# US 2023/0407397 A1 · Methods for identifying drivers of immune responses using molecular expression signatures of immunomodulating agents

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- Number: US 2023/0407397 A1
- Document ID: US20230407397A1
- Status: Published application
- Kind: A1
- Application number: 18/035,269
- Filed: 2021-11-04
- Published: 2023-12-21
- Earliest claimed: 2020-11-04
- Assignees: Massachusetts Institute of Technology; The General Hospital Corporation
- Applicants: Massachusetts Institute of Technology; The General Hospital Corporation
- Inventors: Nir Hacohen; Ang Cui
- Web page: https://patentgrep.com/patent/US20230407397A1

## CPC

- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 37/02: Immunomodulators
- C12N 5/0634: {Cells from the blood or the immune system}
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- A61K 2039/577: {tolerising response}
- C12N 2501/20: Cytokines; Chemokines
- C12Q 2600/136: Screening for pharmacological 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
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure provides methods for determining signatures of immune responses to immunomodulating agents in cells and in tissues. Also provided herein are computational methods for deconvoluting gene expression profiling data. Further provided herein are methods for treating a disease or disorder (e.g, proliferative diseases such as cancer, autoimmune diseases such as rheumatoid arthritis and psoriasis, allergies, etc.) in a subject comprising administering one or more immunomodulating agents to drive an immune response, in combination with a treatment for the disease or disorder (e.g, an anti-cancer therapy, an anti-viral therapy, a vaccination, etc.).

## Claims

### Claim 1

A method for determining signatures of immune responses to immunomodulating agents, the method comprising steps of:
(a) administering an immunomodulating agent into a population of cells, wherein the population of immune cells comprises multiple cell types;
(b) harvesting the population of cells;
(c) profiling gene expression in each cell within the population of cells;
(d) correlating cell type and gene expression to compute cell-type-specific gene expression signatures;
(e) repeating steps (a)-(d) for additional immunomodulating agents; and
(f) comparing the cell-type-specific gene expression signatures across immunomodulating agents.

### Claim 2 (depends on 1)

The method of claim 1, wherein the immunomodulating agent is a cytokine.

### Claim 3 (depends on 2)

The method of claim 2, wherein the cytokine is an IL-1 family cytokine, an IL-6 family cytokine, an IL-10 family cytokine, an IL-12 family cytokine, an IL-17 family cytokine, a common γ chain family cytokine, a common β chain family cytokine, an interferon, or a TNF family cytokine.

### Claim 4 (depends on 1)

The method of claim 1, wherein the immunomodulating agent is a growth factor.

### Claim 5 (depends on 1)

The method of claim 1, wherein the immunomodulating agent is a hormone.

### Claim 6 (depends on 1)

The method of claim 1, wherein the immunomodulating agent is a vaccine adjuvant.

### Claim 7 (depends on 1)

The method of claim 1, wherein the population of cells is a population of human cells.

### Claim 8 (depends on 1)

The method of claim 1, wherein the cell types are selected from the group consisting of monocytes, NK cells, T cells, macrophages, lymphatic cells, innate lymphoid cells, neutrophils, blood endothelial cells, fibroblastic reticular cells, mast cells, basophils, Langerhans cells, and dendritic cells.

### Claim 9 (depends on 8)

The method of claim 8, wherein the T cells are CD4 T cells, CD8 T cells, regulatory T cells, or gamma delta T cells.

### Claim 10 (depends on 7)

The method of claim 7, wherein the dendritic cells are conventional type 1 dendritic cells, conventional type 2 dendritic cells, migratory dendritic cells, or plasmacytoid dendritic cells.

### Claim 11 (depends on 1)

The method of claim 1, wherein the population of cells is in vivo.

### Claim 12 (depends on 1)

The method of claim 1, wherein the population of cells is in a mammal.

### Claim 13 (depends on 12)

The method of claim 12, wherein the mammal is a mouse.

### Claim 14 (depends on 12)

The method of claim 12, wherein the mammal is a human.

### Claim 15 (depends on 1)

The method of claim 1, wherein the step of harvesting is performed at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, or at least 6 hours after the step of administering.

### Claim 16 (depends on 1)

The method of claim 1, wherein the step of harvesting is performed 4 hours after the step of administering.

### Claim 17 (depends on 11, 12, 13, 14)

The method of any one of claims 11-14, wherein the step of harvesting the population of immune cells comprises harvesting and processing cells from draining lymph nodes.

### Claim 18 (depends on 1)

The method of claim 1, wherein the step of profiling gene expression comprises performing single-cell RNA sequencing.

### Claim 19 (depends on 1)

The method of claim 1, wherein the step of profiling gene expression comprises bulk gene expression profiling.

### Claim 20 (depends on 1)

The method of claim 1, wherein the step of profiling gene expression comprises single-cell gene expression profiling.

### Claim 21 (depends on 1)

The method of claim 1, wherein the step of profiling gene expression comprises protein expression profiling.

