# US 11,906,527 B2 · Photocleavable mass-tags for multiplexed mass spectrometric imaging of tissues using biomolecular probes

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- Number: US 11,906,527 B2
- Document ID: US11906527B2
- Status: Granted
- Kind: B2
- Application number: 17/399,390
- Filed: 2021-08-11
- Published: 2022-05-05
- Granted: 2024-02-20
- Earliest claimed: 2020-10-29
- Assignees: AmberGen, Inc.
- Applicants: AmberGen, Inc.
- Inventors: Mark J. Lim; Gargey Yagnik; Kenneth J. Rothschild
- Web page: https://patentgrep.com/patent/US11906527B2

## CPC

- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- G01N 33/6851: {Methods of protein analysis involving laser desorption ionisation mass spectrometry}
- C07C 271/20: to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
- C12Q 1/6841: In situ hybridisation
- G01N 33/5082: {Supracellular entities, e.g. tissue, organisms}
- G01N 33/533: with fluorescent label
- G01N 33/575: for cancer
- G01N 33/57515: of the breast
- G01N 33/6842: {Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins}
- G01N 2333/4724: Lectins
- G01N 2400/00: Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N 2458/15: Non-radioactive isotope labels, e.g. for detection by mass spectrometry
- G01N 2560/00: Chemical aspects of mass spectrometric analysis of biological material
- G01N 2570/00: Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
- Y02P 20/55: Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
- 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)
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

The field of this invention relates to immunohistochemistry (IHC) and in situ hybridization (ISH) for the targeted detection and mapping of biomolecules (e.g., proteins and miRNAs) in tissues or cells for example, for research use and for clinical use such by pathologists (e.g., biomarker analyses of a resected tumor or tumor biopsy). In particular, the use of mass spectrometric imaging (MSI) as a mode to detect and map the biomolecules in tissues or cells for example. More specifically, the field of this invention relates to photocleavable mass-tag reagents which are attached to probes such as antibodies and nucleic acids and used to achieve multiplex immunohistochemistry and in situ hybridization, with MSI as the mode of detection/readout. Probe types other than antibodies and nucleic acids are also covered in the field of invention, including but not limited to carbohydrate-binding proteins (e.g., lectins), receptors and ligands. Finally, the field of the invention also encompasses multi-omic MSI procedures, where MSI of photocleavable mass-tag probes is combined with other modes of MSI, such as direct label-free MSI of endogenous biomolecules from the biospecimen (e.g., tissue), whereby said biomolecules can be intact or digested (e.g., chemically digested or by enzyme).

## Claims

### Claim 1

A multiplex method for co-detecting a plurality of different types of biomarkers in a tissue sample on a single slide, said method comprising:
a) providing a tissue sample on a single slide;
b) contacting said tissue sample with a plurality of different antibodies to effect binding of the antibodies to the tissue sample, each of said antibodies reactive with a different biomarker, and each of said antibodies conjugated to a unique photocleavable mass-tag;
c) illuminating said photocleavable mass-tags with light so as to photocleave at least a portion of said mass-tags prior to step d); and
d) detecting, using mass spectrometric imaging, said mass-tags, or fragments thereof, as molecular ions;
where said antibodies conjugated to a mass-tags have the following general structure

### Claim 2 (depends on 1)

The method of claim 1, further comprising performing, after step a) but before step b), direct mass spectrometric imaging on said tissue sample.

### Claim 3 (depends on 1)

The method of claim 1, wherein said plurality of different antibodies are in a mixture and said tissue sample in step b) is contacted with said mixture.

### Claim 4 (depends on 1)

The method of claim 1, wherein said mass-tags are non-rare-earth-metal mass-tags.

### Claim 5 (depends on 1)

The method of claim 1, wherein said mass-tags comprise a plurality of amino acids.

### Claim 6 (depends on 1)

The method of claim 1, wherein said tissue sample is fresh frozen and thin-sectioned prior to being mounted on said single slide.

### Claim 7 (depends on 1)

The method of claim 1, wherein said slide comprises gold.

### Claim 8 (depends on 7)

The method of claim 7, wherein said slide is a glass slide with a gold layer.

### Claim 9 (depends on 1)

The method of claim 1, wherein said tissue sample was formalin-fixed and paraffin embedded and thin-sectioned prior to being mounted on said single slide.

### Claim 10 (depends on 9)

The method of claim 9, wherein the tissue sample is subjected to a treatment prior to the steps of contacting the sample with antibody, said treatment comprising deparaffinization.

### Claim 11 (depends on 10)

The method of claim 10, wherein said deparaffinization is performed with xylene.

### Claim 12 (depends on 10)

The method of claim 10, wherein the tissue sample is further subjected to a treatment, said treatment comprising rehydration.

### Claim 13 (depends on 12)

The method of claim 12, wherein said rehydration is performed with a series of ethanol/water mixtures and aqueous saline buffers.

### Claim 14 (depends on 12)

The method of claim 12, wherein the tissue sample is further subjected to a treatment, said treatment comprising antigen retrieval.

### Claim 15 (depends on 14)

The method of claim 14, wherein said antigen retrieval is performed by heating in citrate buffer, pH 6.

### Claim 16 (depends on 14)

The method of claim 14, wherein said antigen retrieval is performed with formic acid.

### Claim 17 (depends on 1)

The method of claim 1, wherein a matrix compound is applied to said mass-tags before step d).

### Claim 18 (depends on 17)

The method of claim 17, wherein said matrix compound is selected from the group consisting of alpha-cyano-4-hydroxycinnamic acid (CHCA), 2,5-Dihydroxybenzoic acid (DHB) and 3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid).

### Claim 19 (depends on 17)

The method of claim 17, wherein said matrix compound is applied by sublimation.

### Claim 20 (depends on 17)

The method of claim 17, wherein the tissue sample is subjected to a treatment after the steps of contacting the sample with a matrix compound, said treatment comprising matrix recrystallization.

### Claim 21 (depends on 1)

The method of claim 1, wherein said tissue is from a tumor.

### Claim 22 (depends on 21)

The method of claim 21, wherein said tumor is a breast tumor.

### Claim 23 (depends on 1)

The method of claim 1, wherein at least one of said plurality of different antibodies comprises a fluorescent moiety in addition to said mass-tag.

### Claim 24 (depends on 1)

The method of claim 1, wherein the number of said different antibodies is 8 or more.

### Claim 25 (depends on 24)

The method of claim 24, wherein a subset of said different antibodies reacts with i) the estrogen receptor (ER), ii) the progesterone receptor (PR), iii) human epidermal growth factor receptor 2 (HER2), and iv) Ki67.

### Claim 26 (depends on 24)

The method of claim 24, wherein a subset of said different antibodies reacts with T-cell biomarkers i) CD3 (T-cells), ii) CD4 (T-helper), iii) CDS (cytotoxic T-cells), and iv) CD45RO (memory T-cells).

### Claim 27 (depends on 24)

The method of claim 24, wherein one of said different antibodies reacts with the B-cell biomarker CD20.

### Claim 28 (depends on 24)

The method of claim 24, wherein one of said different antibodies reacts with the macrophage biomarker CD68.

### Claim 29 (depends on 24)

The method of claim 24, wherein one of said plurality of different antibodies reacts with an immune checkpoint molecule.

### Claim 30 (depends on 29)

The method of claim 29, wherein said immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

### Claim 31

A conjugate, having the structure: