CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase of International Application No. PCT/GB2016/051164, filed on Apr. 26, 2016, which claims priority benefit to Application No. 1507104.6, filed in the United Kingdom on Apr. 27, 2015.

FIELD OF THE INVENTION

The present invention relates to constructs and approaches for modulating the relative expression of polypeptides co-expressed from a single vector. In particular, the invention relates to modulating the expression of a transmembrane protein co-expressed from a single vector with a second polypeptide.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application includes a Sequence Listing as a part of the disclosure, submitted as a text file named “52471_Seqlisting.txt”, created on Sep. 25, 2017, 52,273 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.

BACKGROUND TO THE INVENTION

It is often desirable to express different proteins from the same vector since multiple transduction of the same cell is difficult, expensive and unpredictable. Different methods have therefore been developed to allow co-expression of two proteins from a single vector (seeFIG.1).

Initial attempts used two different promoters within the same cassette. This results in two separate transcripts each of which code for a separate protein. This is a difficult approach for a number of reasons. A key problem is “promoter interference” whereby one promoter dominates and causes silencing of the second promoter. In addition, different promoters work differently in different cellular contexts and this makes consistent “tuning” of the relative expression of each transgene difficult to achieve.

An alternative approach is to use an Internal Ribosome Entry sequence (IRES). Here, a single transcript is generated. The IRES sequence in the transcript is placed between the open reading frames for the two transgenes and mimics an mRNA cap structure. Hence, the ribosome either initiates translation at the 5′ cap or the IRES resulting in expression of two separate proteins. A key limitation with this approach is the inability to control relative expression. The 3′ transcript is typically expressed less than the 5′ one, but the ratio of expression is difficult to predict and tune.

A further approach has been provided following characterization of the role of foot-and-mouth-disease virus (FMDV) 2A peptide in allowing FMDV (and related viruses) to express multiple proteins from a single open reading frame (ORF) (Donnelly et al;J. Gen. Virol.;82, 1027-1041 (2001)). The 2A peptide (and homologs) cleaves at very high efficiency immediately after translation of the ORF, enabling the expression of multiple peptides from a single ORF. A problem with the use of the 2A peptide to cleave between different peptides in the same ORF is that expression is limited to a 1:1 ratio.

Thus there is a need for alternative methods for expressing more than one protein from a single vector which are not associated with the disadvantages described above.

SUMMARY OF ASPECTS OF THE INVENTION

The present invention is based on the determination that, when a transmembrane protein is co-expressed with a second protein as a polyprotein which after translation is subsequently cleaved to separate both proteins, the incorporation of an intracellular retention signal in the transmembrane protein allows the cell surface expression of the transmembrane protein to be modulated relative to the second protein by reducing its trafficking to the cell surface and/or by reducing its half-life at the cell surface. This need not be limited to a pair of transgenes, but may be used to allow control of the relative expression of multiple proteins initially translated as a polyprotein.

As used herein, ‘polyprotein’ refers to a polypeptide sequence translated from a single nucleic acid construct as a single entity, but which comprises polypeptide sequences which are subsequently separated and which function as discrete entities (e.g. separate proteins).

Thus in a first aspect the present invention provides a nucleic acid construct comprising the following structure:
A-X-Bin whichA and B are nucleic acid sequences encoding a first and a second protein of interest (POI); andX is a nucleic acid sequence which encodes a cleavage site,wherein either the first or second POI is a transmembrane protein which comprises an intracellular retention signal.

The intracellular retention signal is heterologous to the transmembrane protein i.e. it does not occur in the “wild-type” version of the transmembrane protein (or part thereof) and is introduced by recombinant means. The intracellular retention signal may be synthetic or derivable from another organism or another protein.

The endodomain of the transmembrane protein may comprise the intracellular retention signal.

The intracellular retention signal may direct the transmembrane protein away from the secretory pathway and/or to a membrane-bound intracellular compartment such as a lysozomal, endosomal or Golgi compartment.

The intracellular retention signal may, for example, be a tyrosine-based sorting signal, a dileucine-based sorting signal, an acidic cluster signal, a lysosomal avoidance signal, an NPFX′(1,2)D-Type signal (SEQ ID NO: 50), a KDEL (SEQ ID NO: 51), a KKX′X′ (SEQ ID NO: 52) or a KX′KX′X′ (SEQ ID NO: 53) signal (wherein X′ is any amino acid).

