide," refers to a moiety ¨Se(0)2NR-; the
term
"sulfinamide," refers to a moiety ¨S(0)NR-; the term "disulfinamide," refers
to a moiety
¨S(0)NRS(0)-; the term "seleninamide," refers to a moiety ¨Se(0)NR-; the term
"phosphonamide," refers to a moiety ¨NR-PR(0)NR-; the term "phosphinamide,"
refers
to a moiety ¨PR(0)NR-; and the term "phosphonamidate," refers to a moiety ¨
0-PR(0)NR-; and the term "sultam" refers to a cyclic sulfonamide (e.g.,
wherein the R
group is bonded to the sulfur atom via an alkylene moiety); wherein for each
term the R
group is independently H, alkyl, haloalkyl, or aryl.
[0060] The term "terminus" as used herein refers to termini
on the conjugated
polymer chains that can include a functional group that provides for
bioconjugation. In
some cases, such functionality is referred to as an end linker. The terminus
may be, for
example, hydrogen, halogen, alkyne, optionally substituted aryl, optionally
substituted
heteroaryl, halogen substituted aryl, silyl, diazonium salt, triflate,
acetyloxy, azide,
sulfonate, phosphate, boronic acid substituted aryl, boronic ester substituted
aryl,
boronic ester, boronic acid, optionally substituted tetrahydropyrene (THP),
optionally
substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl
or
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heteroaryl substituted with one or more pendant chains terminated with a
functional
group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide,
activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone,
azide, alkyne,
aldehyde, thiol, and protected groups thereof for conjugation to a substrate,
or a binding
agent
[0061] The term "MdFl" or "MDFI" refers to Median
fluorescence intensity.
[0062] The term "% recruitment" refers to number of gated
cells of relevant
population.
[0063] The term "Multiplexing" herein refers to an assay or
other analytical
method in which multiple analytes can be assayed simultaneously.
[0064] The term "PEG" refers to polyethylene glycol, or
poly(ethylene glycol).
The number after "PEG" refers to the average molecular weight, where Mw refers
to
weight average molecular weight, and Mn refers to number average molecular
weight.
[0065] The term "PBS" refers to phosphate buffered saline
which is an aqueous
buffer which may contain sodium chloride, disodium hydrogen phosphate,
potassium
chloride, and potassium dihydrogen phosphate. For example, PBS may contain
milliQ
water or deionized water and 137 mM NaCI, 2.7 mM KCI, 10 mM Na2HPO4, 1.8 mM
KH2PO4. The pH may be about pH 7.0-7.4. The PBS may or may not be preserved
with
an azide such as sodium azide. PBS is an isotonic solution.
[0066] The acronym "SSC" refers to side scatter.
[0067] The acronym "WBC" refers to white blood cells.
[0068] A "dye" is a moiety that provides a detectable
signal, which can be
attached to or incorporated into a binding partner, either directly or
indirectly. A dye used
in the disclosure can be colored, fluorescent, or luminescent, and is
typically detected
by detector in flow cytometer, e.g., PMT or APD. Fluorescent dyes can be
monomeric
or polymeric. The fluorescent dye may be a fluorescent polymer dye. Polymeric
dyes
are particularly useful for analysis of chemical and biological targets. They
are highly
responsive optical reporters and efficient light absorbers, by virtue of the
multiple
chromophores they comprise. Fluorescent polymer dyes appropriate for use in
the
present disclosure are described herein, for example, in US 2019/0144601 and
US
2020/0190253. Examples of polymeric dyes include, but are not limited to,
conjugated
polymers having repeat units of chromophore, aggregates of conjugated
molecules,
luminescent dyes attached via side chains to saturated polymers, semiconductor
quantum dots and dendritic structures. Polymeric and monomeric dyes disclosed
in U.S.
Pat. Nos. 7,214,489, 8,354,239, 8,575,303 can also be used for the present
invention.
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[0069] As used herein, the term "ammonium" refers to a
cation having the
forrriula NI-IR-3+ where each R group, independently, is hydrogen or a
substituted or
urisubstituted alkyl, aryl, aralkyl, or alko_xy group. Preferably, each of the
R groups is
hydrogen.
[0070] As used herein, "oligoether" is understood to mean an oligomer
containing
structural repeat units having an ether functionality. As used herein, an
"oligorner" is
understood to mean a molecule that contains one or more identifiable
structural repeat
units of the same or different formula.
[0071] The term "sulfonate functional group" or
"sulfonate," as used herein,
refers to both the free sulfonate anion (-S(-0)20-) and salts thereof.
Therefore, the term
sulfonate encompasses sulfonate salts such as sodium, lithium, potassium and
ammonium sulfonate,
[0072] The term "suifonarnicio" as used herein refers to a
group of formula -
S02NR- where R is hydrogen, alkyl or aryl.
[0073] The term "alkyl" as used herein refers to a straight
or branched,
saturated, aliphatic radical having the number of carbon atoms indicated. For
example,
C1-C6alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl,
butyl, isobutyl,
sec-butyl, text-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups
include, but are not
limited to heptyi, octyi, nonyl, decyl, etc. Alkyl can include any number of
carbons, such
as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 243, 3-4, 3-5,
3-6, 4-5, 443
and 5-6. The alkyl group is typically monovalent, but can be divalent, such as
when the
alkyl group links two moieties together.
[0074] The term "cycloalkyl" as used herein refers to a
saturated or partially
unsaturated, rnonocyclic, fused blcyciic or bridged polycyclic ring assembly
containing
from 3 to 12 ring atoms, or the number of atoms indicated monocyclic rings
include, for
example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl.
Bicyclic and
polycyclic rings include., for example, norbornane, decahydronaphthaiene and
adamantane,. For example, C3_8cycloalkyl includes cyclopropyl, cyciobutyl,
cyclopentyi,
cyclohexyl, cyclooctyl, and norbornane.
[0075] The term "haloalkyl" as used herein refers to alkyl
as defined above
where some or all of the hydrogen atoms are substituted with halogen atoms.
Halogen
(halo) preferably represents chloro or fluor , but may also be bromo Of odo.
For
example, haloalkyl includes trifluorornethyl, flouromethyl, 1,2,3,4,5-
pentafluoro-phenyl,
etc. The term "perfluoro" defines a compound or radical which has at least two
available
hydrogens substituted with fluorine. For example, perfluorophenyl refers to
1,2,3,45.-
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pentafluorophenyl; perfluorornethane refers to
1,1 ,14rifluoromethyl, and
perfluoromethoxy refers to 1,1,1-trifluoromethoxy.
[0076]
As used herein, the term "halogen" refers to fluorine, chlorine, bromine
and iodine.
[0077]
The term "aikoxy" as used herein refers to an alkyl group; as defined
above, having an oxygen atom that connects the alkyl group to the point of
attachment.
Alkoxy groups include, for example, rnethoxy, ethoxy, propoxy, iso-propoxy,
butoxy, 2-
butoxy, iso-butoxy. sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxy
groups can
be further substituted with a variety of substituents described within. For
example, the
alkoxy groups can be substituted with halogens to form a "halo-alkoxy" group.
[0078]
The term "alkene" as used herein refers to either a straight chain or
branched hydrocarbon, having at least one double bond. Examples of alkene
groups
include, but are not limited to; vinyl, propenyl, isoprepenyi; 1-butenyl, 2-
butenyl,
isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-peritadienyl,
1,4-
pentadienyl, 1-hexenyl. 2-hexenyl, 3-hexenyl, 1 ,3-hexadienyl, 1 ,4-
hexadienyl, 1 ,5-
hexadienyl, 2,4-hexadienyl, or 1 ,3,5-hexatrienyl. The alkene group is
typically
monovalent, but can be divalent, such as when the alkenyl group links two
moieties
together.
[0079]
The term "aikyne" as used herein refers to either a straight chain or
branched hydrocarbon, having at least one triple bond. Examples of alkynyl
groups
include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl,
isobutynyl, sec-
butynyl, butadiynyi, 1-pentynyl, 2-pentynyl, isopentynyi, 1 ,3-pentadiynyi,
1,4-
pentadiyrtyl, 1.-hexynyl, 2-hexynyl, 3-hexynyl, 1 ,3-hexadiyrtyl, 1,4.-
hexadiynyl, 1,5-
hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. The alkynyl group is
typically
monovalent, but can be divalent, such as when the alkynyl group links two
moieties
together.
[0080]
The term "aryl" as used herein refers to a rnonocyclic or fused
bicyclic,
tricyclic or greater, aromatic ring assembly containing 6 to 16 ring carbon
atoms. For
example, aryl may be phenyl, benzyl or naphthyl, preferably phenyl, "Arylene"
means a
divalent radical derived from an aryl group. Aryl groups can be mono-, di- or
tri-
substituted by one, two or three radicals selected from alkyl, alkoxy, aryl,
hydroxy,
halogen, cyano, amino, arnino-alkyl, trifluoromethyi, alkylenedioxy and oxy-C2-
C3-
alkylene; all of which are optionally further substituted, for instance as
hereinbefore
defined; or 1- or 2-naphthyl; or 1.- or 2-pherianthrenyl. Alkylenedioxy is a
divalent
substitute attached to two adjacent carbon atoms of phenyl; e.g.
methyleriedioxy or
ethylenedioxy. Oxy-C2-C3-alkylene is also a divalent substituent attached to
two
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adjacent carbon atoms of phenyl, e.g. oxyethylene or oxypropylene. An example
for oxy-
02-03-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.
[0081] Preferred as aryl is naphthyl, phenyl or phenyl mono-
or disubstituted by
alkoxy, phenyl, halogen, alkyl or trifluoromethyl, especially phenyl or phenyl-
mono- or
disubstituted by alkoxy, halogen or trifluoromethyl, and in particular phenyl.
[0082] The term "aryloxy" as used herein refers to a 0-aryl
group, wherein aryl
is as defined above. An aryioxy group can be unsubstituted or substituted with
one or
two suitable substituents. The term "phenoxy" refers to an aryloxy group
wherein the
aryl moiety is a phenyl ring. The term "heteroaryloxy" as used herein means an
-0-
heteroaryl group, wherein heteroaryl is as defined below. The term
"(hetero)aryloxy" is
use to indicate the moiety is either an aryioxy or heteroarylox_y group,
[0083] The terms "Polyethylene glycoi" or "PEG" as used
herein refer to the
family of biocompatible water-solubilizing linear polymers based on the
ethylene glycol
monomer unit described by the formula ¨(CH2¨CH2-0¨)n¨ or a derivative thereof.
In some embodiments, "n" is 1000 or less, 500 or less, 200 or less, 100 or
less, 50 or
less, 40 or less, 30 or less, 20 or less, 15 or less, such as 3 to 15, or 10
to 15. It is
understood that the PEG polymeric group may be of any convenient length and
may
include a variety of terminal groups and/or further substituent groups,
including but not
limited to, alkyl, aryl, hydroxyl, amino, acyl, carboxylic acid, carboxylate
ester, acyloxy,
and amido terminal and/or substituent groups.
[0084] The term "heteroaryl" as used herein refers to a
monocyclic or fused
bicyclic or tricyclic aromatic ring assembly containing :5 to 16 ring atoms,
where from 1
to 4 of the ring atoms are a heteroatom each N. 0 or S. For example,
heteroaryl includes
indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl,
benzafuranyl, furanyl, pyrrolyi, thiazolyl, benzothiazolyl, oxazolyl,
isoxazolyl, triazolyi,
tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radicals substituted,
especially
mono- or di-substituted, by e.g. alkyl, nitro or halogen. Pyridyi represents 2-
, 3- or 4-
pyriciyi, advantageously 2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl.
Quinolinyl
represents preferably 2-, 3- or 4-quinolinyl, isoquinolinyl represents
preferably 1-, 3- or
4-isoquinolinyl. Benzopyranyl, benzothiopyranyl represents preferably 3-
benzopyranyi
or 3-benzothiopyranyl, respectively. Thiazolyl represents preferably 2- or 4-
thiazolyl, and
most preferred, 4-thlazolyl. Triazolyl is preferably 1-, 2- or 5-(1,2,4-
triazolyi). Tetrazoiyi
is preferably 5-tetrazolyl.
[0085] Preferably, heteroaryl is pyridyl, indolyl,
quinolinyl, pyrrolyl, thiazolyl,
isoxazolyl, triazolyl, tetraz_olyl, pyrazolyl, imidazolyl, thienyl, furanyl,
benzothiazolyl,
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benzofuranyl, isoquinolinyi, benzothienyl, oxazolyl, indazolyl, or any of the
radicals
substituted, especially mono- or di-substituted.
[0086]
Similarly, substituents for the aryl and heteroaryl groups are varied
and
are selected from: -halogen, -OR', -0C(0)R`, -NR'R",
-R', -ON, -NO2, -CO2R% -
CONR'R", -C(0)R', -0C(0)NR`R", -NR"C(0)R', -NR"C(0)2R', -NR'-C(0)NR"R", -NH-
C(N H2)=N H R'C(N H2)=NH , -N H-O(NH2)=NR', -S(0)R', -S(0)2R',
RR", -N3, -
CH(Ph)2, perfluoro(C1-C4.)alkoxy, and perfluoro(0I-04)alkyl, in a number
ranging from
zero to the total number of open valences on the aromatic ring system; and
where R`,
R" and R`" are independently selected from hydrogen, (Ci-05)alkyl and
heterealkyl,
unsubstitute,d aryl and heteroaryl, (unsubstitute,d aryl)-(01-04)alkyl, and
(unsubstitute,d
aryl)oxy-(Ci-C,1)alkyl.
[0087]
Two of the substituents on adjacent atoms of the aryl or heteroaryl ring
may optionally be replaced with a substituent of the formula -T-C(0)-(CH2),1-
1J-, wherein
T and U are independently -NH-, -0-,
or a single bond, and q is an integer of from
0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl
or heteroaryl
ring may optionally be replaced with a substituent of the formula -A-(0H2),.-B-
, wherein
A and B are independently -CH2-, -0-, -NH-, -S-, -S(0)-, -S(0)2-, -S(0)2NR`-
or a single
bond, and r is an integer of from I to 3. One of the single bonds of the new
ring so
formed may optionally be replaced with a double bond. Alternatively, two of
the
substituents on adjacent atoms of the aryl or heteroaryl ring may optionally
be replaced
with a substituent of the formula -(CH2)s-X-(CH2)1-, where s and t are
independently
integers of from 0 to 3, and X is -0-, -NR`-, -S-, -5(0)-, -S(0)2-, or -
S(0)2NR'-. The
substituent R' in -NR'- and -S(0)2NR'- is selected from hydrogen or
unsubstituted (CI-
C6)alkyl.
[0088]
The term "(hetero)arylamino" as used herein refers an amine radical
substituted with an aryl group (e.g., -NH-aryl). An a.rylarnino may also be an
aryl radical
substituted with an amine group (e.g.,
Arylarninos may be substituted or
unsubstituted,
[0089]
The term "amine" as used herein refers to an alkyl groups as defined
within, having one or more amino groups. The amino groups can be primary,
secondary
or tertiary. The alkyl amine can be further substituted with a hydroxy group.
Amines
useful in the present invention include, but are not limited to, ethyl amine,
propyl amine,
isopropyl amine, ethylene diamine and ethanolamine. The amino group can link
the alkyl
amine to the point of attachment with the rest of the compound, be at the
omega position
of the alkyl group, or link together at least two carbon atoms of the alkyl
group. One of
skill in the art will appreciate that other alkyl amines are useful in the
present invention.
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[0090] The term "carbamate" as used herein refers to the
functional group
having the structure -NR"CO2R', where R' and R" are inde,pende,ntly selected
from
hydrogen, (Ci-C8)alkyl and heteroalkyl, unsubstiti.,ited aryl and heteroaryl,
(unsubstituted
ary1)-(01-04)aikyi, and (unsubstituted aiy)oxy-(0-1-04)alkyl. Exampies of
carbamates
include t-Boc, Fmoc, benzyloxy-carbonyi, alloc, methyl carbarnate.., ethyl
carbarnate, 9-
(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate,
Tbfmoc, Climoc, Bimoc, DBD-Trnoc, Bsrnoc, Troc, Taco, 2-phenylethyl
carbarnate,
Adpoc, 2-chloroethyl carbamate, 1 ,1-dirriethyl-2-haloethyl carbamate, DB-1.-
BOC,
TCBOC, Bpoc, t-Bumeoc, Pyoc, Bripeoc, V-(2-pivaloylamino).1,1-dimethylethyl
carbarnate, NpSSPeoc.
[0091] The term "carboxylate" as used herein refers to the
conjugate base of a
carboxyiic acid, which generally can be represented by the formula R000. For
example,
the term "magnesium carboxylate" refers to the magnesium salt of the
carboxylic acid.
[0092] The term "activated ester" as used herein refers to
carboxyl-activating
groups employed in peptide chemistry to promote facile condensation of a
carboxyl
group with a free amino group of an amino acid derivative. Descriptions of
these
carboxyl-activating groups are found in general textbooks of peptide
chemistry; for
example K. D. Kopple, "Peptides and Amino Acids", W. A. Benjamin, Inc., New
York,
1966, pp. 50-51 and E. Schroder and K. Lubke, The Peptides"; Vol. 1, Academic
Press,
New York, 1965, pp. 77-128.
[0093] The terms "hydrazine" and "hydrazide" refer to
compounds that contain
singly bonded nitrogens, one of which is a primary amine functional group.
[0094] The term "aldehyde" as used herein refers to a
chemical compound that
has an -CHO group,
[0095] The term "thiol" as used herein refers to a compound
that contains the
functional group composed of a sulfur-hydrogen bond. The general chemical
structure
of the thioi functional group is R-SH, where R represents an alkyl, alkene,
aryl, or other
carbon-containing group of atoms.
[0096] The term "silyl" as used herein refers to
Si(Rz)3wherein each
Rz independently is alkyl aryl or other carbon-containing group of atoms.
[0097] The term "diazoniurn salt" as used herein refers to
a group of organic
compounds with a structure of R-N2')(--, wherein R can be any organic residue
(e.g.,
alkyl or aryl) and X is an inorganic or organic anion (e.g., halogen).
[0098] The term "triflate" also referred to as
trifluoromethanesulfonate, is a group
with the formula CF3S0.-5.
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[0099] The term "boronic acid" as used herein refers to a
structure -8(01-1)2. It is
recognized by those skilled in the art that a boronic acid may be present as a
boronate
ester at various stages in the synthesis of the quenchers. Boronic add is
meant to
include such esters. The term "boronic ester" or "boronate ester' as used
herein refers
to a chemical compound containing a -B(Z1)(Z2) moiety, wherein Z1 end Z2
together form
a moiety where the atom attached to boron in each case is an oxygen atom. The
boronic
ester moiety can be a 5-membered ring. The boronic ester moiety can be a 6-
membered
ring. The boronic ester moiety can be a mixture of a 5-membered ring and a 6-
membered
ring.
[00100] Values expressed in a range format should be
interpreted in a flexible
manner to include not only the numerical values explicitly recited as the
limits of the
range, but also to include all the individual numerical values or sub-ranges
encompassed within that range as if each numerical value and sub-range were
explicitly
recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to
5%" should
be interpreted to include not just about 0.1% to about 5%, but also the
individual values
(e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to
2.2%, 3.3%
to 4.4%) within the indicated range. The statement "about X to Y" has the same
meaning
as "about X to about Y," unless indicated otherwise. Likewise, the statement
"about X,
Y, or about Z" has the same meaning as "about X, about Y, or about Z," unless
indicated
otherwise.
[00101] As used herein, "dye conjugate" refers to a binding
partner conjugated to
a non-polymeric or polymeric dye.
[00102] As used herein, "SN" refers to "Super Nova" dyes
commercially available
from Beckman Coulter, Inc.
[00103] As used here "EMPIGEN BR " refers to a zwifterionic
surfactant (GAS
Number 66455.-29-6) comprising N,N--dimethyl-N-dodecylglycine betaine at a
concentration of -30% betaine in aqueous solution.
[00104] The term "about" as used herein can allow for a
degree of variability in a
value or range, for example, within 10%, within 5%, or within 1% of a stated
value or of
a stated limit of a range.
[00105] As used herein, "specific binding" refers to binding
of an antibody or
other binding partner (e.g., in a polymer conjugate dye) to an epitope on a
cell or target
analyte to which the antibody or binding partner is targeted.
[00106] As used herein, "non-specific binding" refers to
binding of an antibody
or other binding partner (e.g., in a polymer conjugate dye) to a cell or
sample
component that does not comprise an epitope to which the antibody or other
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partner is targeted. For example, non-specific binding occurs when an antibody
binds
to a cell that does not have an epitope specifically for that antibody.
[00107] As used herein, "reducing" or "eliminating" of non-
specific binding of the
polymer dye conjugate can refer to when the "negatives" (e.g., negative
granulocyte,
monocyte, and lymphocyte populations) mean fluorescence intensity (MFI), in %,
relative
to when no surfactant is used, is decreased by at least about 50% (e.g., by at
least about
60%, at least about 70%, at least about 80%, at least about 90%, at least
about 95%, at
least 99% or more, from about 50% to about 95%, about 50% to about 75%, about
60%
to about 80% or about 65% to about 90%). In other words, the % reduction of at
least
one of monocyte, granulocyte, and lymphocyte background staining, in %
relative to
when no surfactant is used, is decreased by at ieast about 50% (e.g., by at
least about
60%, at least about 70%, at least about 80%, at ieast about 90%, at least
about 95%, at
least 99% or more; from about 50% to about 95%, about 50% to about 75%, about
60%
to about 80% or about 65% to about 90%).
[00108] The term "substantial" or "substantially" as used
herein refers to a
majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%,
96%, 97%,
98%, 99%, 99.5%, 99.9%, 99.99%,
or at least about 99.999% or more.
[00109] The term "substantially no" or "substantially free
of" as used herein refers
to less than about 1%, 0.5%, 0.1%, 0.05%, 0.001%, or at less than about
0.0005% or
less, about 0%, below quantitation limits, below detectable limits, or 0%.
[00110] In this document, the terms "a," "an," or "the" are
used to include one or
more than one unless the context clearly dictates otherwise. The term "or" is
used to
refer to a nonexclusive "or" unless otherwise indicated. In addition, it is to
be understood
that the phraseology or terminology employed herein, and not otherwise
defined, is for
the purpose of description only and not of limitation. Any use of section
headings is
intended to aid reading of the document and is not to be interpreted as
limiting. Further,
information that is relevant to a section heading can occur within or outside
of that
particular section. Furthermore, all publications, patents, and patent
documents referred
to in this document are incorporated by reference herein in their entirety, as
though
individually incorporated by reference. In the event of inconsistent usages
between this
document and those documents so incorporated by reference, the usage in the
incorporated reference should be considered supplementary to that of this
document;
for irreconcilable inconsistencies, the usage in this document controls.
[00111] In certain embodiments, a dye composition is
provided comprising at
least one polymer dye conjugate and at least one suitable zwitterionic
surfactant.
21
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[00112] In certain embodiments, a dye composition is
provided comprising at
least one polymer dye conjugate with at least one suitable anionic surfactant.
[00113] In some embodiments, a method is provided for
reducing or eliminating
non-specific binding of at least one polymer dye conjugate to a cell in a
biological
sample, such as a blood sample, comprising contacting at least one polymer dye
conjugate with at least one zwitterionic and/or anionic surfactant before,
during, and/ or
after the at least one polymer dye conjugate is contacted with the biological
sample.
The steps can be carried out in any order without departing from the
principles of the
invention, except when a temporal or operational sequence is explicitly
recited.
Furthermore, specified steps can be carried out concurrently unless explicit
claim
language recites that they be carried out separately. For example, a claimed
step of
doing X and a claimed step of doing Y can be conducted simultaneously within a
single
operation, and the resulting process will fall within the literal scope of the
claimed
process. Accordingly, in some instances the at least one polymer dye conjugate
can be
contacted with at the least one zwitterionic or anionic surfactant before the
at least one
polymer dye conjugate is contacted with the blood sample. In some instances,
the at
least one polymer dye conjugate can be contacted with at the least one
zwitterionic or
anionic surfactant at the same time the at least one polymer dye conjugate is
contacted
with the blood sample.
Su rfactant
[00114] Various types of surfactants were explored for
reducing or preventing
non-specific interactions of the polymer dye conjugate with biological samples
[00115] Suitable surfactants may be zwitterionic surfactants
or certain anionic
surfactants. Examples of suitable surfactants include surfactants of the
general formula
[00116] RICO-X(CH2)14N+(R2)(R3)]k-(CH2)r[CH(OH)CH2b-Y,
wherein RI is a
saturated or unsaturated 05-24 alkyl, such as a 06-22, 05-21, 07-19, 011-17,
or 08-18 alkyl, a
saturated 010_16 alkyl or a saturated 012_14 alkyl; X is NH, NR4', wherein R4'
is 01_4 alkyl,
0 or S; j is an integer from 1 to 10, such as from 2 to 5 and 3; g is 0 or 1,
R2' and R3' are
each, independently, a 01-4 alkyl, such as ethyl or methyl; optionally hydroxy
substituted
by a hydroxyethyl group or a methyl; k is 0 or 1; f is an integer from 0 to 4,
such as 0, 1,
2, 3, or 4; h is 0 or 1; and Y is COO, SO3, OPO(0R90 or P(0)(0R90, wherein R5'
is H
or 01-4 alkyl, and when k=0, the surfactant may be in acidic form, or sodium,
or potassium
salts thereof.
22
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[00117]
The surfactant can be present at a concentration in a range of from
about
0.05% to about 0.25%, about 0.06% to about 0.2%, or about 0.08% to about 0.16%
(w/v)
in a buffer or other suitable aqueous composition according to the disclosure.
[00118]
Suitable zwitterionic surfactants that can be used according to the
methods described herein include betaine zwitterionic surfactants such alkyl
betaines,
alkylamidobetaines, amidazoliniumbetaines, sulfobetaines (INCI Sultaines), as
well as
a phosphobetaines.
[00119]
Examples of suitable zwitterionic surfactants include alkyl betaines,
such
as those of the formula:
R1'-N+(CF-13)2-CH2C00,-;
1,:r-CO-NH(CF-12)3-Nr(CH3)2-C1-12C00-;
Ri'--N+(C1-13)2-CH2C1-1(01-1)C1-12S03-: and
Ri'-CO-NH-(CH2)3-NE(CH3)2-CH2CH(OH)CH2S0.3-.
[00120]
Examples of suitable betaines and sulfobetaines are the following
(designated in accordance with INCI): almondamidopropyl betaine,
apricotamidopropyl
betaine, avocadannidopropyl betaine, babassuannidopropyl betaine,
behenannidopropyl
betaine, behenyl betaine, canolamidopropyl betaine, capryl/capramidopropyl
betaine,
carnitine, cetyl betaine, cocamidoethyl betaine, cocamidopropyl betaine,
cocamidopropyl hydroxysultaine, coco betaine, coco hydroxysultaine,
coco/oleamidopropyl betaine, coco sultaine, decyl betaine, di hydroxyethyl
oleyl
glycinate, dihydroxyethyl soy glycinate, dihydroxyethyl stearyl glycinate,
dihydroxyethyl
tallow glycinate, dimethicone propyl of PG-betaine, drucamidopropyl
hydroxysultaine,
hydrogenated tallow betaine, isostearamidopropyl betaine, lauramidopropyl
betaine,
lauryl betaine, lauryl hydroxysultaine, lauryl sultaine, milk amidopropyl
betaine,
milkamidopropyl betaine, myristamidopropyl betaine, myristyl betaine,
oleamidopropyl
betaine, oleamidopropyl hydroxysultaine, leyl betaine, olivamidopropyl
betaine,
palmamidopropyl betaine, palmitamidopropyl betaine, palmitoyl carnitine, palm
kernel
am idopropyl betaine, polytetrafluoroethylene
acetoxypropyl betaine,
rici noleamidopropyl betaine, sesamidopropyl betaine, soyamidopropyl betaine,
stearamidopropyl betaine, stearyl betaine,
tallowamidopropyl betaine,
tallowamidopropyl hydroxysultaine, tallow betaine, tallow dihydroxyethyl
betaine,
undecylenamidopropyl betaine and wheat germ amidopropyl betaine.
[00121]
Suitable betaine zwitterionic surfactants may be N-(alkyl C10_16)-N,N-
dimethylglycine betaine, N-(alkyl C12_14)-N,N-dimethylglycine betaine, N, N-
dimethyl-N-
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dodecylglycine betaine, lauryl dimethyl betaine (also known as lauryl
betaine), myristyl
sulfobetaine, or n-hexadecyl-N,N-dimethy1-3-ammonio-1-propanesulfonate. Lauryl
betaine is commercially available as EMPIGEN BB (Huntsman Corporation) and
has
a CMC of 1.6-2.1 mM (20-25 C). Myristyl sulfobetaine (also known as n-
tetradecyl-
N,N-dimethy1-3-ammonio-1-propanesulfonate, DMMA) is available under the trade
name ZWITTERGENTO 3-14 (Merck KGaA, Darmstadt, Germany), and has a CMC
100-400 uM. n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (also known
as
3-N,N-dimethylpalmitylammonio)propane sulfonate, DMPA) is available under the
tradename ZWITTERGENTO 3-16, and has a CMC 10-60 uM. For example, coconut
dimethyl betaine is commercially available from Seppic under the trade name of
AMONYL 2650; and lauryl betaine is commercially available from Sigma-Aldrich
under
the trade name EMPIGEN BB . A further example betaine is lauryl-imino-
dipropionate
commercially available from Rhodia under the trade name MIRATAINE H2C-HA .
[00122] The zwitterionic surfactant can be present in a
range of from about 0.06%
to about 0.2%, or about 0.08% to about 0.16% in a buffer or other suitable
aqueous
composition according to the disclosure.
[00123] Examples of suitable anionic surfactants include
sarcosinate surfactants
in acidic form or in neutral form. For example, suitable anionic surfactants
may be
sarcosinate surfactants in neutral form. Sarcosinate surfactants may be
alkanoyl
sarcosinate surfactants.
[00124] Examples of suitable anionic surfactants include
surfactants of the
general formula RICO-X(CH2)1-(CH2)f-[CH(OH)CH2b-Y, wherein R1' is a saturated
or
unsaturated C5_24 alkyl, such as a C8-18 alkyl, a saturated C10-16 alkyl or a
saturated C12-
14 alkyl; X is NH, NR4', wherein R4' is C1-4 alkyl, 0 or S; j is an integer
from 1 to 10, such
as from 2 to 5 and 3; g is 0 or 1; f is an integer from 0 to 4, such as 0, 1,
2, 3, 0r4; his
0 or 1; and Y is COO, SO3, OPO(0R5')0 or P(0)(0R5')O, wherein R5' is H or C1-4
alkyl,
and wherein the anionic surfactant may be in acidic form, or sodium, or
potassium salt
forms thereof.
[00125] Suitable anionic surfactants may comprise the
structure
CH3(CH2)aCH2(CH2CH=CH)bCH2(CH2)cCH2(C=0)N(CH3)CH2CO2X, wherein a=1-8;
b=0-2, and c=0-6, and X = H, Na, K.
[00126] Examples of alkanoyl sarcosinates, may include those
of the formulae:
RI¨CO-N(CH3)-CH2-000-; and
R1'¨CO-N(CH3)-CH2-503-, and, for example, sodium or potassium salts thereof,
wherein RI may be saturated or unsaturated C6-24 alkyl, C7-19 alkyl, or C11-17
alkyl.
24
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[00127] Examples of suitable alkanoyl sarcosinates, and
acidic or salt forms
thereof include N-lauroyl sarcosine, sodium lauroylsarcosinate, sodium
palmitoyl
sarcosinate, sodium stearoyl sarcosinate, N-methyl-N-(1-oxotetradecyI)-glycine
sodium
salt, sodium caproyl sarcosinate, sodium capryloyl sarcosinate, N-methyl-N-(1-
oxo-9-
octadecen-1-y1)-glycine, sodium salt, sodium oleoyl sarcosinate, and sodium
linoleoyl
sarcosinate.
[00128] The anionic surfactant can be present in a range of
from about 0.06% to
about 0.2%, or about 0.08% to about 0.16% in a buffer or other suitable
aqueous
composition according to the disclosure.
[00129] The compositions can be used in flow cytometry and,
as such, can
comprise additional components, including, but not limited to, one or more of
any
suitable carrier, stabilizer, buffer, salt, chelating agent (e.g., EDTA) or
preservative. The
compositions can also comprise one or more additional surfactants in addition
to the
zwitterionic surfactants and/or anionic surfactants described herein. Non-
limiting
examples of the one or more additional surfactants includes polysorbates such
as
TWEEN 20 (polyoxyethylene sorbitan monolaurate) and TWEEN 80
(polyoxyethylene sorbitan monooleate). The carrier can be an aqueous solution,
such
as water, saline, alcohol, or a physiologically compatible buffer, such as
Hank's solution,
Ringer's solution, or physiological saline buffer. The carrier may include
formulation
agents, such as suspending agents, stabilizing agents and/or dispersing
agents. The
compositions can also include a buffer or pH adjusting agent, and typically
the buffer is
a salt prepared from an organic acid or base. Representative buffering agents
include
salts of organic acid salts, such as citric acid, ascorbic acid, gluconic
acid, carbonic acid,
tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris tromethamine
hydrochloride,
or phosphate buffer.
[00130] The composition can comprise a protein stabilizer
selected from the
group consisting of a bovine serum albumin (BSA or "Fraction V"), a casein,
and a
gelatin. The protein stabilizer can be BSA, a commercially available bovine
serum
albumin protein derived from cows. The protein stabilizer can be present in
from about
0.1-5 mg/mL, about 0.5-3 mg/mL, or about 2 mg/mL in a buffer or other suitable
aqueous
composition according to the disclosure.
[00131] The stabilizer can be a gelatin, a protein, commonly
derived
from collagen taken from animal body parts. It is brittle when dry and gummy
when
moist. It may also be referred to as hydrolyzed collagen, collagen
hydrolysate, gelatine
hydrolysate, hydrolyzed gelatine, and collagen peptides after it has undergone
CA 03198558 2023- 5- 11
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hydrolysis. Several types of gelatin are commercially available including
gelatin -type A;
gelatin-type B, Prionex highly purified gelatin Type A, and gelatin-cold
water fish.
[00132] The composition can also include any appropriate
preservative. The
preservative can be an antioxidant, biocide, or antimicrobial agent. The
preservative
can be an inorganic salt. The preservative can be sodium azide. The
preservative may
be present in a concentration range of about 0.01 to about 1%, about 0.05% to
about
0.5%, or about 0.1%.
Polymer Dye
[00133] In another embodiment, the composition can be used
with a polymer dye.
The polymer dye may be a fluorescent polymer dye or a fluorescent polymer
tandem
dye. Polymeric dyes are particularly useful for analysis of chemical and
biological target
analytes. They are highly responsive optical reporters and efficient light
absorbers, by
virtue of the multiple chromophores they comprise. The polymer dye conjugate
can
comprise any fluorescent polymer dye or fluorescent polymer tandem dye
previously
disclosed.
[00134] For example, the polymer dye or tandem polymer dye
can be any dye
disclosed in Published POT Appl. No, WO 2017/180998; U.S. Application Na.
2021/0047476; U.S. Application No. 2020/0190253; U.S. Application No.
2020/0147615; U.S. Application No. 2021/0108083; U.S. Application No.
2018/0224460; U.S. Patent No. 11,034,840; U.S. Patent No. 10,228,375; U.S.
Patent
No. 10,545,13762; U.S. Patent No. 10,533,092; U.S, Patent No. 7,214,489; U.S.
Patent
No. 8,354,239; U.S. Patent No. 8,575,303, each of which are incorporated by
reference
as if fully set forth herein in their entirety. The polymer dye conjugate can
have the
structure of any ,,,vater-solubie fluorescent polymer dye disclosed in
Published US Appl,
No. 2020/0190253 Al, which is incorporated by reference as if fully set forth
herein in
its entirety. The polymer dye conjugate can have the structure of any water-
soluble
fluorescent polymer dye disclosed in Published US Appl. No. 2019/0144801,
which is
incorporated by reference as if fully set forth herein in its entirety.
[00135] The polymer dye or polymer dye conjugate can be any
commercially
available polymer dye or polymer dye conjugated to a binding partner. The
polymer dye
or polymer dye conjugate may comprise a polymer dye excitable by a violet
laser. The
polymer dye or polymer dye conjugate may comprise a polymer dye excitable by a
violet
laser, for example, at 405 nm. The polymer dye or polymer dye conjugate may
comprise
a violet laser (405 nm)-excitable polymer dye.
[00136] In some embodiments, the polymer dye or polymer dye
conjugate may
comprise a SuperNovaTM dye (Beckman Coulter, Inc.). SuperNovaTM polymers are a
26
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new generation of polymer dyes useful for flow cytometry application. The
polymer dye
or polymer dye conjugate may comprise SuperNovaTM v428, SuperNovaTM v605 or
SuperNovaTm v786 (Beckman Coulter, Inc.). SuperNovaTm v428 has unique photo-
physical properties leading to extremely bright conjugates when conjugated to
antibodies or other binding partners. For example, SuperNovaTM v428 (SN v428)
(Beckman Coulter, Inc.) is a polymer dye optimally excited by the violet laser
(e.g., 405
nm) with an excitation maximum of 414 nm, an emission peak of 428 nm, and can
be
detected using a 450/50 bandpass filter or equivalent.
[00137] SuperNovaTM v428 is one of the brightest dyes
excitable by the violet
laser, so it is particularly suited for assessing dimly expressed markers.
SuperNovaTM
conjugated antibodies may include anti-CD19 antibody-SuperNova TM v428, anti-
CD22
antibody-SuperNova v428, anti-CD25 antibody-SuperNovaTM v428, and anti-CD38
antibody-SuperNova TM v428 antibody-polymeric dye conjugates.
[00138] SuperNovaTM v605 and SuperNova TM v786 (Beckman
Coulter, Inc.) are
tandem polymer dyes, derived from the core SuperNovaTM v428 polymer dye. Both
share same absorbance characteristics, with maximum excitation at 414 nm. With
SuperNova TM v605 and SuperNovaTm v786 having emission peak's at 605 nm and
786
nm, respectively, they are optimally detected using the 610/20 and 780/60 nm
bandpass
filters of the flow cytometer. SuperNovaTm v605 and SuperNovaTm v786 may be
conjugated, for example, with anti-CD19 antibody, anti-CD22 antibody, anti-
CD25
antibody, and anti-CD38 antibody.
[00139] The polymer dye or polymer dye conjugate may
comprise a polymer dye
excitable by an ultra-violet ("UV") laser. The polymer dye or polymer dye
conjugate may
comprise a polymer dye excitable by a UV laser at a wavelength of 320 nm to
380 nm,
340 nm to 360 nm, 345 nm to 356 nm, or less than or equal to 380 nm but
greater than
or equal to 320 nm. The polymer dye or polymer dye conjugate may comprise a UV-
excitable polymer dye. The UV-excitable polymer dye or polymer dye conjugate
may
emit light typically at a wavelength of 380 nm to 430 nm, 406 nm to 415 nm, or
less than
or equal to 430 nm but greater than or equal to 380 nm.
[00140] The polymer dye or polymer dye conjugate can
comprise a Brilliant
VioletTmdye (BioLegende/Sirigen Group Ltd.), such as Brilliant Violet
4211m(excitation
max. 405 nm, emission max. 421 nm, 450/50 filter), Brilliant Violet
510Tm(excitation max
405 nm, emission max 510 nm, 510/50 filter), Brilliant Violet 570TM
(excitation max 405
nm, emission max 570 nm, 585/42 filter), Brilliant Violet 605TM (excitation
max 405 nm,
emission max 603 nm, 610/20 filter), Brilliant Violet 650TM (excitation max
405 nm,
27
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emission max 645 nm, 660/20 filter), Briliant Violet 711 TM (excitation max
405 nm,
emission max 711 nm, 710/50 filter), Brilliant Violet 750TM (excitation max
405 nm,
emission max 750 nm, 780/60 filter), Brilliant Violet 785TM (excitation max
405 nm,
emission max 785 nm, 780/60 filter). The polymer dye or polymer dye conjugate
may
comprise a Spark VioletTM 538 (BioLegend, Inc.)(excitation max 405 nm,
emission max
538 nm).
[00141] The polymer dye or polymer dye conjugate may
comprise a Super Bright
dye (Invitrogen, ThermoFisher Scientific). Super Bright dyes may be excited by
the
violet laser (405 nm). The Super Bright dye may be Super Bright 436
(excitation max
414 nm, emission max 436 nm, 450/50 bandpass filter), Super Bright 600
(emission max
600 nm, 610/20 bandpass filter), Super Bright 645 (emission max 645 nm, 660/20
bandpass filter), or Super Bright 702 (emission max 702 nm, 710/50 bandpass
filter).
pm 42] The polymer dye or polymer dye conjugate may
comprise a BD Horizon
BrilliantTM Violet polymer dye (Becton, Dickinson and Co., BD Life Sciences).
The
polymer dye may be a BD Horizon BrilliantTM BV421 (450/40 or 431/28 filter),
BV480
(525/40 filter), BV510 (525/40 filter), BV605 (610/20 filter), BV650 (660/20
filter), BV711
(710/50 filter), BV786 (786/60 filter).
[00143] The polymer dye may be prepared synthetically by
polymerization of
monomers, which leads to formation of a highly conjugated fluorescent
backbone.
Capping may be carried out on the polymer by activation using appropriate
functionalities, which results in a polymer capable of being conjugated to a
binding
partner. Alternatively, the polymer may be activated for conjugation by
attaching
appropriate functionalities off the polymer backbone. The activated polymers
may be
conjugated to a binding partner. Any appropriate binding partner may be
employed, for
example, an antibody, followed by purification, for example, by using standard
procedures. Functional groups can be selected from the group consisting of
amine,
carbarnate, carboxylic acid, carboxylate, maleimide, activated ester, N-
hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde,
thiol,
and protected groups thereof for conjugation to a substrate or binding
partner.
[00144] The polymer dye conjugate can comprise fluorescent
polymers having
monomer subunits including, but not limited to, dihydrophenanthrene (DHP),
fluorene,
and combinations thereof. In some embodiments, the polymer dye conjugate can
comprise a polymer dye having the structure of Formula III:
28
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PCT/IJS2021/059254
G1 [ Aa ( \\\) m
L ] G2
b c d
m (III)
[00145] Each A is independently selected from the group
consisting of an
aromatic co-monomer and a heteroaromatic co-monomer. Each A can be substituted
with a functional group that will be conjugated with a binding partner
[00146] Each optional M is independently selected from the
group consisting of
an aromatic co-monomer, a heteroaromatic co-monomer, a bandgap-modifying
monomer, optionally substituted ethylene, and ethynylene, and is evenly or
randomly
distributed along the polymer main chain. Each M may be independently selected
from
the group consisting of
, R&
.,,,, a WO OR" = 3D
"------.., r-----; ss,,..,..2. õ
==,............, P.C"), ORf, i......-
...=, .N..._..s.,
1-4 =-=======::,=,
:. ,..., ..ej s , .: f, ' \ s... 1
....,.......c= ........
