Tricistronic Anti-GPC3 CAR Constructs for Balanced Protein Expression

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Solution Overview

Problem

Current T cell therapies face challenges in achieving consistent expression levels of multiple proteins, such as chimeric antigen receptors and cytokines, leading to variable therapeutic efficacy due to differing translation efficiencies in bicistronic and multi-cistronic vectors.

Innovation Solution

Engineering immune cells to express a chimeric antigen receptor (CAR) along with a TGF-beta dominant negative receptor (TGFβ DNR) and a membrane-bound IL15 protein (mbIL15) using tricistronic constructs, which include specific linkers and self-cleaving peptides to ensure balanced protein expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If bicistronic or multi-cistronic vectors are used to express multiple proteins in T cells, then the ability to express multiple therapeutic proteins is improved, but the translation efficiency varies drastically leading to highly variable expression levels

Engineering Contradiction:

Improveability to express multiple proteinsVSAvoidexpression level consistency

Core Design Contradiction:

Adaptability or versatilityVSManufacturing precision

Solution Approach 1:

The patent divides the multi-cistronic mRNA into separate transcriptional units by introducing self-cleaving peptide sequences (such as 2A peptides) between coding sequences. This segmentation allows each protein to be translated from effectively separate open reading frames, reducing the variability in translation efficiency while maintaining the ability to express multiple proteins from a single vector.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent uses self-cleaving peptide sequences as intermediary elements between different protein coding sequences. These peptides facilitate the separation of translation events while maintaining the single-mRNA structure, acting as mediators that resolve the conflict between multi-protein expression and uniform translation efficiency.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Device complexity

If multiple coding sequences are transcribed into a single mRNA, then the complexity of introducing multiple expression vectors is reduced, but the translation efficiency of different proteins varies drastically

Engineering Contradiction:

Improvenumber of expression vectorsVSAvoidprotein expression uniformity

Core Design Contradiction:

Device complexityVSManufacturing precision

Solution Approach 1:

The patent segments the single mRNA into functionally independent translation units by inserting self-cleaving peptide sequences between coding sequences. This maintains the simplicity of using one expression vector while achieving uniform translation efficiency comparable to separate vectors by creating distinct open reading frames within the single transcript.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent combines multiple protein coding sequences into a single mRNA transcript while using self-cleaving peptides to maintain functional independence of each coding sequence. This merging approach reduces vector complexity while preserving translation uniformity through the cleavage mechanism.

Inventive Principle:

Principle #5Merging (Combining)

Data Source

PatentUS20250382383A1Tricistronic constructs for Anti-GPC3 car

Publication Date: 2025.12.18 KITE PHARMA INC

AI summary

Immune cells engineered to express a chimeric antigen receptor (CAR) along with a TGF-beta dominant negative receptor (TGFβ DNR) and/or a membrane-bound IL15 protein (mbIL 15) are provided which are suitable for the treatment of diseases such as cancer.