Modified Lymphocytes for Solid Tumor Immunotherapy
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Solution Overview
Problem
Current cellular immunotherapies, such as CAR-T cell therapies, face challenges in effectively targeting solid tumors due to T cell dysfunction in the tumor microenvironment, and existing genome engineering methods are limited in performing gain-of-function screens in human cells.
Innovation Solution
The development of modified lymphocytes expressing specific genes like LTBR, ADA, IFNL2, IL12B, and others, combined with chimeric antigen receptors (CARs) or T cell receptors (TCRs), to enhance T cell proliferation, activation, and cytokine production, and the use of mRNA or viral vectors for gene delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CAR-T cell therapy is used to target solid tumors, then T cell efficacy should improve, but T cell dysfunction in the tumor microenvironment suppresses effector function
Solution Approach 1:
The patent performs genome-wide CRISPR screens to identify genes that, when modified, change the functional parameters of T cells. By systematically knocking out or modifying genes, the invention discovers genetic factors that enhance T cell effector functions and overcome suppression in the tumor microenvironment, thereby improving reliability while addressing harmful dysfunction.
Solution Approach 2:
The patent uses CRISPR-Cas9 technology to create isogenic T cell lines with specific gene modifications. By copying and comparing T cell populations with and without specific gene edits, the invention identifies genetic modifications that confer resistance to tumor microenvironment suppression, enabling selection of T cell variants with improved efficacy.
2Ease of manufacture
If CRISPR genome engineering is used to knock out genes, then gene modification capability improves, but delivery of Cas9 via lentivirus to primary T cells becomes challenging due to large size
Solution Approach 1:
The patent extracts the essential gene-modifying function from the complete CRISPR-Cas9 system by focusing on delivering only the minimal necessary components (Cas9 protein or mRNA along with guide RNA) rather than attempting to deliver the entire plasmid construct. This extraction approach reduces delivery complexity while maintaining gene modification capability.
Solution Approach 2:
The patent uses alternative delivery intermediaries such as electroporation, lipid nanoparticles, or protein transfection reagents to deliver Cas9 protein or mRNA to primary T cells, bypassing the limitations of lentiviral delivery. These intermediary methods enable efficient gene modification without requiring large viral vectors.
3Measurement precision
If loss-of-function screens are used to identify T cell dysfunction genes, then screening capability is established, but gain-of-function screens in human cells remain limited
Solution Approach 1:
The patent inverts the traditional loss-of-function screening approach by implementing gain-of-function screens using CRISPR activation (CRISPRa) or overexpression libraries. Instead of knocking out genes to identify dysfunction, the invention activates or overexpresses genes to discover those that enhance T cell function, thereby expanding screening versatility while maintaining measurement precision.
Data Source
AI summary
Provided herein are nucleic acids, expression cassettes, modified lymphocytes and compositions comprising the same which include a sequence encoding a gene of Table 1. In some embodiments, the gene is LTBR. In certain embodiments, the cell is a T cell. In certain embodiments, the cell further comprises a CAR or engineered TCR. Methods of treatment using the provided compositions are also described.