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

[0058]As used herein, a “regulatory sequence” also named an “expression control sequence” refers to polynucleotide sequences which affect the expression of coding sequences to which they are operatively linked. Expression control sequences or regulatory sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term “control sequences” (also interchangeably named regulatory sequences) is intended to encompass, at a minimum, any component whose presence is essential for expression, and can also encompass an additional component whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0209]A particular advantage of the immunogenic compositions of the invention is that they can be used to redirect the specificity and function of T lymphocytes and other immune cells against multiple antigens against which the CAR in the vector or vector particles are directed. As a result, the invention encompasses a composition that could be used in therapeutic vaccination protocols. In particular, it can be used in combination with adjuvants, other immunogenic compositions, chemotherapy, or any other therapeutic treatment.

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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