Pct/us22/79410

A method for generating a pre-effector gene signature for determining the cytotoxic effector potential of a preparation of chimeric antigen receptor (CAR) T cells, the method comprising:

• a) measuring single-cell gene expression data and endogenous T cell receptor (TCR) sequencing data of the preparation of CAR T cells;
• b) administering the preparation of CAR T cells to a subject;
• c) isolating post-infusion CAR T cells at one or more time points after the administration step b);
• d) identifying cytotoxic effector CAR T cells within the post-infusion CAR T cells isolated in step c);
• e) acquiring single-cell gene expression data and endogenous TCR sequencing data of the cytotoxic effector CAR T cells identified in step d);
• f) using the endogenous TCR sequencing data obtained in steps a) and e) to determine which CAR T cells from the preparation of CAR T cells are cytotoxic effector precursors by determining which of the CAR T cells of the preparation of CAR T cells share a clonally related TCR with the post-infusion cytotoxic effector CAR T cells;
• and
• g) generating a pre-effector gene signature by determining which set of genes are differentially expressed in the cytotoxic effector precursors as compared to other or all other CAR T cells from the preparation.

Non-transitory computer-readable medium configured to communicate with one or more processor(s) of a computational system, the non-transitory computer-readable medium including instructions thereon, that when executed by the processor(s), cause the computational system to:

• a) receive, as inputs,
• a preparation of CAR T cell gene expression dataset comprising single cell gene expression data of CAR T cells,
• TCR sequencing data each associated with respective single cell gene expression data of the preparation of CAR T cell single cell gene expression dataset,
• a post-infusion CAR T cell gene expression dataset comprising single cell gene expression data of CAR T cells originating from subjects post-infusion, and
• endogenous TCR sequencing data each associated with respective single cell gene expression data of the post-infusion CAR T cell gene expression dataset;
• b) identify, based on the post-infusion CAR T cell gene expression dataset, single cell gene expression data of cytotoxic effector CAR T cells;
• c) identify clonal TCR sequencing data such that the clonal TCR sequencing data is (i) associated with single cell gene expression data of the preparation of CAR T cell gene expression dataset, and is (ii) clonally related to endogenous TCR sequencing data associated with each of the single cell gene expression data of cytotoxic effector CAR T cells;
• d) identify cytotoxically effective precursor gene expression data such that the cytotoxically effective precursor gene expression data comprises single cell gene expression data of the preparation of CAR T cell gene expression dataset associated with the clonal TCR sequencing data;
• e) identify, based at least in part on the cytotoxically effective precursor gene expression data, a pre-effector gene signature comprising markers for determining a cytotoxic effector potential of CAR T cells;
• and
• f) provide, as an output, the pre-effector gene signature.

Non-transitory computer-readable medium configured to communicate with one or more processor(s) of a computational system, the non-transitory computer-readable medium including instructions thereon, that when executed by the processor(s), cause the computational system to:

• a) receive, as an input, a preparation of CAR T cell gene expression dataset comprising single cell gene expression data of CAR T cells of a preparation of CAR T cells;
• b) determine a cytotoxic effector potential for each single cell gene expression data of the preparation of CAR T cell gene expression dataset,
• wherein the cytotoxic effector potential indicates likelihood of a CAR T cell, when administered, to give rise to a lineage that results in functional effector post-infusion CAR T cells;
• and
• c) calculate, based on the cytotoxic effector potentials, a gene set module score for the preparation of CAR T cells,
• wherein the gene set module score indicates likelihood of the preparation of CAR T cells, when administered, to give rise to a clinically effective quantity of functional effector post-infusion T cells.