# US 11,079,372 B2 · Polymers and conjugates comprising the same

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- Number: US 11,079,372 B2
- Document ID: US11079372B2
- Status: Granted
- Kind: B2
- Application number: 15/476,904
- Filed: 2017-03-31
- Published: 2017-07-20
- Granted: 2021-08-03
- Earliest claimed: 2014-10-02
- Assignees: Ventana Medical Systems, Inc.
- Applicants: Ventana Medical Systems, Inc.
- Inventors: Lei Tang; Wenjun Zhang
- Web page: https://patentgrep.com/patent/US11079372B2

## CPC

- G01N 33/533: with fluorescent label
- G01N 33/532: Production of labelled immunochemicals
- G01N 2458/10: Oligonucleotides as tagging agents for labelling antibodies
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Polymers and conjugates comprising polymers are disclosed herein. In some embodiments, the conjugates disclosed are suitable for use as detection probes in immunohistochemical assays, including multiplex immunohistochemical assays.

## Claims

### Claim 1

A polymer of Formula (V),
wherein
‘Olig’ is a single-stranded oligonucleotide sequence having between 1 and about 50 nucleotides, wherein the oligonucleotide sequence has a Tm of less than 70° C.;
‘Spacer’ is a saturated straight chain aliphatic group having between 4 and 32 carbon atoms, wherein the aliphatic group is unbranched and unsubstituted, and optionally includes one or more oxygen atoms;
‘Linker’ is a saturated straight chain aliphatic group having between 4 and 18 carbon atoms, wherein the aliphatic group is unbranched and unsubstituted, and optionally includes one or more oxygen atoms;
‘Label’ is selected from the group consisting of haptens, chromogens, and enzymes;
R is H, a hydroxyl group, an amino group, a carbonyl group, a phosphate group, a phosphodiester group, or a cation;
T is a group having a terminal reactive moiety;
x is 1 or 2;
y is 1, or 2;
z is an integer ranging from 2 to 18;
m is 1, or 2;
n is 1 or 2;
a is an integer ranging from 1 to 8;
b is 1 or 2; and
wherein any of the ‘Olig’, ‘Spacer,’ ‘Linker’ or ‘Label’ may be bonded directly to each other or through an optional group; and
wherein at least one of the ‘Spacer’ and the ‘Linker’ comprises a moiety having Formula (IX):
where d and e are each independently an integer ranging from 1 to 32,
wherein the ‘Label’ is selected from the group consisting of a di-nitrophenyl, biotin, digoxigenin, fluorescein, a fluorescein derivative, a rhodamine, an oxazole, a pyrazole, a thiazole, a nitroaryl, a benzofuran, a triterpene, a urea, a thiourea, a rotenoid, a coumarin, a cyclolignan, 5-nitro-3-pyrazole carbamide, 2-(3,4-dimethoxyphenyl)quinoline-4-carboxylic acid), 3-hydroxy-2-quinoxalinecarbamide, 2,1,3-benzoxadiazole-5-carbamide, and 2-acetamido-4-methyl-5-thiazolesuifonamide.

### Claim 2 (depends on 1)

The polymer of claim 1, wherein the ‘Olig’ comprises between 2 and about 50 nucleotides.

### Claim 3 (depends on 1)

The polymer of claim 1, wherein the ‘Olig’ comprises between 2 and about 24 nucleotides.

### Claim 4 (depends on 1)

The polymer of claim 1, wherein the ‘Olig’ comprises between 4 and about 24 nucleotides; x is 1; y is 1; and a is 1 or 2.

### Claim 5 (depends on 1)

The polymer of claim 1, wherein d ranges from 1 to 4; and e ranges from 2 to 8.

### Claim 6 (depends on 1)

The polymer of claim 1, wherein the ‘Linker’ includes a group derived from a poly(alkylene)glycol.

### Claim 7 (depends on 1)

The polymer of claim 1, wherein the ‘Label’ is selected from the group consisting of di-nitrophenyl, biotin, digoxigenin, fluorescein or a derivative thereof, or rhodamine.

### Claim 8 (depends on 1)

The polymer of claim 1, wherein the ‘Label’ is selected from the group consisting of oxazoles, pyrazoles, thiazoles, nitroaryls, benzofurans, triterpenes, ureas, thioureas, rotenoids, coumarins, or cyclolignans.

### Claim 9 (depends on 1)

The polymer of claim 1, wherein the ‘Label’ is selected from the group consisting of 5-nitro-3-pyrazole carbamide, 2-(3,4-dimethoxyphenyl)quinoline-4-carboxylic acid), 3-hydroxy-2-quinoxalinecarbamide, 2,1,3-benzoxadiazole-5-carbamide, and 2-acetamido-4-methyl-5-thiazolesulfonamide.

### Claim 10 (depends on 1)

The polymer of claim 1, wherein x is 1; y is 1 or 2; a is 1 or 2; and z ranges from between 3 to 18.

### Claim 11 (depends on 10)

The polymer of claim 10, wherein x is 1; y is 1; a is 1 or 2; and z ranges from between 3 to 9.

### Claim 12 (depends on 11)

The polymer of claim 11, wherein a is 2; and m, n, and b are 1.

### Claim 13 (depends on 12)

The polymer of claim 12, wherein the ‘Label’ is fluorescein or a fluorescein derivative.