### Claim 22 (depends on 1)

The method of claim 1, wherein the step of profiling gene expression comprises chromatin accessibility profiling.

### Claim 23 (depends on 1)

The method of claim 1, wherein the method further comprises identifying polarization states of cell types within the population of immune cells in response to immunomodulating agents.

### Claim 24 (depends on 1)

The method of claim 1, further comprising:
determining a target gene expression profile; and
determining, using at least some of the signatures of immune response, at least one immunomodulating agent associated with the target gene expression profile.

### Claim 25 (depends on 24)

The method of claim 24, wherein determining the at least one immunomodulating agent associated with the target gene expression profile is performed by regressing the at least some of the signatures of immune response against the target gene expression profile using a regression technique.

### Claim 26 (depends on 25)

The method of claim 25, wherein the regression technique is a regularized linear regression technique.

### Claim 27 (depends on 26)

The method of claim 26, wherein the regularized linear regression technique is a ridge regression technique, a least absolute shrinkage and selection operator (LASSO) regression technique, or an elastic net regression technique.

### Claim 28 (depends on 24)

The method of claim 24, wherein determining the at least one immunomodulating agent associated with the target gene expression profile is performed by correlating at least one signature of immune response with the target expression profile.

### Claim 29

A method for treating a disease or disorder in a subject in need thereof, the method comprising administering one or more immunomodulating agents that drive an immune response within the subject in combination with a treatment for the disease or disorder, wherein the immune response is associated with increased efficacy of the treatment.

### Claim 30 (depends on 29)

The method of claim 29, wherein the disease or disorder is a proliferative disease, an infectious disease, an inflammatory disease, an autoimmune disease, or allergies.

### Claim 31 (depends on 30)

The method of claim 30, wherein the disease or disorder is cancer

### Claim 32 (depends on 31)

The method of claim 31, wherein the immune response is induced in NK cells.

### Claim 33 (depends on 32)

The method of claim 32, wherein the immune response comprises enhancing NK cell proliferation.

### Claim 34 (depends on 33)

The method of claim 33, wherein the immunomodulating agent is a cytokine.

### Claim 35 (depends on 33)

The method of claim 33, wherein the immunomodulating agent is selected from the group consisting of IL18, IL15, IFNb, IL2, IL12, IL7, IL33, IL27, IFNk, Cardiotrophin-1, IL36α, GM-CSF, IFNe, IL3, IL11, OSM, 41BBL, IL9, M-CSF, CD40L, Neuropoietin, TL1A, IL21, LIF, IL4, FasL, IL17F, IL17A, IL10, IL17E, IL5, IFNI2, LIGHT, IFNg, Noggin, IL24, IL17C RANKL IL13, BAFF, Persephin, IL23, Flt3I, G-CSF, APRIL, IL36RA, VEGF, GDNF, IL31, LTA1-B2, GITRL, IL6, CD30L, HGF, OX40L, CD27L, FGF-basic, Resistin, IL17B, Leptin, IL30, TRAIL, SCF, IL17D, IL-Y, IGF-I, EGF, IL20, and LTA2-B1.

### Claim 36 (depends on 33)

The method of claim 33, wherein the immunomodulating agent is IL18.

### Claim 37 (depends on 31)

The method of claim 31, wherein the immune response is induced in dendritic cells.

### Claim 38 (depends on 37)

The method of claim 37, wherein the immune response comprises induction of Ccr7, MHC-II, Cd80, Cd83, Cd86, or Mfge8 expression.

### Claim 39 (depends on 31)

The method of any one of claim 31, wherein the immune response is induced in macrophages.

### Claim 40 (depends on 39)

The method of claim 39, wherein the immune responses comprises induction of iNos or MHC-II expression in M1-like macrophages.

### Claim 41 (depends on 39)

The method of claim 39, wherein the immune response comprises elevated expression of Arg1 or IL10 in M2-like macrophages.

### Claim 42 (depends on 39)

The method of claim 39, wherein the immune response comprises decreased expression of M1-like macrophages.

### Claim 43 (depends on 39)

The method of claim 39, wherein the immune response comprises decreased expression of M1-like macrophages.

### Claim 44 (depends on 30)

The method of claim 30, wherein the immune response comprises decreased antigen presentation molecules on B cells.

### Claim 45 (depends on 30)

The method of claim 30, wherein the disease or disorder is rheumatoid arthritis.

### Claim 46 (depends on 45)

The method of claim 45, wherein the immune response comprises reduction of TNF expression.

### Claim 47 (depends on 30)

The method of claim 30, wherein the disease or disorder is psoriasis.

### Claim 48 (depends on 47)

The method of claim 47, wherein the immune response comprises reduction of IL23 expression.

### Claim 49 (depends on 29)

The method of claim 29, wherein the disease or disorder is an allergic reaction.

### Claim 50 (depends on 49)

The method of claim 49, wherein the immune response comprises decreased expression of IL4, IL5, IL13, or IL33.

