US-12625140-B2 - Multiplexed imaging with nanobody probes

Abstract

Methods include (a) contacting a biological sample with a composition featuring a plurality of different types of probes, where each type of probe of the plurality of different types of probes includes a detection moiety that selectively binds to a different type of protein target in the sample, a nanobody bound to the detection moiety, and an oligonucleotide linked to the nanobody and featuring an oligonucleotide sequence, where the oligonucleotide sequence of each type of probe is different from the oligonucleotide sequences of each of the other types of probes of the plurality of probes; and (b) contacting the sample with a set of one or more different types of optical labels, where each different type of optical label of the set of optical labels includes an oligonucleotide that selectively hybridizes to only one type of probe.

Inventors

• Yi Zheng
• Julia Kennedy-Darling
• Peter J. Miller

Assignees

• AKOYA BIOSCIENCES, INC.

Dates

Publication Date

20260512

Application Date

20211118

Claims (20)

1. 1 . A method, comprising: (a) contacting a biological sample with a composition comprising a plurality of different probes, wherein each different probe of the plurality of different probes comprises: a detection moiety that selectively binds to a different protein in the sample; a nanobody bound to the detection moiety; and an oligonucleotide linked to the nanobody and comprising an oligonucleotide sequence, wherein the oligonucleotide sequence of each different probe of the plurality of different probes is different from the oligonucleotide sequences of each of the other different probes of the plurality of different probes; (b) contacting the biological sample with a set of one or more different optical labels, wherein each different optical label of the set of one or more different optical labels comprises an oligonucleotide that selectively hybridizes to only one probe among the plurality of different probes, and an optical moiety; and (c) obtaining at least one image of the biological sample, wherein the at least one image comprises optical signals generated by at least one of the optical moieties of the set of one or more different optical labels.
2. 2 . The method of claim 1 , further comprising identifying one or more protein targets in the sample based on the optical signals in the at least one image of the sample.
3. 3 . The method of claim 2 , further comprising determining an amount of at least one of the one or more protein targets in the sample based on the optical signals in the at least one image of the sample.
4. 4 . The method of claim 1 , wherein the set of one or more different optical labels comprises multiple different optical labels.
5. 5 . The method of claim 4 , wherein the set of one or more different optical labels comprises at least three different optical labels.
6. 6 . The method of claim 1 , wherein the detection moiety of at least one probe of the plurality of different probes comprises an antibody or antibody fragment.
7. 7 . The method of claim 1 , wherein the composition comprises at least 10 different probes.
8. 8 . The method of claim 7 , wherein the composition comprises at least 30 different probes.
9. 9 . The method of claim 1 , wherein the at least one of the optical moieties of the set of one or more different optical labels comprises a fluorescent dye.
10. 10 . The method of claim 1 , wherein for one or more of the different probes among the plurality of different types, the nanobody is bound to the detection moiety with a dissociation constant K d that is 1.0×10 −9 mol/L or less.
11. 11 . The method of claim 1 , wherein the composition comprises multiple different probes comprising antibody binding moieties of a common species and isotype.
12. 12 . The method of claim 1 , wherein the composition further comprises at least one fragment of a binding target of at least one of the nanobodies among the plurality of different probes.
13. 13 . The method of claim 12 , wherein the composition further comprises multiple different fragments of binding targets of multiple nanobodies among the plurality of different probes.
14. 14 . The method of claim 1 , further comprising, prior to contacting the sample with the composition, forming each of the different probes of the plurality of different probes, wherein each different probe of the plurality of different probes is formed by: contacting the detection moiety for the different probe of the plurality of different probes with a labeling moiety comprising the nanobody for the different probe of the plurality of different probes linked to the oligonucleotide for the different probe of the plurality of different probes; and incubating the binding moiety and the labeling moiety to bind the nanobody to the binding moiety.
15. 15 . The method of claim 14 , wherein the nanobody undergoes site-specific binding to the binding moiety.
16. 16 . The method of claim 1 , further comprising, following step (a), contacting the sample with at least one fixative.
17. 17 . The method of claim 1 , further comprising, following step (a), washing the sample to remove unbound probes from the sample.
18. 18 . The method of claim 1 , wherein at least one optical label of the set of one or more different optical labels comprises the oligonucleotide of the at least one optical label of the set of one or more different optical labels linked to the optical moiety of the at least one optical label of the set of one or more different optical labels through a streptavidin-biotin linkage.
19. 19 . The method of claim 1 , wherein the at least one image comprises at least one fluorescence image of the sample.
20. 20 . The method of claim 1 , further comprising repeating steps (b)-(c) with at least one additional set of one or more different optical labels, to obtain at least one additional image of the sample comprising optical signals generated by at least one of the optical moieties of the at least one additional set of one or more different optical labels.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63/115,593, filed on Nov. 18, 2020, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD This disclosure relates to multiplexed imaging of biological samples, probes for use in multiplexed imaging, and imaging kits. BACKGROUND Immuno-labeling can be used to target molecules in samples such as cells, tissue, and other biological specimens. High sensitivity and specificity enables reliable detection of low-abundance targets. SUMMARY The disclosure relates to multiplexed analysis of tissue samples. Methods, compositions, and reagent kits for labeling target molecules with oligonucleotide-barcoded detection molecules are described. High-performance barcoded antibodies can be generated using oligonucleotide-conjugated nanobodies that selectively bind to antibodies with high affinity. A biological sample is incubated with a cocktail of detection molecules labeled with oligonucleotides containing barcode sequences. Imaging is performed by introducing a readout moiety conjugated to an oligonucleotide sequence that contains a countersense sequence to one of the barcode sequences. In an aspect, the disclosure features methods for detecting multiple target compounds in a sample that include contacting a sample with a cocktail containing a plurality of detection moieties, each of which includes a detection molecule and a nanobody conjugated to an oligonucleotide label with a barcode sequence, and subjecting it to an incubation period, subjecting the sample to one or more wash steps, introducing a readout moiety featuring a readout molecule coupled to an oligonucleotide that includes a countersense sequence to a barcode sequence associated with one of the detection moieties, hybridizing the readout moiety with the detection moiety based on the barcode sequence, and imaging the sample. Embodiments of the methods can include any one or more of the following features. The nanobody species can bind to targets in the detection molecules with high affinity. The detection molecules can be antibodies. The cocktail can contain multiple antibody species of the same species and isotype. The cocktail can also contain fragments of the nanobody target for one or more nanobody species. The nanobodies can be conjugated using site-specific conjugation. The sample can be subjected to fixation after the wash steps. The readout moiety can include barcoded fluorescent dyes. The readout moiety can include an enzyme. The readout moiety can include biotin. The sample imaging can include fluorescent imaging. The methods can include dehybridizing the readout moiety from the detection moiety and repeating at least some of the steps for additional barcode sequences. Multiple species of readout moiety can be introduced at the same time. Embodiments of the methods can also include any of the other features described herein, including any combinations of features individually described in connection with different embodiments, except as expressly stated otherwise. In another aspect, the disclosure features reagent kits that include a nanobody moiety conjugated to an oligonucleotide label with a barcode sequence, where the nanobody binds with high affinity to a target in the antibody. Embodiments of the reagent kits can include any one or more of the following features. The target can be one of rabbit IgG, mouse IgG1, mouse IgG2a, mouse IgG2b, mouse IgG2c, and mouse IgG3. The kits can include fragments of the target. The kits can include barcoded readout molecules. Embodiments of the reagent kits can also include any of the other features described herein, including any combinations of features individually described in connection with different embodiments, except as expressly stated otherwise. In another aspect, the disclosure features methods that include: (a) contacting a biological sample with a composition featuring a plurality of different types of probes, where each type of probe of the plurality of different types of probes includes a detection moiety that selectively binds to a different type of protein target in the sample, a nanobody bound to the detection moiety, and an oligonucleotide linked to the nanobody and featuring an oligonucleotide sequence, where the oligonucleotide sequence of each type of probe of the plurality of probes is different from the oligonucleotide sequences of each of the other types of probes of the plurality of probes; (b) contacting the sample with a set of one or more different types of optical labels, where each different type of optical label of the set of optical labels comprises an oligonucleotide that selectively hybridizes to only one type of probe among the plurality of different types of probes, and an optical moiety; and (c) obtaining at least one image of the sample, where the at least one image includes optical signals generated by at least one of the optical moiet