Hook fusion protein for regulating the cellular trafficking of a target protein

Abstract

The present invention relates to a hook fusion protein comprising a hook domain and at least one cytoplasmic carboxy terminal endoplasmic reticulum (ER) retention signal and / or at least one cytoplasmic amino terminal endoplasmic reticulum (ER) retention signal; wherein the hook fusion protein is a soluble protein that localizes in the cytoplasm. The present invention also relates to a nucleic acid system for intracellular targeting control comprising a nucleic acid encoding a hook fusion protein as herein disclosed, and a nucleic acid encoding a target fusion protein comprising a hook-binding domain; wherein said target fusion protein in a membrane protein; and wherein the hook fusion protein localizes in the ER when bound to the target fusion protein. The invention also encompasses a vector system, viral particle system, host cell and kit comprising said nucleic acids. The invention also includes the vector system, viral particle system, host cell or kit for use as a medicament, in particular for immunotherapy.

Description

INTRODUCTION[0001]The inventors of the present application have previously described a molecular system named RUSH (Retention Using Selective Hooks) capable of controlling the intracellular trafficking of a target fusion protein and in particular that can be used to control the targeting of a target protein in a given cellular compartment.[0002]RUSH was described as a two state system based on the reversible interaction of a hook protein fused to streptavidin and stably anchored in the donor compartment with a target fusion protein fused to a streptavidin-binding peptide (SBP), which is therefore immobilized in said donor compartment. Addition of biotin caused a synchronous release of the target protein from the hook which is therefore free to resume its journey to its final compartment.[0003]The RUSH has been successfully applied to Chimeric Antigen Receptors (CARs) as a tool to selectively control the expression of the CARs at the cell surface, to prevent adverse effects (see WO20...

AI Technical Summary

Benefits of technology

The patent text describes a way to control the expression of genes in an organism. This can be useful in creating immunogenic compositions that can be used to redirect the function of immune cells against multiple antigens. The invention can be combined with other therapies like chemotherapy or vaccination protocols. The technical effect of this patent is the ability to control gene expression and create targeted immune responses to multiple antigens.

Problems solved by technology

However these hooks had some limitations.

Consequently higher size inserts, such as the ones containing the hook and the reporter (above 4 kb) impaired the production of lentivirus required for the transduction and / or generation of stable cell lines, namely modified immune cells containing CAR.

It has been reported that transduction of the proteins through IRES can impair their expression (Jones, Peng et al.

Moreover, the large size of the IRES sequence and differences in gene expression levels before and after IRES are other limiting factors.

Therefore such hooks do not support a fully reversible system, wherein the target membrane protein could be retrieved from the membrane.

Similarly such hooks do not allow preventing the target membrane protein to leak out from the ER, and in particular the leaking of the target membrane protein to the cell membrane.

Lastly, the hooks that were stably anchored in the membrane from the ER can only retain in the ER proteins that are in their close vicinity.

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