PREDICTIVE RESPONSE BIOMARKER DISCOVERY PROCESS

1 . A method of identifying a predictive response biomarker (PRB) for treating a disease (e.g., a cancer) using a T/B-cell-targeted immunomodulatory therapy, the method comprising the following steps:

(a) in a pre-treatment sample of the disease (e.g., cancer), identifying response-capable T/B cells having TCR (T Cell Receptor)/BCR (B Cell Receptor) clonotypes identical to TCR/BCR clonotypes of clonally expanded T/B cells in a matching post-treatment sample of the disease (e.g., cancer), wherein said clonally expanded T/B cells in the matching post-treatment sample are clonally expanded following the treatment; and,

(b) generating a list of genes upregulated and/or down-regulated in said response-capable T/B cells in the pre-treatment sample, to create the PRB;

(c) optionally, each gene in said PRB is weighed using a logistic regression coefficient obtained by fitting said each gene as predictor with expansion status and/or clinical response status as outcome.

2 . The method of claim 1 , wherein step (a) comprises:

(1) generating said matching post-treatment sample by contacting an ex vivo culture of an untreated sample of the disease (e.g., cancer) with a T-cell-targeted immunomodulatory compound, under a condition and for a time period sufficient for an immunomodulatory effect of the compound on a T cell population within the ex vivo culture to manifest;

(2) encapsulating individual T cells isolated, purified, or enriched from said ex vivo culture into picoliter droplets for single-cell profiling of a functional property, thereby separating each encapsulated individual T cells into a first pool of responder T cells and a second pool of non-responder T cells based on the presence or absence, respectively, of said functional property;

(3) determining TCR clonotype for each encapsulated individual T cells in the first pool of responder T cells and the second pool of non-responder T cells, thereby identifying TCR clonotypes of the responder T cells as the TCR clonotypes of clonally expanded T cells in said matching post-treatment sample;

(4) encapsulating individual T cells isolated, purified, or enriched from said pre-treatment sample into picoliter droplets to identify said response-capable T cells having TCR clonotypes identical to TCR clonotypes of said clonally expanded T cells in said matching post-treatment sample; or,

(1) obtaining, from a suitable donor, said pre-treatment sample and said matching post-treatment sample of the disease (e.g., cancer), wherein said matching post-treatment sample has been contacted with the compound, under a condition and for a time period sufficient for an immunomodulatory effect of the compound on a T cell population within the post-treatment sample to manifest;

(2) encapsulating individual T cells isolated, purified, or enriched from said post-treatment sample into picoliter droplets for single-cell profiling of a functional property, thereby separating each encapsulated individual T cells into a first pool of responder T cells and a second pool of non-responder T cells based on the presence or absence, respectively, of said functional property;

(3) determining TCR clonotype for each encapsulated individual T cells in the first pool of responder T cells and the second pool of non-responder T cells, thereby identifying TCR clonotypes of the responder T cells as the TCR clonotypes of clonally expanded T cells in said matching post-treatment sample;

(4) encapsulating individual T cells isolated, purified, or enriched from said pre-treatment sample into picoliter droplets to identify said response-capable T cells having TCR clonotypes identical to TCR clonotypes of said clonally expanded T cells in said matching post-treatment sample.

3 . The method of claim 2 , wherein step (b) comprises:

(5) using single cell RNA sequencing (scRNA-seq) to identify said list of genes upregulated in said response-capable T cells in the pre-treatment sample, wherein each gene in said PRB has a log2-fold change of >0.2, and is expressed in <40% of T cells with TCR clonotypes that are not clonally expanded in the matching post-treatment sample;

optionally, wherein said scRNA-seq is carried out using a gene panel specifically designed for a mechanism of action of the compound, or a pre-determined gene panel designed to assess immuno-modulation.

4 . The method of any one of claims 1 - 3 , wherein

(1) said pre-treatment sample is a blood sample; and/or

(2) the disease is a cancer, such as a solid tumor.

5 . The method of any one of claims 1 - 4 , wherein said ex vivo culture is:

(1) freshly isolated from a disease tissue;

(2) established from a stored (e.g., a frozen) disease tissue;

(3) a single cell suspension; and/or,

(4) an adherent culture.