### Claim 51 (depends on 50)

The method of claim 50, wherein the immune response comprises decreased activation of mast cells or basophils.

### Claim 52 (depends on 29)

The method of claim 29, wherein the immune response comprises induction of Granzyme B (GZMB).

### Claim 53 (depends on 52)

The method of claim 52, wherein Granzyme B is induced in T cells and NK cells.

### Claim 54 (depends on 29)

The method of claim 29, wherein the immune response comprises suppression of transforming growth factor beta (TGF-β).

### Claim 55 (depends on 52, 53, 54)

The method of any one of claims 52-54, wherein the treatment is an anti-tumor therapy.

### Claim 56 (depends on 52, 53, 54)

The method of any one of claims 52-54, wherein the treatment is an anti-viral therapy.

### Claim 57 (depends on 52, 53, 54, 55, 56)

The method of any one of claims 52-56, wherein the treatment is checkpoint blockade therapy.

### Claim 58 (depends on 29)

The method of claim 29, wherein the immunomodulating agent is a vaccine.

### Claim 59 (depends on 58)

The method of claim 58, wherein the immune response comprises regulation of IFNa, IFNb, TFNa, IL15, IL18, IL1a, IFNk, IL36α, IFNg, Flt3I, OX40L, BAFF, IFNg, Flt3I, IL6, IL27, IL36-RA, IL34 or GM-CSF.

### Claim 60

A method of polarizing a population of cells, the method comprises stimulating the cells with IL7r, Gzmb, Isg15, Klra4, Srm, or Mcm5.

### Claim 61 (depends on 60)

The method of claim 60, wherein the cell are NK cells.

### Claim 62

A method of expanding a population of cells, the method comprising stimulating the cells with an immunomodulating agent.

### Claim 63 (depends on 62)

The method of claim 62, wherein the cells are NK cells.

### Claim 64 (depends on 62)

The method of claim 62, wherein the immunomodulating agent is a cytokine.

### Claim 65 (depends on 62)

The method of claim 62, wherein the immunomodulating agent is selected from the group consisting of IL18, IL15, IFNb, IL2, IL12, IL7, IL33, IL27, IFNk, Cardiotrophin-1, IL36α, GM-CSF, IFNe, IL3, IL11, OSM, 41BBL, IL9, M-CSF, CD40L, Neuropoietin, TL1A, IL21, LIF, IL4, FasL, IL17F, IL17A, IL10, IL17E, IL5, IFNI2, LIGHT, IFNg, Noggin, IL24, IL17C RANKL IL13, BAFF, Persephin, IL23, Flt3I, G-CSF, APRIL, IL36RA, VEGF, GDNF, IL31, LTA1-B2, GITRL, IL6, CD30L, HGF, OX40L, CD27L, FGF-basic, Resistin, IL17B, Leptin, IL30, TRAIL, SCF, IL17D, IL-Y, IGF-I, EGF, IL20, and LTA2-B1.

### Claim 66

A method of treating cancer, the method comprising administering IL18 to a subject in need thereof in combination with a cancer therapy.

### Claim 67

A method for treating cancer, the method comprising administering an anti-cancer therapy in combination with inducing GZMB expression in a subject in need thereof.

### Claim 68 (depends on 67)

The method of claim 67, wherein GZMB is induced in T cells and NK cells.

### Claim 69

A method for treating a viral infection, the method comprising administering an anti-viral treatment in combination with inducing GZMB expression in a subject in need thereof.

### Claim 70 (depends on 69)

The method of claim 69, wherein GZMB is induced in T cells and NK cells.

### Claim 71

A method of vaccination, the method comprising administering a vaccine and stimulating IFNa, IFNb, TFNa, IL15, IL18, IL1a, IFNk, IL36α, IFNg, Flt3I, OX40L, BAFF, IFNg, Flt3I, IL6, IL27, IL36-RA, IL34, or GM-CSF in a subject in need thereof

### Claim 72 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)

The method of any one of claims 1-28, further comprising using the cell-type-specific gene expression signatures obtained in steps (a)-(f) to identify cells responding to these agents in single cell expression profiles from tissues of individuals with a disease or undergoing therapy for a disease.

### Claim 73 (depends on 1)

The method of claim 1, wherein the cell types are selected from the group consisting of B cells, monocytes, NK cells, T cells, macrophages, lymphatic cells, innate lymphoid cells, neutrophils, blood endothelial cells, fibroblastic reticular cells, mast cells, basophils, Langerhans cells, and dendritic cells.

### Claim 74 (depends on 1)

The method of claim 1, wherein step (d) further comprises calculating expression signatures associated with each immune-modulating agent relative to a control population of immune cells that has not been administered an immunomodulating agent.

### Claim 75 (depends on 24)

The method of claim 24, wherein determining the at least one immunomodulating agent associated with the target gene expression profile is performed using a Wilcoxon rank-sum test.