.i., =s. S. < \;..,......S. 1 ="'k ...t¨ S.,
\
! ev<:;:s.s.1=3 \:i.....;51 NZZUSAI '
_.., / ''''''C's, l'XL.:' \ .... Vs., ; N.S. ......N.Z.
. WO OR' ' k: , . µ /* '-'.
= . .i----4,:,õ 7¨""=':µ,õ f'---.) 'e.' O.>. ". `--- .'.
.
0 ,.....t '...1 ,...1.:-..:;.0
RFi
3
- :' == 0 t L,K., 1., õ1
.= is
.:=,, ,...r......,,,,õ ,-.N., I,. t li
[I. Y ..'>---
-. =) ,,,,,.. ...:?' "..\
1,...., `......,,,:, ss....i., SN,
1 L --3, =$,.
O 'N- "0
o'''' ls,i, -. ';',.)
, ........................... ,:,4---.4 , W' re=
R6 A.
...
f.z..., 0, 0 re
R5
i .. =:, -,,,: -, ...
/ \ 0 0
..........................................................................
Is.'s.*
== .4
.."*".'-' '' )(:' i µ..>"""4;, 1 ..., ....,+=::::rs- N., 'Cc.
. ..> \ / i = Al i. ';,-- = , ,...-t=
.¨......../1 .S.. el "",....._
glf"'S Cf" Ns
P!:. ,e (.- /...../ \----
="'"--.' i \kg- \-..:õ.....-;"
stratte
,
Rs Rs
R:5
sO C.; k...
. i
i =c---...s. õ...-.:7,..-",.. .......-,... .., = . , =
1 I ,. /1/ ''.;µ, 2.," 'Ns, i......,
0 ORE'y
--.'..,s..,.......1,,,..) ...,,,, ,
6 1 b T ,.....¨:¨....e.. e!,.....-:-..< ' s
/ ................................................................... `.
=1== 4
')'''''''4
k... .....*: .., ,..,õ .......;,--:=.,, si,
, µ2'-'''' .S ,f,'" .. =:s. fe- s'.'= ', .>.:
\..,. ....;/ .4
)
(...1. b ." '' = , = .,---
. ,' .f.......-,-. --......, ,. .., ,...t.
R.4 R-' R'-'0
29
CA 03198558 2023- 5- 11
WO 2022/104147 PCT/IJS2021/059254
F:}R.'4."
. ..
''::. .... .'''. 0 R,' ',....."
i .................................................... \ ......,/
.., , ._ N NI
..........= ..õ.....,,,,.....,õ,,,
\ is-- ...., -:=.' k
s....'.::õ ,....fl¨ `,,, 4::=:7-
.., ..õ ,,,
R60----<= ==¨ < .. z= .-> < .. ;= =¨= ). =õ;
,..= ='," = s ==,, ........ ,...., ,s ,..:
p............. it 'µ',
. = \ \,= -1/ s>. c:, s=Pt=--
S....,... .---..s,
Rr-'0
i.. .,
e
,
,
A).
S
......, .....1".õ.,.õ .t ,
, ./.., r.
, ,. :;-- . ....)
...........,...,-, N:3_1 ;iõ......õ;,.., =-
=,........ ,i I i
= ,.. e. === , / 40,.
.."=,.. ...-1:'
, .1. ===== 4... 11 ': \ Ar
!.$ ,...; i.f /1 : ':.:, õ..-c:' , ......).-.õ,;$
N N N N 4.=
,4,s =-=-=== ' ,,.....--'---- N ''. i!, =si
,....,,.....õ=:,- ======.F
sta'
e R5
'''µ J.).. I"'
0..* ;===== `-...In 0 klir'
i.: F
..,,k ....,.. -:,....,,õ:, õCI .3,. ....,
õ.
( 1 I ,. ,
'...: -----4.
= : . ............... `,.... i \ 1 1
< s.A..
...,........,.. , .
.5x, e" '=:-.; `,..,,, -....:........ .,,N,
i.,"....1.7 .t.r... ..,...s,.... .....,,,,,
.,..., I
:$..õ ...vs.'
1 F:
I 1 ,
'
N.
'\#'4'
F I
P r .1. z:
..,....1.
)1*
( ,..t.
.s....,a
F., --.1/4:,,. õ-.F I .' i ,
1 ..................s
õ
....., , .. ....../
, ........,,,,, c
?== ,
, ..,,,..
\\ k A ../..5> =
==;.....---õ., ,..=-=====.õ=-= ..4, z. p,,
i -N ..,-4.,k....."`Z.,... =,'
ss-,*"...'st=''' 0" s'N,' "cst;'' N.,' 10" N.a- - -,
=-= .{.:. z
H f
... =
.,..,, 0 . .0 ''' " 11
l'...õ:f.:',C, ./.....0,-, ....,,,,,,,, = fq .,,,ind,40,
,..,.., I
1 ''s
O.'S. e=-... 1,.(,)
¨ ti =$ ====-= tt...?
, 0 1 ' O.."' PI
"....i' =====' t.r.
1
1
e....' ,I. ,0 s ' -- ' = s
Mea L.= : ki Me0' is¨ µ-1, 0
, le ;
.., i
.., (
1.======== ........................................... =
1---.. õ..N......4
,., ..................................................
....
....
, ...,
,
0, f...õ.., ...-1,. =OP,fie b ? -
y -...- OMe
(.; .f.
.riZr.ef.)
eX.F.::=0
/ tttttt
i .
=
.:
" \',. ...p
õ.. =
'
1 ..
, ; ..... i.------4\ . µ s
aik1.1
.õ t/
i... ,..: z ..,... ..7
. .....s.,. ..../ , = , it .0
õ i and =========¨ PE'Gro-
1 ;
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tit4' t'l
z
SO?
=fti=;z:Cv =
f;;.:
x X
,- -µ, ....
.
ir=== .,;:=-4,. .. ,,s.,, ,,,, ,¨- "--
....,:. c
%-----d 'sµ =1 N---1 ....
.. . / s
..7--..... ,\i'e"'""t ............... .õ "--- k )5-- \. 1 '=
if S ,> '
so.z......, \v \;=-õ-=;=-=,,, .,¨,- ,k::::.,,,,
...:;;:,s =,;,=....õ4, ==== it
,
,
Wi
(Per:qt le le'
f re s, i
"N¨N
m).
.õ,; '%0 = ¨, -t.===Nt+ -=
i
= =,$.0z,: ;-..);A , ========$1 =
:: k.),...: l'", .
, 1
01,=C).X." i?.s.ki. t It 2C.3', fe.11,) / =
_ = .,... ....,=
:Z'
le .4.'''.k.''W , 5c A,=:x-V-4:z,
A ___________________________________________ ):1'¨',¨"4
'<,4r.s1
,
i=-=e, W
\========<'
k - ,
N \ 4
0-A .... ':V:12 N11-i J-ii4
...--
I's'. /
,. .
õ.
<,:,' e=
0
.........;< .,..---N. . , .,. ,
,, ,::,- ',,,, == .. ==,;, ,, , !,-* ==,:s.
,..., x. ,
-
',¨...,.......,- - '-::::::, , and
,
wherein, each M can be substituted, and terminated with a functional group
selected from amine, carbamate, carboxylic acid, carboxylate, maleimide,
activated
ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne,
aldehyde, thiol, amide, sulfonamide, ether, thioether, thiocarbamate,
hydroxyl,
iodoacetyl, hydrazido, hydrazino, ketone, phosphine, epoxide, urea, thiourea,
thioester, imine, disulfides, and protected groups thereof for conjugation to
another
substrate, acceptor dye, molecule or binding agent, and
wherein, each R5 is independently selected from the group consisting of
halogen, hydroxyl, 01-012 alkyl, 02-012 alkene, 02-012 alkyne, 03-012
cycloalkyl, Cl-
012 haloalkyl, 01-012 alkoxy, a 02-018 (hetero)aryl group, 02-018
(hetero)aryloxy, 02-
018 (hetero)arylamino, carboxylic acid, carboxylate ester,
(CH2)x,(OCH2¨CH2)y,OCH3,
and (CH2)x,(OCH2¨CH2)õ,0CF3 where each x' is independently an integer from 0-
20
and each y' is independently an integer from 0-50; wherein
each RI is independently an ammonium alkyl salt, an ammonium alkyloxy salt,
an ammonium oligoet her sait, a suiforiate alkyl salt, a sulfenate alkoxy
salt, a suifonate
oligoether salt, a sulfonamide oligoether, or a moiety:
31
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so2
(C1-1-,r
f'ffjrrj-
each R2 is independently H, alkyl, akenyl, akynyl, cycloalkyl, haloalkyl,
alkoxy,
(hetero)aryloxy, aryl, (hetero)aryiarnino, a PEG group, an ammonium alkyl
salt, an
ammonium alkyloxy salt, an ammonium oligoether salt; a sulfonate alkyl salt, a
sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamido oligoether,
or a moiety
R3
so2
(CH2r
f'f'fj\rff ; and
each R3 is independently selected from the group consisting of H, alkyl,
alkene,
alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl,
(hetero)arylamino, and a
PEG group;
each Z is independently selected from the group consisting of 0, 0, and N:
each 0 is independently selected from the group consisting of a bond, NH, NR4,
and
CH2; and
each subscript n is independently an integer from 0 to 20.
[00147] Linkers are represented in Formula ill as L. Each
optional linker L may
be an aryl or heteroaryl group evenly or randomly distributed along the
polymer main
chain and can be substituted with one or more pendant chains terminated with a
functional group selected from the group consisting of amine, carbamate,
carboxylic
acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl,
hydrazine,
hydrazide, hydrazone, azide., alkyne, aldehyde, thiol, and protected groups
thereof for
conjugation to a substrate or binding partner.
[00148] The polymers complexes of the disclosure also
contain terminus
represented in Formula Ill as each G1 and G2. The terminus may be modified or
32
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unmodified. The terminus may each independently selected from the group
consisting
of hydrogen, halogen, alkyne, optionally substituted aryl, optionally
substituted
heteroaryl, halogen substituted aryl, silyl, cfiazoniurn salt, trifiate,
acetyloxy, azide,
sulfonate, phosphate, boronic acid substituted aryi, boronic ester substituted
aryi,
boronic ester, boronic acid, optionally substituted dihydrophenanthrene (DHP),
optionally substituted fluorene, aryl or heteroaryi substituted with one or
more pendant
chains terminated with a functional group selected from amine, carbamate,
carboxyiic
acid, carboxylate, maleirnide, activated ester, N-hydroxysuccinimidyl,
hydrazine,
hydrazide, hydrazone, azide, aikyne, aldehyde, thiol, and protected groups
thereof that
may be conjugated to a substrate or binding partner.
[00149] In the structure of Formula III, a, c, and d define
the mol % of each unit
which each can be evenly or randomly repeated and where each a is a mol % from
10
to 100%, each c is a mol % from 0 to 90%, and each d is a mol % from 0 to 25%;
each
b is independently 0 or 1; and each m is an integer from 1 to about 10,000.
[00150] In some embodiments, the polymer dye conjugate can
have the structure
of Formula
(I)
L2E
(,
11111 B ci2,
((W)t----L1)
wherein:
each A is independently selected from the group consisting of an aromatic co-
monomer
and a heteroarornatic co-monomer;
L1, L2, and L3 are linker moieties;
W is a water-solubilizing moiety;
each E is an independently selected chromophore, functional moiety, or binding
partner;
each B is independently selected from the group consisting of an aromatic co-
monomer,
a heteroarornatic co-monomer, a bandoap-modifying monomer, optionally
substituted
ethylene, and ethynylene;
Gland G2are independently selected from an unmodified polymer terminus and a
modified polymer terminus;
subscripts n and m are independently integers ranging from 1 to 10,000,
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subscript p is an integer ranging from 0 to 10,000, and
the sum of subscripts n, rn, and p ranges from 2 to 10,000;
subscript q is 1, 2, 3, or 4;
subscript r is 1, 2, 3, 0r4;
subscript s is 0, 1, 2, or 3;
subscript t is 1 or 2
the sum of subscript r and s ranges from 1 to 4; and
A and B are distributed randomly or non-randomly in the conjugated polymer.
L, can be a sulfonamide, a sulfonamide, a sultam, a disulfinamide, an amide, a
phosphonamide, a phosphonamidate, a phosphinamide or a secondwy amine. Or
Ll can be a sulfonamide, an amide, a phosphonarnide, or a secondary amine.
The subscript q can be equal to the sum of subscripts r and s, subscript r can
be 1 or 2,
if subscript r is 1, then subscript s is 0 or 1, and if subscript r is 2, then
subscript s is 0.
Each L3 can be a covalent bond.
[001511 The conjugated polymer can have a structure
according to Formula
o 0 0 0 0 0
%s//
R1)
(RS)
'1,1c) (L1'
w w
w
G _______________________ A _________________________ G2,
- - - -P
wherein:
L1''' is a linker moiety; and
R1 is selected from the group consisting of H and an amine protecting group.
A variety of linkers L13 and L2, as described herein, can be employed for
synthesis of
polymers according to Formula I and Formula IL For example:
Lla can be selected from the group consisting of a covalent bond, C1..6
alkylene, 2- to 8-
membered heteroalkylene (e.g., a divalent aikoxy linker), C3_8cycloalkylene,
aryiene, 5- to 12-membered heteroarylene, 5- to 12-membered heterocyc.,Iyiene,
¨
NHC(0)L1-, ¨C(0)NhiLa-, --C(0)L-, and combinations thereof;
L2can be selected from the group consisting of a covalent bond, C1-3alkylene,
2- to 8-
membered heteroalkylene (e.g., a divalent alkoxy linker), C,Bcycioalkylene, C6-
.10 arylene, 5- to 12-membered heteroarylene, 5- to 12-membered
heterocyclylene,
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LbNIFIC(0)¨, -LbC(0)1',1H¨, -L1C(0)¨, ¨C(0)NHL, ¨C(0)L, and combinations
thereof;
Land Lb can be independently selected from the group consisting of C/-8
alkyiene and
2- to 8-membered heteroalkylene; and
FR' can be selected from the group consisting of H and an amine protecting
group.
Polymers according to Formula II are provided wherein:
Lla is selected from the group consisting of a covalent bond, C1_,9aikyiene, 2-
to 8-
membered heteroalkylene, ¨NHC(0)L3-, ¨C(0)NHL-, and -CL-,
L2is selected from the group consisting of a covalent bond, Ci_s alkylerie; 2-
to 8-
membered heteroalkylene, -LbNHC(0) ____________ ,-LC(0)NH __ , -LbC(0)
_____________ , C(0)NHL1-, and
¨C(0) Lb;
Land L" are independently selected from the group consisting of C1-8 alkylene
and 2- to
8-membered heteroalkylene; and
F1 is selected from the group consisting of H and an amine protecting group.