### Claim 14 (depends on 13)

The polymer of claim 13, wherein ‘Olig’ comprises between 4 and 18 nucleotides; and wherein ‘Linker’ comprises between 4 and 12 carbon atoms.

### Claim 15 (depends on 11)

The polymer of claim 11, wherein a is 1; and m, n, and b are 1.

### Claim 16 (depends on 15)

The polymer of claim 15, wherein the ‘Label’ is di-nitrophenyl.

### Claim 17 (depends on 16)

The polymer of claim 16, wherein ‘Olig’ comprises between 4 and 18 nucleotides; and wherein ‘Linker’ comprises between 4 and 12 carbon atoms.

### Claim 18 (depends on 1)

The polymer of claim 1, wherein a ratio of a:b is 2:1 or 3:1; and wherein a number of ‘Label’ groups present per polymer ranges from 3 to 9.

### Claim 19 (depends on 1)

The polymer of claim 1, where the terminal reactive moiety of T is an amino group, a carboxyl group, or a sulfhydryl group.

### Claim 20 (depends on 1)

The polymer of claim 1, wherein the polymer has the structure of Formula (VIa):

### Claim 21 (depends on 1)

The polymer of claim 1, wherein the polymer has the structure of Formula (VIb):

### Claim 22 (depends on 1)

The polymer of claim 1, wherein the polymer has the structure of Formula (VIc):

### Claim 23

A polymer of Formula (V),
wherein
‘Olig’ is an oligonucleotide sequence having between 1 and about 50 nucleotides, wherein the oligonucleotide sequence has a Tm of less than 70° C.;
‘Spacer’ is a saturated straight chain aliphatic group having between 4 and 32 carbon atoms, wherein the aliphatic group is unbranched and unsubstituted, and optionally includes one or more oxygen atoms;
‘Linker’ is a saturated straight chain aliphatic group having between 4 and 18 carbon atoms, wherein the aliphatic group is unbranched and unsubstituted, and optionally includes one or more oxygen atoms;
‘Label’ is selected from the group consisting of haptens, fluorophores, chromogens, enzymes and quantum dots;
R is H, a hydroxyl group, an amino group, a carbonyl group, a phosphate group, a phosphodiester group, or a cation
T is a group having a terminal reactive moiety;
x is 1 or 2;
y is 1, or 2;
z is an integer ranging from 2 to 18;
m is 1, or 2;
n is 1 or 2;
a is an integer ranging from 1 to 8;
bis 1 or 2; and
wherein any of the ‘Olig’, ‘Spacer,’ ‘Linker’ or ‘Label’ may be bonded directly to each other or through an optional group, and wherein ‘Spacer’ and ‘Linker’ are different; and
wherein at least one of the ‘Spacer’ or the ‘Linker’ comprises a moiety having Formula (IX):
where each d is independently an integer ranging from 1 to 32, and where e is an integer ranging from 2 to 32; and where the haptens are selected from the group consisting of pyrazoles, nitrophenyl compounds, benzofurazans, triterpenes, ureas, thioureas, rotenone, rotenone derivatives, oxazole s, thiazoles, coumarins, coumarin derivatives, nitroaryls, triterpenes, and cyclolignans.

### Claim 24 (depends on 23)

The polymer of claim 23, wherein both the ‘Spacer’ and the ‘Linker’ comprise a moiety having Formula (IX):
Wherein each d is independently an integer ranging from 1 to 32, and wherein e is an integer ranging from 2 to 32.

### Claim 25 (depends on 1)

The polymer of claim 1, wherein both the ‘Spacer’ and the ‘Linker’ comprise a moiety having Formula (IX):
where d and e are each independently an integer ranging from 1 to 32.

### Claim 26

A polymer of Formula (V),
wherein
‘Olig’ is a single-stranded oligonucleotide sequence having between 1 and about 50 nucleotides, wherein the oligonucleotide sequence has a Tm of less than 70° C.;
‘Spacer’ is a saturated straight chain aliphatic group having between 4 and 32 carbon atoms, wherein the aliphatic group is unbranched and unsubstituted, and optionally includes one or more oxygen atoms;
‘Linker’ is a saturated straight chain aliphatic group having between 4 and 18 carbon atoms, wherein the aliphatic group is unbranched and unsubstituted, and optionally includes one or more oxygen atoms;
‘Label’ is a fluorophore or a hapten;
R is H, a hydroxyl group, an amino group, a carbonyl group, a phosphate group, a phosphodiester group, or a cation;
T is a group having a terminal reactive moiety;
x is 1 or 2;
y is 1, or 2;
z is an integer ranging from 3 to 6;
m is 1, or 2;
n is 1 or 2;
a is an integer ranging from 1 to 8;
bis 1 or 2; and
wherein any of the ‘Olig’, ‘Spacer,’ ‘Linker’ or ‘Label’ may be bonded directly to each other or through an optional group; and
wherein at least one of the ‘Spacer’ and the ‘Linker’ comprises a moiety having Formula (IX):
where d and e are each independently an integer ranging from 1 to 32.

### Claim 27 (depends on 26)

The polymer of claim 26, wherein both the ‘Spacer’ and the ‘Linker’ comprise a moiety having Formula (IX):
where d and e are each independently an integer ranging from 1 to 32.