6 . The method of any one of claims 2 - 5 , wherein:

(1) the time period is about 12-24 hrs, 24-36 hrs, 36-48 hours, up to 3 days, up to 4 day, or up to 5 days; and/or

(2) the condition comprises: one of a series of concentrations of the compound, and/or with or without combination with a second therapeutic agent;

optionally, the second therapeutic agent comprises a cytokine (e.g., IL-2, TNF), TCR stimulation (e.g., by CD3 cross-linking) and/or co-stimulation (e.g., CTLA, B7, CD28 etc).

7 . The method of any one of claims 1 - 6 , wherein the compound is an immuno-modulatory antibody.

8 . The method of any one of claims 2 - 7 , wherein step ( 2 ) is carried out by a microfluidic device.

9 . The method of any one of claims 2 - 8 , wherein said individual T cells are isolated, purified, or enriched by:

(1) isolating, purifying, or enriching a specific T cell subset or population (such as CD4 + or CD8 + T cells); and/or

(2) generally isolating, purifying, or enriching for T cells.

10 . The method of any one of claims 2 - 9 , wherein said functional property comprises: IFN-γ secretion and/or upregulation of an activation marker.

11 . The method of any one of claims 3 - 10 , wherein said gene panel comprises a gene for proliferation (such as Ki67); a gene for stem-like feature (such as TCF7); a gene for T-cell activation (such as CD25, Granzyme B, Perforin, CD28, TNF, IL2, IFNG, 4-1BB, CD38, CD69, OX-40, GITR, IL4, IL6); a gene for T cell exhaustion (CD39, CTLA-4, EOMES, PD-1, TIGIT, 2B4, LAG-3, T-bet, TIM3, TOX, CD160, IL-10); a gene for migration (such as CD31, CD103, CXCR5, CXCR4, CCR7, CCR3, CCR4, CCR8, CCR5, CXCR3); and a combination thereof; optionally, the gene panel further comprises CD4, CD8, FOXP3, CD62L, CD44, CD127, CD27, IL33R, and/or PTPRC.

12 . The method of any one of claims 2 - 11 , wherein in step (2), said function property is

(1) used to physically separating said responder T cells and non-responder T cells using a microfluidic device based on the presence or absence of said functional property; or,

(2) an activation marker or a response signature (either or both of which can be determined by, e.g., single-cell RNA sequencing), and wherein said responder T cells and non-responder T cells are not physically separated but are virtually distinguished based on the expression or lack of expression of said activation marker or response signature.

13 . The method of any one of claims 2 - 12 , further comprising profiling the pre-treatment sample and/or the post-treatment sample to characterize the samples for, e.g., cell type composition (via, e.g., flow cytometry) and target expression on T cells.

14 . A method for selecting a patient for treatment of a disease (e.g., a cancer) using a T-cell-targeted immunomodulatory compound, said method comprising: obtaining a clinical response score for the patient, by assessing the expression status and/or expression level of genes in a predictive response biomarker (PRB) of the disease (e.g., cancer) obtained by the method of any one of claims 1 - 13 , to determine whether said clinical response score for the patient exceeds a pre-determined threshold clinical response score;

wherein patients having clinical response scores exceeding the threshold score are identified as being beneficial for said treatment and are selected for said treatment, and/or,

wherein patients having clinical response scores below a second threshold score are identified as not being beneficial for said treatment and are not selected for said treatment.

15 . A method for treating a patient for a disease (e.g., a cancer) using a T-cell-targeted immunomodulatory compound, said method comprising:

(i) obtaining a clinical response score for the patient, by assessing the expression status and/or expression level of genes in a predictive response biomarker (PRB) of the disease (e.g., cancer) obtained by the method of any one of claims 1 - 24 , to determine whether said clinical response score for the patient exceeds a pre-determined threshold clinical response score; wherein patients having clinical response scores exceeding the threshold score are identified as being beneficial for said treatment and are selected for said treatment; and,

(ii) administering a therapeutically effective amount of the compound to the patient identified in (i) as being beneficial for and being selected for said treatment.