W can comprise one or more ethylene glycol monomers. Or VV can comprise
poly(ethylene glycol).
L3 can be a trivalent arylalkyl moiety having: a first point of attachment to
a first L' moiety
(or a first Lia moiety); a second point of attachment to a second L1 moiety
(or a second
Lla moiety); and a third point of attachment to an A monomer.
[00/52]
For example, the disclosure provides conjugated polymers having two or
more chromophores attached as shown in Formula VI;
(w)
E o 0 0 0 E
1 %// 1
L2
,L2
L'a N
w1 \0
0
3a
JW.M6/1M
Wherein
Lla is as previously defined;
L2 is as previously defined;
W is as previously defined;
L3a is selected from the group consisting of a covalent bond, C1.8alkyiene, 2-
to 8-
membered heteroalkylene, ¨NHC(0)L3-, ¨C(0)N1-11.."-, and ¨C(0)L ______ ;
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La is selected from the group consisting of C=1.3alkylene and 2- to 8-membered
heteroalkyle,ne; and the wavy line is the point of the attachment to the a
monomer.
Each A monomer in polymers having a structure of Formula I, H or IH can be the
same
monomer_ Each A monomer in polymers having a structure of Formula I, H or H
can be
a different monomer. A can be a fluorescent monomer. A can be a 9,10-
phenanthrenedione-hased monomer (e.g., a dihydrophenanthrene (DHP)-based
monomer), a fiuorene-based monomer, or a fluorenoexepine-based monomer.
[00153] Monomers A in polymers having a structure of Formula
l, H or can be
DHP-based monomers such as:
R2¨x¨ Y
wherein:
each X is independently C or Si;
each Y is independently CR1R2or Sili1R2;
each R1 is independently an ammonium alkyl salt, an ammonium alkyloxy salt, an
ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a
sulfonate
oligoether salt, a sulfonarnido oligoether, or a moiety:
=
2
(CH2)"
r>fssZ
each R2is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl,
alkoxy,
(hetero)aryloxy, aryl, (hetero)arylarnino, a PEG group, an ammonium alkyl
salt, an
ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a
sulfonate
alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, or a
moiety
36
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Q----R3
' I
so,
(CH21., ---- -
I
z
rrsj\rffl .
each R3is independently selected from the group consisting of H, alkyl,
aikene, alkyne;
cycloaikyi, haloalkyl, aikoxy, (hetero)aryloxy, aryl, (hetero)aryiai-nino, and
a PEG group;
each Z is independently selected from the group consisting of C. 0, and N;
each 0 is independently selected from the group consisting of a bond, NH, NR4,
and
CH2; and
each subscript n is independently an integer from 0 to 20.
[00154] R1 can have the structure shown below, wherein 0 is
NH:
...--R,
Q
I
,....,, SO2
(CH2r
z1
each R3 is independently selected from the group consisting of H, aikyi,
alkene, aikyne, cycloalkyl, halcalkyl, alkoxy, (hetero)aryloxy, aryl,
(hetero)anylamino,
and a PEG group: and each Z is independently selected from the group
consisting of C,
0, and N.
[00155] The DHP-based monomer can have the structure:
RI,
(PEG) f ¨ N
\ õ,--- N---õ,.., /
SO2 02S (PEG)/
'
/ \
(H2C)n
/ (CH2)"
\
Z Z
R2 R2
wherein:
each subscript f is independently an integer from 0 to 50;
each subscript n is independently an integer from 0 to 20;
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each R2 is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl,
alkoxy,
(hetero)aryloxy, aryl, (hetero)arylamino, a PEG group, an ammonium alkyl salt,
an
ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a
sulfonate
aikoxy salt, a sulfonate olic.loether salt, a suifonamido oligoether, or a
moiety
OS
(CH2Y:
z
rs'rj
each R5 is inde.pendently H, i-C20 alkyl, C2-C2oalkenyl, CCo
al kynyl,
020 cycloalkyl, C1-C20 haloalkyl, Ci-C20 alkoxy, 02-025 aryloxy, C-C25
heteroaryloxy, 02-
026 arylamino, or C2-C26 heteroarylamino; and
each Z is independently selected from the group consisting of 0, 0, and N.
[00156] The DHP monomer
can have the structure:
R5, R5
H ===
(PEG)f ¨N (PEG)f ¨N R5
R5
\ SO2 \ SO2 02
S 02S (PEG)/
(142C) (CE12)"
(H2C)1 (CII2r
Z Z
R2 R2
wherein.
each subscript f is independently an integer from 0 to 50;
each subscript n is independently an integer from 0 to 20;
each R2 is independently H, alkyl, alkenyl, aikynyl, cycloaikyi, haloalkyl,
alkoxy,
(hetero)aryloxy, aryl, (hetero)arylamino, a PEG group, an ammonium alkyl salt,
an
ammonium alkyloxy salt, an ammonium oligoether salt; a sulfonate alkyl salt, a
sulfonate
alkoxy salt, a sulfonate oliooether salt, a sulfonamido oligoether, or a
moiety
38
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R3
¨s o2
(CHir
zI
rfrj\fµi'cl
each R5 is independently H, C1-.020 alkyl, C2.-C20 alkenyl, C2.-C20 al kynyl,
C3¨
C20 cycloalkyl, C1-C20 haloalkyl, C1C2c.alkoxy, C2-C26 aryioxy, C2-C26
heteroaryloxy, C2
C26 arylamino, or C2-C26 heteroai ylamino; and
each Z is independently selected from the group consisting of C, 0, and N.
[00157] Monomers A in polymers having a structure of
Formulas 1, H or H can be
fluorene-based monomers such as:
R' R2
\x/
Rs Rs
(PEG)f (PEG)f
HN¨S02 02S-1\-1-1
(CH2) ¨ZN Z¨(CH2)In
wherein X, Z, R1, R2, R5, subscript n, subscript fare as defined herein.
[00158] R1 groups and R2 groups such as ammonium alkyl
salts, ammonium
alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate
alkoxy salts,
sulfonate oligoether salts, sulfonamido oligoethers, or moieties having the
structure:
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so,
$-r-ri\j'rjj
can impart solubility in water/buffer. In some embodiments, for example, the
polymer is
soluble at levels in excess of 10 mg/nil_ in excess of 15 rngirnL, in excess
of 20 mg/iTiL,
in excess of 25 rnglmL, in excess of 30 mo/mL, in excess of 35 mgirnL, in
excess of 40
mg/mL, in excess of 45 mg/nit_ in excess of 50 mgin-IL, in excess of 60 mgimL,
in excess
of 70 mgimL, in excess of 80 maimL, in excess of 90 mg/n1L or in excess of 100
mg/mL
[00159] Monomers A also include bridged monomers. For
example, bridged
monomers of the present invention include:
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IR5
02S SO2
0\ /
SO2
02S
= and
R5 R5
NH HN
025 802
0\ /0
wherein, X, Y, R2, and R5 are as previously defined.
[00160] Monomers A in polymers having a structure of Formula
H or can be
oxepine-based monomers (e.g., fluorenooxepine-based monomers), such as:
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R!,\ /R2
X
0
R2
, and
0
R2
o
wherein X, R1. and R2 are as defined herein.
Tandem Polymer Dyes
[00161] The polymer can have acceptor dyes attached to the
backbone that will
provide for monitoring the emission of the acceptor dyes attached to the
backbone
through energy transfer. Acceptor dyes useful in the tandem polymer dyes
include, for
example, FITC, CY3B, Cy55, Alexa 488, Texas red, Cy5, Cy7, Alexa 750, and
800CW.
For example, acceptor dyes can be attached to the polymer through a linker L:
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R1
G2 and
a
energy transfer
.40 is an acceptor dye
R2 R1
\ /
X
01
a G2 .
energy transfer
40 is an acceptor dye
As described in US Published Application No. 2020/0190253, which is
incorporated
herein by reference in its entirety, acceptor dyes can also be attached
directly to
monomer A as group E in the structures of Figure I or II above. SuperNova
tandem
dyes SuperNova v605 and SuperNova v786 (Beckman Coulter, Inc.) are tandem
polymer dyes, derived from the core SuperNova v428. Both SuperNova v605 and
SuperNova v786 share the same absorbance characteristics, with maximum
excitation
at 414 nm. With emission peak respectively at 605 nm and 786 nm, they are
optimally
detected using the 610/20 and 780/60 nm bandpass filters of the flow
cytometer.
Conjugate Dyes
[00162] The polymer dyes may be conjugated to different
specificities of binding
partners, e.g., target-analyte specific antibodies, in order to synthesize a
binding
partner-dye conjugate such as CD19- SN v428, CD20- SN v605, etc.
[00163] The polymer dye and polymer dye conjugates may be
formulated with an
aqueous buffer. Any appropriate aqueous buffer may be employed, for example,
an
isotonic aqueous buffer such as a PBS buffer. The aqueous buffer may include
43
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additives. For example, the aqueous buffer may include BSA, sodium azide, a
non-ionic
surfactant, e.g. PF-68, and a zwitterionic surfactant, e.g., Empigen BB , or
anionic
surfactant, e.g., NLS, as described herein. BSA helps in stabilizing the
conjugate,
sodium azide prevents from any microbial contamination, and the surfactant,
such as
Empigen BB , significantly reduces or eliminates non-specific binding on the
monocytes
& granulocytes. The BSA may be present in a range of from 0-3 mg/mL, 0.5-2.5
mg/mL
or about 2 mg/mL. The sodium azide may be present in a range of from 0-0.05%,
0.05-
0.03%, or about 0.01% (w/v).
Binding partner
[00164] As used herein, "binding partner" refers to any
molecule or complex of
molecules capable of specifically binding to a target analyte. The binding
partner may
be, for example, a protein (e.g., an antibody or an antigen-binding antibody
fragment),
a small organic molecule, a carbohydrate (e.g., a polysaccharide), an
oligonucleotide, a
polynucleotide, a lipid, an affinity ligand, an aptamer, or the like. In some
embodiments,
the binding partner is an antibody or fragment thereof. Specific binding in
the context of
the present invention refers to a binding reaction which is determinative of
the presence
of a target analyte in the presence of a heterogeneous population. Thus, under
certain
assay conditions, the specified binding partners bind preferentially to a
particular protein
or isoform of the particular protein and do not bind in a significant amount
to other
proteins or other isoforms present in the sample.
[00165] In some cases, the antibody includes intravenous
immunoglobulin (IVIG)
and/or antibodies from (e.g., enriched from, purified from, e.g., affinity
purified from)
IVIG. IVIG is a blood product that contains IgG (immunoglobulin G) pooled from
the
plasma (e.g., in some cases without any other proteins) from many (e.g.,
sometimes
over 1,000 to 60,000) normal and healthy blood donors. IVIG is commercially
available.
Aspects of IVIG are described, for example, in US. Pat. Appl. Pub. Nos.
2010/0150942;
2004/0101909; 2013/0177574; 2013/0108619; and 2013/0011388, which are
incorporated herein by reference.
[00166] When the binding partners are antibodies, they may
be monoclonal or
polyclonal antibodies. The term "antibody" as used herein refers to
immunoglobulin
molecuic and immunologically active portions of immunoglobulin (1g) molecules,
for
example, which specifically bind to an antigen in a target analyte. Such
antibodies
include, but are not limited to, polyclonal, monoclonal, mono-specific
polyclonal
antibodies, antibody mimics, chimeric, single chain, Fab, Fab' and F(ab')2
fragments, Fv,
and an Feb expression library. In some cases, the antibody is a monoclonal
antibody of
44
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a defined sub-class (e.g., IgG1, IgG2, IgG3, or IgG4, IgA, IgD, IgE, IgG2a,
IgG2b, IgG3,
and IgM). If combinations of antibodies are used, the antibodies can be from
the same
subclass or from different subclasses. For example, the antibodies can be IgG1
antibodies. In some embodiments, the monoclonal antibody is humanized.
Antibody
fragments may include molecules such as Fab, scFv, F(ab')2, and Fab'
molecules.
Antibody derivatives include antibodies or fragments thereof having additions
or
substitutions, such as chimeric antibodies. Antibodies can be derived from
human or
animal sources, from hybridomas, through recombinant methods, or in any other
way
known to the art.
[00167] Binding partners other than antibodies or target
analyte specific antibody
fragments or derivatives can also be used in the present system and methods.
For
example, binding partners may be nucleic acids or nucleic-acid analogs, such
as
oligonucleotides or PNA probes. In one embodiment, aptamers can be used as
specific
binding partners. Aptamers are single-stranded DNA or RNA (ssDNA or ssRNA)
molecules that can bind to pre-selected targets including proteins and
peptides with high
affinity and specificity. Other binding partners that can bind to target
analyte to form pairs
of receptor-ligand, enzyme-substrate, enzyme-inhibitor, and enzyme-cofactor
pairs can
also be used. Specific examples of such binding partner pairs include
carbohydrate and
lectin, biotin and avidin or streptavidin, folic acid and folate binding
protein, vitamin B12
and intrinsic factor, Protein A and immunoglobulin, and Protein G and
immunoglobulin.
Also included are binding partners that form a covalent bond with the target
analytes.
Conjugation
[00168] A polymer dye conjugate can comprise any known
polymer dye
conjugated to a binding partner using techniques known to those of skill in
the art. In
some embodiments, a polymer dye can be conjugated to a binding partner to form
a
polymer dye conjugate using the method of direct modification of core polymers
described in US Published Application No. 2020/0190253, which is incorporated
herein
by reference in its entirety.
[00169] In some instances, a polymer dye can be conjugated
to a binding partner
to form a polymer dye conjugate using the method described in US Published
Application No. 2019/0144601, which is incorporated herein by reference in its
entirety.
The method can be depicted as follows:
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<*.ROw oxoww .
POystw . ________________________________ 0,* Pz#ytraersQ,X.Nti
Poirtxro:',<:4
MAirt pk0i6L4 >MOW kai:t&
{;;OON groozn
TV#11,os. Ciac.;N
akzoi*,1 fto
:ftsb=mes.-eleey d' ______ = Putmer-ws%e*-,µ,e ___ ftetel,emotf.*$4,0,
Mtiitsedy, buffer
maw:sr u?Ogscly
[00170] SuperNova v428 (SN v428 (Beckman Coulter) is a
bright polymer dye
that can be activated with amine for tandem dyes, followed by maleimide
activation for
tandem conjugates. The rigidity of the polymer dye structure may help reduce
rotational
energy leading to brighter emissions. SuperNova v428 is one of the brightest
dyes
excitable by the violet laser, so it is particularly suited for assessing
dimly expressed
markers. SuperNova conjugated antibodies may include anti-CD19 antibody-
SuperNova v428, anti-0D22 antibody-SuperNova v428, anti-0D25 antibody-
SuperNova
v428, and anti-CD38 antibody-SuperNova v428 antibody-polymeric dye conjugates.
Target Analyte
[00171] The disclosure also relates to a method for
detecting a target analyte in
a sample, wherein the target analyte comprises a target antigen and can be a
substance,
eq., molecule, whose abundance/concentration is determined by some analytical
procedure. The present invention is designed to detect the presence, and in
some cases
the quantity of specific target analytes. The term "target analyte" refers to
a target
molecule containing a target antigen to be detected in a biological sample,
for example,
peptides, proteins, polynucleotides, organic molecules, sugars and other
carbohydrates,
lipids, and small molecules. It is an important aspect of the disclosure that
the target
analytes are comprised in a liquid sample and are accessible, or made
accessible at
some point, to bind target analyte-specific binding partners of the instant
invention.
Target analytes may be found in a biological sample, such as a blood sample, a
cell line
development sample, a tissue culture sample, and the like.
[00172] The target analyte may be, for example, nucleic
acids (DNA, RNA,
rriRNA, tRNA, or rRNA), peptides, polypeptides, proteins, lipids, ions,
monosaccharides,
oligosaccharides, polysaccharides, lipoproteins, olycoproteins, glycolipids,
or fragments
thereof. The target analyte can be a protein and can be, for example, a
structural
microfilarnent, microtubule; and intermediate filament proteins, organelle-
specific
markers, proteasomes, transmernbrane proteins, surface receptors, nuclear pore
proteins; protein/peptide translocases, protein folding chaperones, signaling
scaffolds,
46
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on channels and the like. The protein can be an activatable protein or a
protein
differentially expressed or activated in diseased or aberrant cells, including
but not
limited to transcription factors, DNA and/or RNA-binding and modifying
proteins, nuclear
import and export receptors, regulators of apoptosis or survival and the like.
[00173] Target analytes can be present and accessible on the
surface of cells.
Illustrative examples of useful analytes include, but are not limited to, the
following: 1)
specific cell surface macromolecules and antigens (including hormones, protein
complexes, and molecules recognized by cell receptors) and 2) cellular
proteins, DNA
or RNA in permeabilized cells including abnormal DNA or RNA sequences or
abnormal
amounts of certain messenger RNA. Detection of these analytes may be
particularly
useful in situations where they are contained in and/or are identifiers of
rare cells such
as are found in the early stages of a variety of cancers.
[00174] In some examples, the target analyte may be a CD2,
CD3, CD4, CD8,
CD10, CD11c, CD14, CD15, CD16, CD19, CD20, CD22, CD25, CD27, CD38, CD45,
CD45RA, CD56, CD62L, CD64, CD95, CD103, HLA-DR, IFN-y, TNF-a, or ZAP-70, or
other target analyte of interest.
Biological Sample
[00175] Non-limiting examples of the biological sample
include blood, serum,
plasma, urine, semen, milk, sputum, mucus, a buccal swab, a vaginal swab, a
rectal
swab, an aspirate, a needle biopsy, a section of tissue obtained for example
by surgery
or autopsy, plasma, serum, spinal fluid, lymph fluid, the external secretions
of the skin,
respiratory, intestinal, and genitourinary tracts, tears, saliva, tumors,
organs, samples of
in vitro cell culture constituents (including but not limited to conditioned
medium resulting
from the growth of cells in cell culture medium, putatively virally infected
cells,
recombinant cells, and cell components).
[00176] The sample in the methods of the disclosure can be,
for example, blood.
The blood sample can be whole blood. The whole blood can be obtained from the
subject using standard clinical procedures. The sample can be a subset of one
or more
cells of whole blood (e.g,, erythrocyte, leukocyte, lymphocyte (e.g., T cells,
B cells or NK
cells), phagocyte, monocyte, macrophage, granulocyte, basophil, neutrophil,
eosinophil,
platelet, or any cell with one or more detectable markers). The sample can be
from a
cell culture. The sample may comprise a target analyte naturally or may be
prepared
through synthetic means, in whole or in part.
47
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Subject
[00177] The subject can be a human (e.g., a patient
suffering from, or suspected
of suffering from, a disease), a commercially significant food chain mammal,
including,
for example, a cow, steer, pig, goat, sheep, bird, fish, or horse. Samples can
also be
obtained from household pets or companion animals, including, for example, a
dog, cat,
rabbit, bird, or ferret. The subject can be a laboratory animal used as an
animal model
of disease or for drug screening, for example, a monkey, mouse, a rat, a
rabbit, or guinea
pig. The subject can be an exotic animal, such as a zoo animal or a wild
animal, such
as an elephant, antelope, zebra, bison, giraffe, lion, tiger, panther,
orangutan, gorilla,
whale, dolphin, shark, or reptile.
Reaction Vessel
[00178] A reaction vessel disclosed herein can be any
container where reactions
between the binding partners or polymer dye conjugates thereof and the target
analytes
can occur. For example, a reaction vessel can be a tube, a plate, a well of a
microtiter
plate, a chamber, and a slide. In a preferred embodiment, a reaction vessel
has a lid or
cap such that the binding reaction can occur in a closed environment.
Substrate
[00179] A reaction vessel comprises one or more substrates.
The substrate can
be any suitable surface, including but not limited to, plastic,
nitrocellulose, cellulose
acetate, quartz, and glass. Non-limiting examples of plastic may include
polystyrene,
polypropylene, cyclo-olefin, and polycarbonate. In some embodiments, the
substrate is
a membrane. The substrate can be the inside surface of the body of a reaction
vessel,
e.g., a plastic tube or well of a microtiter plate. The substrate can also be
a bead. In
some embodiments, at least one of the substrates receiving the labeled binding
partners
(e.g., a membrane) is bonded to an inside surface of the body of the reaction
vessel. In
some embodiments, the membrane substrate is a sheet or roll, which makes it
easier to
deposit the solutions and easier to dry. In some embodiments, the membrane can
be
cut to separate individual dried reactant spots. In some embodiments, the cut
membrane
is simply dropped into the reaction vessel. In some preferred embodiments, the
cut
membranes are bonded to the surface of the reaction vessel, so that the spots
do not
escape the vessel when liquid is pipetting into or out of the reaction vessel.
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Liquid Sample
[00180] The reaction vessel is configured to receive a
liquid sample. Liquid
samples used in the invention typically comprise target analytes obtained as
or
dispersed in a predominantly aqueous medium.
[00181] The sample can be, for example, a biological sample,
such as a blood,
bone marrow, spleen cells, lymph cells, bone marrow aspirates (or any cells
obtained
from bone marrow), urine (lavage), serum, plasma, saliva, cerebral spinal
fluid, lymph
fluid, urine, amniotic fluid, interstitial fluid, feces, mucus, milk, semen,
buccal swab,
nasopharangial swab, a vaginal swab, a rectal swab, an aspirate, a needle
biopsy, a
section of tissue obtained for example by surgery or autopsy, or tissue (e.g.,
tumor
samples, disaggregated tissue, disaggregated solid tumor) sample. The sample
can be
a blood sample. The blood sample can be a whole blood sample. The whole blood
can
be obtained from the subject using standard clinical procedures. The sample
can be a
subset of one or more cells of whole blood (e.g., erythrocyte, leukocyte,
lymphocyte
(e.g., T cells, B cells or NI< cells), phagocyte, monocyte, macrophage,
granulocyte,
basophil, neutrophil, eosinophil, platelet, or any cell with one or more
detectable
markers). The sample can be from a cell culture, in vitro cell culture
constituents
(including but not limited to conditioned medium resulting from the growth of
cells in cell
culture medium, putatively virally infected cells, recombinant cells, and cell
components).
[00182] Samples can be any source of biological material,
and may include
proteins, carbohydrates, and/or polynucleotides that can be obtained from a
living
organism, directly or indirectly. Samples can include, e.g., cells, tissue, or
fluid, and the
deposits left by that organism, including viruses, mycoplasma, and fossils.
The sample
may comprise a target analyte. The target analyte may be naturally occurring
in a
biological sample, or may be prepared through synthetic means, in whole or in
part.
Labeled Binding partner
[00183] Dyes can be conjugated to binding partners by
various linking chemistry
between reactive pairs located in the binding partners and the labels. The
reactive
pairs can include, but not limited to, maleimide/thiol, succimidylester (NHS
ester)/annine, azide chemistry, carboxy/EDC (1-Ethyl-343-
dimethylaminopropyl]carbodiimide Hydrochloride)/amine, amine/Sulfo-SMCC
(Sulfosuccinimidyl 4-[N-maleimidom ethyl]cyclohexane-1-carboxylate)/thiol, and
amine/BMPH (N-[-Maleimidopropionic acid]hydrazide.TFA)/thiol. Methods for
49
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performing the conjugation are well known in the art. Commercial kits for
performing
the conjugation are also readily available, e.g., from Innova biosciences
(Cambridge,
UK), Novus Biologicals (Littleton, Colo.), Thermo Fisher Scientific (Waltham,
Mass.).
[00184] Either a dry or liquid polymer dye conjugate can be
used in the methods
and compositions. Dried polymer dye conjugate can be prepared using any
technique
known in the art. The techniques can be as described in US 2019/0242882, which
is
incorporated herein by reference.
[00185] A polymer dye conjugate may be employed in a
composition according
to the disclosure that may be used directly to stain blood and analyze it in a
flow
cytometer.
Assay Systems
[00186] Assay systems utilizing a binding partner and a
fluorescent lab& to
quantify bound molecules are well known. Examples of such systems include flow
cytometers, scanning cytometers, imading cytometers, fluorescence microscopes,
and
confocal fluorescent microscopes.
[00187] Flow cytornetry is used to detect fluorescence. A
number of devices
suitable for this use are available and known to those skilled in the art.
Examples include
BC i Navios, Gallios, Aquios, and CytoFLEXTM flow cytometers
[00188] The assay can be an immunoassay. Examples of
immunoassays useful
in the invention include, but are not limited to, fiuoroluminescence assay
(FLA), and the
like. The assays can also be carried out on protein arrays.
[00189] When the binding partners are antibodies, antibody
or multiple antibody
sandwich assays can also be used. A sandwich assay refers to the use of
successive
recognition events to build up layers of various binding partners and
reporting elements
to signal the presence of a particular analyte. Examples of sandvvich assays
are
disclosed in US. Pat. No. 4,486,530 and in the references noted therein.
[00190] A light source is applied to the sample that can
excite the polymer and
light emitted from the conjugated polymer complex is detected, In the typical
assay,
fluorescent polymer dye conjugates for use in the invention are excitable with
a light
having wavelength between about 395 nm and about 415 nm. The emitted light is
typically between about 415 nm and about 475 nm. Alternatively, excitation
light can
have a wavelength between about 340 nrn and about 370 nm and the emitted light
is
between about 390 nm and about 420 nm.
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Applications
[00191] Compositions according to the disclosure may include
a single-color, i.e.,
a single polymer dye conjugate, such as a single SN polymer dye conjugate. For
example, biological samples may be stained using SN conjugates to monitor or
identify
particular cell populations, depending on the antibody conjugated to the
polymer dye.
[00192] In some embodiments, compositions according to the
disclosure may
include a single color polymer dye conjugate along with conventional non-
polymeric dye
conjugates. For example, SN conjugates can be used along with non-polymeric
dye
conjugates such as CD4-FITC, CD7-PE, CD25-ECD, CD56-PC5.5, etc., in a panel to
identify cell subpopulations in human whole blood samples by flow cytometry.
[00193] in some embodiments, one or a plurality of the
compositions according
to the disclosure may be contacted with a biological sample, such as a blood
sample.
For example, biological samples may be stained with a composition comprising a
plurality of SN conjugates to monitor or identify particular cell populations,
depending on
the antibody conjugated to the polymer dye. In some embodiments, 2 or more, 3
or
more, or 4 compositions according to the invention may be contacted with a
biological
sample. In some embodiments, compositions comprising a plurality of polymer
dye
conjugate compositions may further comprise non-polymeric dye conjugates such
as
CD4-FITC, CD7-PE, CD25-ECD, CD56-PC5.5, etc., in a panel to identify cell
subpopulations in human whole blood samples by flow cytometry.
EXAMPLES
[00194] The present invention can be better understood by
reference to the
following examples which are offered by way of illustration. The present
invention is not
limited to the examples given herein.
Example I: Preparation of DHP Polymer Complex
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//),- 0
OH
0 0
0 --.
S
Me0 o õ 0 NH 0 OMe
n-11
[00195] Method 1: In a round bottom flask dibromo DHP
monomer and diboronic
DHP monomers; as described in WO 2017/180908, (1:1) were taken in (DMF-water)
mixture and purged with nitrogen for 10 minutes, Under nitrogen about 20
equivalent of
CsF and 10% of Pd(OAc)2 were mixed and heated at 80 deg Celsius.
Polymerization
was monitored using UV-Vis spectroscopy and SEC chromatography. Later to the
reaction mixture, a capping agent (selected from Gl) containing appropriate
functional
group was added and 3 hours later the second capping agent (selected from G2)
added,
After the reaction the crude reaction mixture was evaporated off and passed
through a
gel filtration column to remove small organic molecules and low MW oligomers.
Later
the crude polymer passed through a Tangential flow filtration system equipped
with a
100K MWCO membrane, it is washed using 20% ethanol until the absorption of the
Filtrate diminishes,
[00196] Method 2: Alternatively, the polymerization can be
done by self-
polymerizing a brorno-boronic ester of DHP molecule. In a round bottom flask
DHP
brornoboronic ester was taken in (DMF-water) mixture and purged with nitrogen
for 10
minutes. Under nitrogen about 10 equivalent of CsF and 5% of Pd(OAc)2were
mixed
and heated at 80deg Celsius. Polymerization was monitored using UV-Vis
spectroscopy
and SEC chromatography. Later to the reaction mixture, a capping agent
(selected from
Gl) containing appropriate functional group was added and 3 hours later the
second
capping agent (selected from G2) added. After the reaction the crude reaction
mixture
was evaporated off and passed through a gel filtration column to remove small
organic
molecules and low MWoligorners. Later the crude polymer passed through a
Tangential
flow filtration system equipped with a 100K IVIVVCO membrane. It is washed
using 20%
ethanol until the absorption of the filtrate diminishes,
[00197] Method 3: In a round bottom flask both the dibromo
dihydrophenanthrene
and diboronic dihydrophanenthrene monomers (1:1) were taken and dissolved in
THF-
water (4:1) mixture containing 10 equivalent of K2CO3 and 3% Pd(PPh3)4. The
reaction
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mixture was put on a Schenk line and was degassed with three freeze-purnp-thaw
cycles and then heated to 80cle,g C. under nitrogen with vigorous stirring for
18 hours.