The intracellular retention signal may comprise a sequence selected from the group of: NPX′Y (SEQ ID NO: 54), YX′X′Z′ (SEQ ID NO: 55), [DE]X′X′X′L[LI] (SEQ ID NO: 56), DX′X′LL (SEQ ID NO: 57), DP[FW] (SEQ ID NO: 58), FX′DX′F (SEQ ID NO: 59), NPF, LZX′Z[DE] (SEQ ID NO: 60), LLDLL (SEQ ID NO: 61), PWDLW (SEQ ID NO: 62), KDEL (SEQ ID NO: 51), KKX′X′ (SEQ ID NO: 52) or KX′KX′X′ (SEQ ID NO: 53);

wherein X′ is any amino acid and Z′ is an amino acid with a bulky hydrophobic side chain.

The intracellular retention signal may comprise any of the sequences shown in Tables 1 to 5.

The intracellular retention signal may comprise the Tyrosinase-related protein (TYRP)-1 intracellular retention signal. The intracellular retention signal may comprise the TYRP-1 intracellular domain. The intracellular retention signal may comprise the sequence NQPLLTD (SEQ ID NO: 35).

The intracellular retention signal may comprise the Adenoviral E3/19K intracellular retention signal. The intracellular retention signal may comprise the E3/19K cytosolic domain. The intracellular retention signal may comprise the sequence KYKSRRSFIDEKKMP (SEQ ID NO: 36); or DEKKMP (SEQ ID NO: 37).

The intracellular retention signal may be proximal or distal to a transmembrane domain of the transmembrane protein.

X may be a nucleic acid sequence encoding a self-cleaving peptide, a furin cleavage site or a Tobacco Etch Virus cleavage site.

X may be a nucleic acid sequence encoding a 2A self-cleaving peptide from an aphtho- or a cardiovirus or a 2A-like peptide.

The transmembrane protein may be any transgenically expressed transmembrane protein.

The transmembrane protein may be selected from a list of: excitatory receptors such as 41 BB, OX40, CD27, CD28 and related molecules; or inhibitory receptors such as PD1, CTLA4, LAIR1, CD22 and related molecules; or cytokine receptor molecules such as IL1R, IL2R, IL7R, IL15R and related molecules; or homing molecules such as N-CAM, V-CAM, L1-CAM, LFA-1, CDH1-3, Selectins or Integrins;

The transmembrane protein may be a synthetic protein such as a suicide gene or a marker gene.

The transmembrane protein may be or comprise the α and/or β chains of a T-cell receptor.

The transmembrane protein may be a chimeric-antigen receptor (CAR).

Either or both of the first and second POIs may be a transmembrane protein; and either or both of the transmembrane proteins may comprise an intracellular retention signal as defined herein.

Either or both of the first and second POIs may be a single pass transmembrane protein, such a type I transmembrane protein.

The amount of a transmembrane protein which comprises an intracellular retention signal which is expressed at the cell surface may be, for example, less than 90%, 70%, 50% or 30% compared to a transmembrane protein expressed from the same nucleic acid construct which does not comprise an intracellular retention signal.

The invention also provides nucleic acid construct encoding more than two proteins of interest. For example, in one embodiment, the invention provides a nucleic acid construct comprising the following structure:
A-X-B-Y-Cin which
A, B and C are nucleic acid sequences encoding first, second and third polypeptides of interest (POIs); and
X and Y are nucleic acid sequences which may be the same or different, each of which encodes a cleavage site,
wherein at least two of the POIs are transmembrane proteins which comprise an intracellular retention signal.

In this embodiment, the at least two POIs may:

(a) comprise different intracellular retention signals; and/or

(b) have the intracellular retention signal located at a different position in the POI,

such that when the nucleic acid is expressed in a cell, there is differential relative expression of the at least two POIs at the cell surface.

In a second aspect the present invention provides a vector comprising a nucleic acid construct according to the first aspect of the invention.

The vector may be a retroviral vector or a lentiviral vector or a transposon.

In a third aspect the present invention provides a cell comprising a nucleic acid construct according to the first aspect of the invention or a vector according to the second aspect of the invention.

The invention further relates to a method for modulating the relative cell surface expression of a transmembrane protein expressed as a polyprotein from a single nucleic acid with a second protein by including an intracellular retention signal in the nucleic acid sequence which encodes the transmembrane protein.

The inclusion of an intracellular retention signal in a transmembrane protein reduces the amount of the transmembrane protein expressed on the cell surface. As such, the relative expression level of a transmembrane protein derived from a polyprotein including a second polypeptide can be modulated. Where the transmembrane protein is only active at the cell surface (or predominantly active at the cell surface), reducing the relative cell surface expression of the protein also reduces its relative activity.

This invention can be extended to modulate the relative expression of three or more proteins expressed as a concatenated polypeptide, separated by cleavage sites and relative surface expression dictated by retention signals of differing activity.

