JPWO2020141204A5

JPWO2020141204A5 -

Info

Publication number: JPWO2020141204A5
Application number: JP2021537878A
Authority: JP
Publication date: 2022-11-08

Claims

A method for identifying an agent that can induce respiratory sensitization in a mammal, comprising:
(a) providing a population of dendritic cells or a population of dendritic-like cells;
(b) exposing the cells provided in step (a) to a test agent;
(c) measuring the expression of two or more biomarkers selected from the group defined in Table A in said cells of step (b);
A method, wherein expression of said two or more biomarkers measured in step (c) is indicative of a respiratory sensitizing effect of said test agent of step (b).

step (c) comprises two or more biomarkers selected from the group defined in Table A(i), such as 3, 4, 5, 6 of the biomarkers listed in Table A(i) , 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24, or 25, or 2. The method of claim 1, comprising measuring.

3. The method of claim 1 or 2 , wherein step (c) comprises or consists of measuring the expression of all of the biomarkers listed in Table A.

d) exposing said separate population of dendritic or dendritic-like cells to one or more negative control agents that are not respiratory sensitizers in a mammal;
e) measuring expression of said two or more biomarkers measured in step (c) in said cells of step (d);
identifying said test agent as a respiratory sensitizer if the expression of said two or more biomarkers measured in step (e) is different than the expression of said two or more biomarkers measured in step (c); to be
and/or
f) exposing said separate population of dendritic or dendritic-like cells to one or more positive control agents that are respiratory sensitizers in mammals;
g) measuring expression of said two or more biomarkers measured in step (c) in said cells of step (f);
if the expression of the two or more biomarkers measured in step (f) corresponds to the expression of the two or more biomarkers measured in step (c), then the test agent is a respiratory sensitizer identified ,
The method according to any one of claims 1-3 .

step (c) comprises measuring the expression of one or more nucleic acid molecules of said biomarkers;
optionally, said nucleic acid molecule is a cDNA molecule or an mRNA molecule,
Optionally, measuring the expression of one or more of said biomarkers in step (c) comprises Southern hybridization, Northern hybridization, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR). ), quantitative real-time PCR (qRT-PCR), nanoarray, microarray, macroarray, autoradiography, and in situ hybridization, and
optionally, measuring the expression of one or more of said biomarkers in step (c) is determined using a DNA microarray;
The method according to any one of claims 1-4 .

measuring the expression of one or more of said biomarkers in step (c) selectively binds to nucleic acid molecules each encoding one of the biomarkers identified in Table A. is carried out using one or more binding moieties capable of
optionally, said one or more binding moieties each comprise or consist of a nucleic acid molecule,
The method according to any one of claims 1-5 .

each of said one or more binding moieties comprises or consists of DNA, RNA, PNA, LNA, GNA, TNA, or PMO;
optionally, said binding moiety comprises a detectable moiety,
optionally, said detectable moiety is selected from the group consisting of a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, a radioactive moiety (e.g. a radioactive atom), or an enzymatic moiety;
7. The method of claim 6 .

step (c) is performed with an array ,
optionally, said array is a bead-based array,
optionally, said array is a surface-based array,
and/or said array is selected from the group consisting of macroarrays, microarrays, nanoarrays,
The method according to any one of claims 1-7 .

said population of dendritic cells or said population of dendritic-like cells comprises or consists of immortal cells and/or non-naturally occurring cells;
and/or the population of dendritic cells or the population of dendritic-like cells is a population of dendritic-like cells,
optionally, said dendritic-like cells are myeloid dendritic-like cells,
optionally, said myeloid dendritic cells are derived from myeloid dendritic cells,
Optionally, said myeloid dendritic cell-derived cells are in the group consisting of KG-1, THP-1, U-937, HL-60, Monomac-6, AML-193, MUTZ-3, and SenzaCell are myeloid leukemia-derived cells such as those selected from
The method according to any one of claims 1-8 .

to identify agents that may induce respiratory hypersensitivity responses;
and/or said hypersensitivity response is a humoral hypersensitivity response,
and/or to identify agents that can induce type I hypersensitivity responses in mammals,
and/or to identify drugs that may induce respiratory allergies,
and/or the method indicates the relative sensitizing effect of the sample tested;
The method according to any one of claims 1-9 .

said method comprising:
(i) culturing dendritic or dendritic-like cells;
(ii) seeding the cells of (i) into one or more wells, such as one or more wells of a multiwell assay plate;
(iii) adding the agent(s) to be tested to one or more wells of (ii);
(iv) adding one or more positive controls to one or more separate wells of (ii);
(v) adding one or more negative controls to one or more separate wells of (ii);
(vi) incubating the cells in the wells of (iii)-(v), preferably for about 24 hours;
(vii) isolating purified total RNA from the cells of (vi) and optionally converting the mRNA to cDNA;
(viii) quantifying the expression levels of individual mRNA transcripts from (vii) using arrays, e.g., Affymetrix Human Gene 1.0 ST arrays, and/or Nanostring code sets;
(ix) exporting and normalizing the expression data from (viii);
(x) separating data from (ix) derived from GARD Prediction Signature biomarkers (i.e., biomarkers of Table A);
(xi) applying a predictive model to the data from (x), e.g. applying a frozen SVM model previously established and trained on historical data, e.g. and predicting the respiratory sensitizing effect of the negative/positive control(s).

An array for use in the method of any one of claims 1-11 , comprising:
said array consists of binding moieties of 2 to 28 biomarkers defined in Table A;
the binding portion of the array comprises one or more binding portions as defined in any one of claims 6-7;
Optionally, an array comprising one or more binding moieties for each of the biomarkers defined in any one of claims 1-11 .

Use of two or more biomarkers selected from the group defined in Table A to identify respiratory sensitizers, preferably one or more biomarkers defined in Table A(i) selected from the group identified, use.

Use of two or more binding moieties each having specificity for a biomarker selected from the group defined in Table A to identify respiratory sensitizers, preferably one or more binding moieties A use wherein the portion has specificity for a biomarker selected from the group defined in Table A(i).

(a) an array according to claim 12 ;
(b) (optionally) one or more control agents;
(c) (optionally) instructions for performing the method as defined in any one of claims 1-11 . Analysis kit for use in the method.