Later to the reaction mixture, a capping agent (seiected from Cl) containing
appropriate
functional group was added via a cannula under excess nitrogen pressure and 3
hours
later the second capping agent (selected from G2) added. After the reaction
the crude
reaction mixture was evaporated off and passed through a gel filtration column
to
remove small organic molecuies and low MW oligorners. Later the crude polymer
passed through a Tangential now filtration system equipped \A/U.11 a 100K MWCO
membrane. It is washed using 20% ethanol until the absorption of the filtrate
diminishes.
[00198] Method 4: Alternatively the polymerization can be
done by se-lf-
polymerng a bromo-boronic ester of dihydrophenanthrene molecule, in a round
bottom flask dihydrophenanthrene brornoboronic ester was taken and dissolved
in THF-
water (4:1) mixture containing 10 equivalent of K2.0O3 and 3% Pd(PPh3)4. The
reaction
mixture was put on a Schenk line and was degassed with three freeze-pump-thaw
cycles and then heated to 80deg C. under nitrogen with vigorous stirring for
18 hours.
Later to the reaction mixture, a capping agent (selected from G1) containing
appropriate
functic3nal group was added via a cannula under excess nitrogen pressure and 3
hours
later the second capping agent (selected from G2) added. After the reaction
the crude
reaction mixture was evaporated off and passed through a gel filtration column
to
remove small organic molecules and low MW oiigorners. Later the crude polymer
passed through a Tangential flow filtration system eoLlipped with a 100K MWCO
membrane. It is washed using 20% ethanol until the absorption of the filtrate
diminishes.
Examp/e 2: Preparation of Fiuorene-DHP Copolymer Complex
/
0
OH
-
MK) 0 TIN NH 0 OMe
(/ __________________
le0 0 HN/ s=
0" \
NH 0 OMe
(/\) (/ __
n 11
[00199] Method 1: in a round bottom flask both the dibrorno
DI-IP and diboronic
fluorene monomers (1:1) were taken in (DMF-water) mixture and purged with
nitrogen
for 10 minutes. Under nitrogen about 20 equivalent of CsF and 10% of Pd(OAc)2
were
mixed and heated at 80cleg Celsius. Polymerizz:Aion was monitored using UV-Vis
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spectroscopy and SEC chromatography. Later to the reaction mixture, a capping
agent
(selected from Cl) containing appropriate functional group was added and 3
hours later
the second capping agent (selected from G2) added. After the reaction the
crude
reaction mixture was evaporated off and passed through a gel filtration column
to
remove small organic molecules and low MW oligomers. Later the crude polymer
passed through a Tangential flow filtration system equipped \rvith a 100K MWCO
membrane. It is washed using 20% ethanol until the absorption of the filtrate
diminishes:
[00200] Method 2: In a round bottom flask both the dibromo
fluorene and
diboronic DHP monomers (1:1) were taken in (DMF-water) mixture and purged with
nitrogen for 10 minutes. Under nitrogen about 20 equivalent of CsF and 10% of
Pd(OAc)2 were mixed and heated at 80deg ceicius. Polymerization was monitored
using
UV-Vis spectroscopy and SEC chromatography. Later to the reaction mixture, a
capping
agent (selected from Cl) containing appropriate functional group was added and
3
hours later the second capping agent (selected from G2) added. After the
reaction the
crude reaction mixture was evaporated off and passed through a gel filtration
column to
remove small organic molecules and low MW oligomers. Later the crude polymer
passed through a Tangential flow filtration system equipped with a 100K MWCO
membrane. It is washed using 20% ethanol until the absorption of the filtrate
diminishes.
[00201] Method 3: In a round bottom flask both the dibromo
dihydrophenanthrene
and diboronic fluorene monomers (1:1) were taken and dissolved in THE-water
(411)
mixture containing 10 equivalent of K2CO3 and 3% Pd(PPI13)4. The reaction
mixture was
put on a Schlenk line and was degassed with three freeze-pump-thaw cycles and
then
heated to 80deg C. under nitrogen with vigorous stirring for 18 hours. Later
to the
reaction mixture, a capping agent (selected from Gl) containing appropriate
functional
group was added via a cannula under excess nitrogen pressure and 3 hours later
the
second capping agent (selected from G2) added. After the reaction the crude
reaction
mixture was evaporated off and passed through a gel filtration column to
remove small
organic molecules and low MW oligomers. Later the crude polymer passed through
a
Tangential flow filtration system equipped with a 100K MWCO membrane. It is
washed
using 20% ethanol until the absorption of the filtrate diminishes.
[00202] Method 4: In a round bottom flask dibromo fluorene
and diboronic
dihydrophenanthrene monomers (1;1) were taken and dissolved in THF-water (4:1)
mixture containing 10 equivalent of K2CO3and 3% Pd(PPh3)4. The reaction
mixture was
put on a Schlenk line and was degassed with three freeze-pump-thaw cycles and
then
heated to 80deg C. under nitrogen with vigorous stirring for 18 hours. Later
to the
reaction mixture, a capping agent (selected from G1) containing appropriate
functional
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group was added via a cannula under excess nitrogen pressure and 3 hours later
the
second capping agent (selected from G2) added. After the reaction the crude
reaction
mixture was evaporated off and passed through a gel filtration coiurnn to
remove small
organic molecules and iow MV\I oligomers. Later the crude polymer passed
through a
Tangential flow filtration system equipped with a 100K WACO membrane. It is
washed
using 20% ethanol until the absorption of the filtrate diminishes.
Example 3: Comparison oF Fluorescence Emission Spectra
[00203] Comparison of fluorescence emission spectra of
fluorene (A-FE),
dihydrophenanthrene (DHP-DHP) and fluorene-DHP (DHP-FE) polymers were
undertaken. After excitation at 405 rim, DHP containing polymers show a marked
difference in their fluorescence maxima which is at 426-426 nm, whereas the
fiuorene
based polymers show a maxima of 421 nm, as shown in FIG. 1A.
Example 4: Comparison of Absorption Spectra
[00204] The absorption spectra of both fluore..ne (FE-FD
polymer and
dihydrophenarithrene (DHP-DHP) polymer were measured. The DHP-DHP polymer
(black curve) exhibits lambda max (Amax) at 390 and 410 rim, whereas the Fl-Fl
(grey
curve) poiyrner shows lambda max (Amax) at about 400 nm, as shown in FIG. 18.
Samples were measured under different concentrations.
Examp/e 5: Poiymtw dye properties
[00205] Polymer dyes of the disclosure were found to possess
certain physical
and chemical characteristics of absorption, fluorescence, brightness,
molecular weight,
poiydispersity, dye to protein ratio when conjugated to an antibody etc. The
preferred
ranges of these parameters are shown in Table 1A.
[00206] Table 1A. Polymer Dye Characteristics
Abs/Em Max MW (Mn) PD (I) Brightness
F/P
2rnax395-415 nm 20K-70K Between 1.5 300K to 0.4 to 0.75
150K to 1900K 1 to 12
kern 420-430 nm to 2.5 2500K
2rnax340-370 nm 20K-70K Between 1.5 300K to 0.1 to 0.75
30K to 1900K Ito 12
kern 390-420 nm to 2.5 2500K
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[00207] The excitation and emission spectra of tandem
polymers was measured.
Excitation was carried out at the polymer maxima (405 nrn) and the emissions
observed
from the various acceptor dyes attached to the backbone.
Example 6: Experiments with unconjugated dye
[00208] A blood sample was stained with unconjugated polymer
dye SN v605
(without antibody), with and without EmpigEm BE , and analyzed in a flow
cytorrieter.
As shown in FIG, 2, lower right, presence of EMPIGEN BB showed effective
decrease
in the non-specific interactions of the fluorescent polymers conjugated to
binding
partners to white blood cells in blood. In FIG. 2, the fluorescent polymer dye
SN v605
without antibody was used to stain a blood sample and analyzed in a flow
cytorneter. It
is evident in FIG. 2, lower left, that the polymer dye without Ernpigen BB is
binding to
the monocytes/granulocytes non-specifically, 'Arnile not wishing to be bound
by any
specific theory, it is thought that the polymer is likely adsorbing on the
cell surface of
monocytes and granulocytes. When EMPIGEN BE is added, the surface of cells is
blocked by EMPIGEN BE molecules and non-specific binding of polymers to
monocytes and granulocytes is substantially reduced.
[00209] With the addition of EMPIGEN BB to the polymer, the
dot plots appear
like that of an unstained sample of FIG. 2, upper panel, which does not
contain any
polymer. The spread of the granulocyte population is comparable to unstained
tube
suggesting that when EMPIGEN BB is added to the polymer dye the non-specific
interaction of the polymer dye with monocytes and granulocytes is reduced
drastically.
Example 7: Experiments with conjugated dye SN 605-0O20
[00210] EMPIGEN BS was formulated with the conjugates
described herein
(e.g., 5N605-CD20, SN786-0D103, and SN428 conjugates), bovine serum albumin
(BSA; 2 mgirnif), sodium azide (0.1%), and piuronic F-68 (polyethylene oxide-
polypropylene oxide-polyethylene oxide nonionic triblock copolymer) to a dose
of 0.12%
per 10 pi of conjugate.
[00211] CD20 is a B-lineage cell marker expressed during pre-
B lymphocyte
development, persists in B-lymphocyte expression, and losses its expression
while
plasma cell differentiation. 0D20 is not expressed on other leukocyte
population
including monocytes, granulocytes and NK cells. FIG. 3 gives the performance
of SN
605-0020 conjugate without EMPIGEN BE and in the presence of EMPIGEN BB .
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[08212] The percentage of non-specifically bound
granulocytes was reduced (see
the "P2" gate in the dot plot) with the usage of EMPIGEN BE . Also, the
functional
aspect of the conjugate also did not change (see the "P1" gate in the dot
plot), since the
percentage of the positive population is similar in both the cases.
[00213] in order to confirm the effect of EMPIGEN BE , two
lots of SN 605-CD20
conjugates were tested in the presence and absence of EMPIGEN BE . The mean
fluorescence intensity (MR) was compared to the autofluoreseence (negative
population
median fluorescence intensity (MdFI)) of the monocytes from the unstained
sample. The
results shown in FIG. 4 demonstrate that in the presence of surfactant the non-
specific
interaction on monocytes were reduced to 75% and 67% for Lot-1 and Lot-2 5N605
0D20 conjugates, respectively. A similar effect (reduction of non-specific
binding), was
also observed in granulocytes too, but the percent of reduction is not as
pronounced
(13.7% and 17.7% for Lot-1 and Lot-2 respectively, FIG. 5).
Example 8: Experiments with conjugated dye SN 786-CD103
[00214] 0D103 conjugates are tested on cell line (MOLT16) as
they are not
usually expressed in normal whole blood. Since there is no expression of 0D103
in
normal blood, there should be no positive signal. But due to the non-specific
interaction,
SN786 CD103 conjugates tend to bind to whole blood as well. Addition of
EMPIGEN
BE to this formulation helps to contain this non-specific interaction. This
is illustrated
in FIG. 6,
[00215] Two lots of SN 786-CD103 conjugates were tested in
presence and
absence of EMPIGEN BE and the autofluorescence (negative population MdFI) of
the
monocyte and granulocyte population is compared against an unstained sample.
As
shown in FIG. 7, the signal from non-specific binding of conjugates to
monocytes
reduced 149.3% and 202.1% for Lot-1 and Lot-2 of SUPERNOVATM conjugates
respectively. The same effect (reduction of non-specific binding), was also
observed in
the granulocyte population as well, with a 150,8% and 253% reduction for Lot-1
and Lot-
2 respectively as shown in FIG, 8.
Example 9: Zwitterionic Surfactant effect on monocyte background reduction
with SN
428 conjugates
[00216] The efficiency of zwitterionic, surfactant EMPIGEN
BE on the reduction
of non-specific binding with monocytes was evaluated. Other populations were
also
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studied, namely lymphocytes and granulocytes, to evaluate the non-impact of
the
detergent on MR and percentage of cells.
[00217] The experimental conditions were generally as
foliows:
o CD19-SN 428 (lot D19-094, polymer lot RDS-042919 (82.7 kD), 1 dose (0.5
pg/test).
o 0D22-SN 428 (lot D19-109, polymer lot VVX-20190624 (86.4 kD), 1 dose (0.5
pg/test).
o CD25-SN 428 (lot D19-107, polymer lot RDS-062419 (72.8 kD), 1 dose (0.5
pg/test).
o CD19, CD22 and CD25-BV 421 from Becton Dickinson at 1X commercial dose.
o Three doses of EMPIGEN BB 0.06%, 0.12%, and 0.2% prepared in conjugate
final formulation.
o Added 10 pL sample with 100 pL whole blood.
o Tested on 2 donors lysing with VersaLyse (lysing solution, used to lyse
red blood
cells, Beckman Coulter, Inc.) + Fix, one wash.
o Navios flow cytometer acquisition on FL9.
[00218] The required x number of tubes were prepared,
wherein "x number of
tubes" depends on the performance tested. A calculated volume of conjugated
antibody
(at required dose) was added to each tube. Whole blood (100 pL) was added in
each
tube. The tubes were gently vortexed for 15 seconds and incubated for 15 to 20
Minutes
at room temperature at 18-25 C and protected from light. VersaLyse and 10Test3
Fixative mixture (2 rnL Versalyse Ref. A09777+ 50 pi 10Test3 fixative 10X Ref.
A07800)
were added to the tubes. The tubes were immediately vortexed for 1 second and
incubated for 10 minutes at room temperature (18-25'C), protected from light.
The tubes
were centrifuged for 5 minutes at 300 g at room temperature, the supernatant
removed
by aspiration, and the cell pellet resuspended using 3 mL of PBS 'IX. The tube
was
again centrifuged for 5 minutes at 300 g at room temperature (18-25'C), the
supernatant
removed by aspiration and the cell pellet resuspended using: 0,5 ml PBS 1X or
PBS 1X
Formaldehyde 0.1% (can be obtained by diluted 1 rni PBS IX + 12.5p110Test3
fixative
10X).
[00219] In cytorrietry, compensation is a mathematical
correction of a signal
overlap between the channels of the emission spectra of different
fiuorochrornes.
Therefore, this correction factor was used to eliminate the bleeding of
signals into other
unwanted channels. Manual compensation was performed to assess the conjugate
performance.
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[00220] The raw data, normalized data to the condition
without EM PIGEN TM, and
finally the % reduction of monocyte background staining that were obtained on
2 donors
with the CD19, CD22 and CD25 SUPERNOVA v428 conjugates are shown in Table
1B. The data show that in the presence of Empigen BB the non-specific
background
monocyte binding was substantially reduced by between 52 and 73 % compared to
the
condition without EMPIGENO where the monocyte background is maximal.
[00221] Table 1B. Analysis of background reduction of
Empigen on monocytes
Donor 1 Donor
2
MEI Normalized `)/0 reduction MEI
Normalized to % reduction
raw to condition of raw condition
w/o of
data w/o background data Empigen
background
Empigen on
on
monocytes
monocytes
CD19 w/o 3,380 100,0 0,0 2,290 100,0 0,0
Empigen
w/ 0.06% 1,600 47,3 52,7 1,250 54,6
45,4
Empigen
w/0.12% 1,090 32,2 67,8 0,950 41,5
58,5
Empigen
w/ 0.20% 0,890 26,3 73,7 0,770 33,6
66,4
Empigen
CD22 w/o 2,520 100,0 0,0 2,47 100,0 0,0
Empigen
w/ 0.06 /0 1,860 73,8 26,2 1,69 68,4
31,6
Empigen
w/ 0.12% 1,280 50,8 49,2 1,28 51,8
48,2
Empigen
w/ 0.20% 0,870 34,5 65,5 0,93 37,7
62,3
Empigen
CD25 w/o 2,500 100,0 0,0 2,020 100,0 0,0
Empigen
WI 0.06% 1,350 54,0 46,0 1,230 60,9
39,1
Empigen
w/ 0.12% 0,940 37,6 62,4 0,940 46,5
53,5
Empigen
w/ 0.20% 0,730 29,2 70,8 0,700 34,7
65,3
Empigen
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MFI = Mean Fluorescence Intensity
w/o = without
w/ = with
[00222] Data showing the effect of Empigen BB on
granulocyte background
reduction is shown in Table 2. Granulocyte background reduction was found to
range
from 4 to 21% compared to the condition without EMPIGENO where the granulocyte
background is maximal.
[00223] Table 2. Analysis of background reduction of Empigen
on granulocytes
Donor 1 Donor 2
MFI Normalized % reduction MFI
Normalized % reduction
raw to condition of raw to
condition of
data w/o background data w/o
background
Empigen on Empigen
on
monocytes
monocytes
CD19 w/o 1,070 100,0 0,0 0,990 100,0 0,0
Empigen
w/ 0.06% 0,890 83,2 16,8 0,860 86,9
13,1
Empigen
w/ 0.12% 0,900 84,1 15,9 0,870 87,9
12,1
Empigen
w/ 0.20% 0,920 86,0 14,0 0,820 82,8
17,2
Empigen
CD22 w/o 1,070 100,0 0,0 0,970 100,0 0,0
Empigen
w/ 0.06% 1,00 93,5 6,5 0,920 94,8
5,2
Empigen
w/ 0.12% 0,930 86,9 13,1 0,880 90,7
9,3
Empigen
w/ 0.20% 0,900 84,1 15,9 0,930 95,9
4,1
Empigen
CD25 w/o 0,930 100,0 0,0 0,880 100,0 0,0
Empigen
w/ 0.06% 0,890 95,7 4,3 0,840 95,5
4,5
Empigen
w/ 0.12% 0,750 80,6 19,4 0,810 92,0
8,0
Empigen
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w/ 0.20% 0,730 78,5 21,5 0,730 83,0
17,0
Empigen
[00224] Table 3 shows the effect of EMPIGEN on the positive
lymphocyte
population: the presence of EMPIGENO did not induce a significant variation of
the
positive signal on lymphocytes when compared to the condition without
EMPIGENO.
[00225] Table 3. Analysis of Empigen effect on positive
lymphocytes
Donor 1 Donor 2
MFI Normalized ')/0 variation MFI
Normalized % reduction
raw to condition of positive
raw to condition of
data w/o signal on data w/o
background
Empigen lymphocytes Empigen
on
monocytes
CD19 w/o 83,25 100,0 0,0 85,06 100,0 0,0
Empigen
w/ 0.06`)/0 81,91 98,4 -1,6 88,21 103,7
3,7
Empigen
w/0.12% 83,28 100,0 0,0 91,85 108,0 8,0
Empigen
w/ 0.20% 82,36 98,9 -1,1 96,72 113,7
13,7
Empigen
CD22 w/o 64,98 100,0 0,0 81,95 100,0 0,0
Empigen
w/ 0.06`)/0 66,47 102,3 2,3 91,72 111,9
11,9
Empigen
w/0.12% 72,93 112,2 12,2 94,85 115,7 15,7
Empigen
w/ 0.20`)/0 71,56 110,1 10,1 98,59 120,3
20,3
Empigen
CD25 w/o 6,79 100,0 0,0 5,77 100,0 0,0
Empigen
w/ 0.06% 6,86 101,0 1,0 5,96 103,3
3,3
Empigen
w/0.12% 6,69 98,5 -1,5 6,02 104,3 4,3
Empigen
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w/0.20% 6,65 97,9 -2,1 6,29 109,0 9,0
Empigen
[00226] The data in Tables 1-3 are summarized in FIGS. 11-
15.
[00227] Table 4 describes additional experiments showing
percent reduction of
background on monocytes and granulocytes.
[00228] Table 4. Percent reduction of background on
rnonocytes and
granulocytes
% Reduction in
MFI Normalization
PBS
Monocytes
605-CD20 w/o Empigen_Lot #1 790
605-CD20 with Empigen_Lot #1 1720.3 930.3
605-CD20 w/o Empigen_Lot #2 978.2 188.2 80%
605-CD20 with Empigen_Lot #2 1524.3 734.3
PBS 911.3 121.3 83%
786-CD103 w/o Empigen_Lot #1 169.9
786-CD103 with Empigen_Lot #1 706.4 536.5
786-CD103 w/o Empigen_Lot #2 283.7 113.8 79%
786-CD103 with Empigen_Lot #2 790.3 620.4
261.6 91.7 85%
% Reduction in Granulocytes
Test
% Reduction in
MFI Normalization
PBS
Granulocytes
605-CD20 w/o Empigen_Lot #1 1241.8
605-CD20 with Empigen_Lot #1 1545.3 303.5
605-CD20 w/o Empigen_Lot #2 1358.7 116.9 61%
605-CD20 with Empigen_Lot #2 1491.7 249.9
PBS 1267.3 25.5 90%
786-CD103 w/o Empigen_Lot #1 180.9
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786-CD103 with Empigen_Lot #1 561.7 380.8
786-CD103 w/o Empigen_Lot #2 223.9 43 89%
786-CD103 with Empigen_Lot #2 719.9 539
Example 10: Effect of EMPIGEN BB on sample cell integrity
[00229] EMPIGEN BB is a surfactant and, as such, could
cause a
permeabilization of cell membranes, leading to cell death. From the studies
described
herein, it was concluded the concentration at which EMPIGEN BB that is used
with the
polymer dye conjugate does not induce whole blood cell pemieabilization or
death and
does not affect performance of the conjugate
[00230] The rnicellar concentration was studied in samples
and during the
staining to be sure not to exceed critical micellar concentration (CMC). See
Table 5,
which shows the evaluation of the CMC in the conjugate formulation and during
staining.
CMC was studied in the conjugate formulation and during the staining in 100
pi., whole
blood. Experiments were conducted to evaluate the impact of addition of
EMPIGEN BBC)
on whole blood cell integrity and also on peripheral blood mononuclear cells
(PMBCs).
[00231] Table 5. Ernpigen BB Concentration in Conjugate
Formulation and
during Staining
Formulation in the vial 0,2 %
2 g/L
279 g/nnol
0,007168 M
7,2 mM > CMC within the conjugate
formulation
During the staining 100 IILWB
p.L conjugate
11 Dilution factor
0,65 nnM <CMCduringthestaining
[00232] The percentage of dead cells in whole blood sample
with the 7-AAD was
evaluated. 7-AAD is a DNA marker, the staining is positive when the cellular
membrane
is permeabilized. CD19-SNv428 D19-094 without EMPIGEN BB (negative control)
and
with 0.06%, 0.12%, and 0.2% EMPIGEN BB , was tested on four donors' whole
blood,
with the 7-ADD, to evaluate the percentage of dead cells in each condition.
The goal of
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the experiment was to verify if the percentage of dead cells is not increased
by the
EMPIGEN BE concentration.
[00233] Two whole blood samples that had been preserved for
greater than 24
hours were added as positive control of 7-AAD staining.
[00234] Protocol for 7-AAD staining:
o 100 pl whole blood + 10 pl CD19-SNv428 +/-Empigen, incubation 20 minutes.
o Lyse with Versalyse, one wash,
o Resuspension in 500 pl PBS 1X
o Add 20 pl of 7-AAD (Ref B88526), incubation 15-20 minutes
o Navios flow cytometer acquisition: FL4 for 7-AAD and FL9 for CD19-SNv428
[00235] The data are presented in FIG. 16.
[00236] In conclusion, EMPIGEN BE was proven to be
effective in reducing
non-specific interaction of the polymer dye conjugates described herein (SN
605-0D20,
SN786-00103, 5N428-0D25, 5N428-0D19 and 5N428-0D22). EMPIGEN BE
efficiently reduces non-specc background binding with the rnonocyte and
granulocyte
population when tested on five specificities of conjugates. This efficiency of
EMPIGEN
BB is not donor dependent. Wien the conjugates were compared with BV786-0D103
and BV 421, there was clear differentiation of reduction of rnonocyte non-
specific pullout.
In addition to its performance, the presence of EMPIGEN BB with the polymer
dye
conjugate did not induce whole blood cell membrane perrneabilization and
didn't induce
whole blood cell death at a concentration of up to at least 0.2% in the
compositions.
Example 11: Effect of Nonionic surfactants
[00237] Nonionic surfactants Tween-20, tergitol, NP-40 and
Pluronic F-68 (PF-
68) were additional detergents/surfactants that were tested to remove non-
specific
binding of the conjugates described herein on rnonocytes. FIG. 9 shows the
inefficiency
of Tween-20 and PF-68 in avoiding non-specific binding of conjugates on
rnonocytes.
Example 12: Effect of protein blockers
[01323a] The issue of non-specific interaction on nionocytes
was also observed
with conventional tandem dyes (e.g., P05, P05.5, P07, AA700 available from
Beckman
Coulter, Inc.). To overcome this issue, it was thought that BSA-ox (oxidized
BSA) and
BSA-Cy5-ox (oxidized 0y5-.BSA), which are known protein biockers, might
prevent non--
specific binding. But it was found that BSA, BSA-ox, and BSA-Cy5-ox were all
inefficient
in controlling the non-specific interactions of polymer dye conjugates with
the
dranulocytes/monocytes. See FIG. 10.
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Example 13: Effect of Anionic surfactant on Non-specific binding
[00239] Anionic surfactant N-lauryi sarcosine (NLS) was
found to be effective at
reducing non--specific staining on monocytes and granulocytes at 0.16% and
0.08%
(w/v).
CH3 0
HaC N
0- Na
9
0
[00240] NLS is an anionic surfactant having a CMC of 14.57
mM (30 *C). NLS
sodium was evaluated to determine effective concentration for preventing non-
specific
binding of polymer dye conjugates (SN v605-CD20) on monocytes and
granulocytes,
[00241] In this example, different concentrations (0.16%,
0.08%, 0.04% and
0.02% w/v) of NLS was formulated with SN v605 CD20 conjugate in the presence
of
BSA and sodium azide and stained peripheral blood samples. Flow cytometry was
performed following sample staining.
[00242] FIG. 17A-E show the dot-plots of blood samples in
the absence and
presence of conjugates with Empigen/without Empigen /NLS at different
concentrations.
A dot plot of a peripheral blood sample without single color conjugate is
shown in FIG
17A, evident as there is no population in the CD20+ gate.
[00243] A positive control dot plot of a peripheral blood
sample in the presence
of CD2O-SN v605 single-color conjugate in a buffer composition containing BSA,
sodium
azide, and zwitterionic surfactant Empigen BB as additives is shown in FIG.
17B. When
compared to negative control dot plot (FIG. 17C), the % population in the gate
"Mons
Non-specific binding" (0.64%) and "Grans Non-specific binding" (0.68%) are
each
considerably reduced, indicating the effectiveness of Empigen BB in
preventing non-
specific binding to monocytes and granulocytes.
[00244] A negative control dot plot of a peripheral blood
sample in the presence
of CD2O-SN v605 single-color conjugate in a buffer composition containing only
BSA
and sodium azide as additives is shown in FIG. 17C. The granulocyte non-
specific
staining was 1.20%, and the monocyte non-specific staining was 1.63% without
surfactant.
[00245] A test dot plot of a peripheral blood sample in the
presence of CD2O-SN
v605 single-color conjugate in a buffer composition containing BSA, sodium
azide, and
NLS (0.16% w/v) as additives is shown in FIG. 17D. Non-specific staining of
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granulocytes was substantially reduced to 0.60%, and monocyte non-specific
staining
was substantially reduced to 0.39%, compared to negative control (FIG. 170).
[00246] A test dot plot of a peripheral blood sample in the
presence of CD2O-SN
v605 single-color conjugate in a buffer composition containing BSA, sodium
azide, and
NLS (0.08% w/v) as additives is shown in FIG. 17E. Non-specific staining of
granulocytes was reduced to 0.76%, and monocyte non-specific staining was
reduced
to 0.93%, compared to negative control (FIG. 170).
[00247] The dot plot of FIG. 17B (Empigen BB ) when compared
to FIGs. 17D
and 17E (NLS) shows the % population in the gate "Mons Non-specific binding"
and
"Grans Non-specific binding" are very similar, indicating the effectiveness of
NLS
equivalent to Empigen in preventing non-specific binding on cells at 0.16% and
0.08%
[00248] The effective concentration of NLS was found to be
0.16% to 0.08% w/v
to reduce or eliminate non-specific staining on monocytes and granulocytes in
0D20-
SN v605, therefore this concentration range of NLS anionic surfactant was
demonstrated to be effective to reduce non-specific binding in single color
fluorescent
polymer dye conjugate compositions.
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