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Title:
FUSION PROTEINS FOR THE DETECTION OF APOPTOSIS
Document Type and Number:
WIPO Patent Application WO/2019/111194
Kind Code:
A1
Abstract:
The present invention relates to fusion proteins which are capable of binding to phosphatidylserine comprising a phosphatidylserine binding ligand and a modified O6-alkylguanine-DNA alkyltransferase which is capable of autoconjugation to an O6-benzylguanine-modified label, the fusion proteins being capable of binding to phosphatidylserine on the surface of a cell undergoing apoptosis. The invention also relates to recombinant polypeptide precursors of the fusion proteins which comprise a secretion leader sequence, purification tag, protease cleavage site and the fusion protein. Also included in the scope of the invention are nucleic acids encoding the recombinant polypeptide precursor, vectors comprising the nucleic acids, host cells comprising the vectors, methods of production of the fusion proteins, kits and assays for detecting apoptosis.

Inventors:
PADAYACHEE, Eden (916 Durham Square, Salt River, 7925 Cape Town, 7925, ZA)
BITEGHE, Fleury Augustin Nsole (48 Central Square, Conradie StreetBellville, 7925 Cape Town, 7925, ZA)
AKINRINMADE, Olusiji Alex (20 George Street, Observatory, 7925 Cape Town, 7925, ZA)
JORDAAN, Sandra (109 The Centurion, 1 Frere RoadSea Point, 8005 Cape Town, 8005, ZA)
BARTH, Stefan (115A Ocean View Drive, 8005 Green Point, 8005, ZA)
Application Number:
IB2018/059704
Publication Date:
June 13, 2019
Filing Date:
December 06, 2018
Export Citation:
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Assignee:
UNIVERSITY OF CAPE TOWN (Lovers Walk, Rondebosch, 7701 Cape Town, 7701, ZA)
International Classes:
C07K14/47; C12N15/62; C12Q1/48; G01N33/50
Domestic Patent References:
WO1999019470A21999-04-22
WO2017118764A12017-07-13
Other References:
VERMES I ET AL: "A novel assay for apoptosis Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V", JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM, NL, vol. 184, no. 1, 17 July 1995 (1995-07-17), pages 39 - 51, XP004021001, ISSN: 0022-1759, DOI: 10.1016/0022-1759(95)00072-I
ALINE TIRAT ET AL: "Evaluation of two novel tag-based labelling technologies for site-specific modification of proteins", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES., vol. 39, no. 1-3, 1 August 2006 (2006-08-01), NL, pages 66 - 76, XP055567319, ISSN: 0141-8130, DOI: 10.1016/j.ijbiomac.2006.01.012
XIAOLI SUN ET AL: "Development of SNAP-Tag Fluorogenic Probes for Wash-Free Fluorescence Imaging", CHEMBIOCHEM, vol. 12, no. 14, 19 September 2011 (2011-09-19), pages 2217 - 2226, XP055069445, ISSN: 1439-4227, DOI: 10.1002/cbic.201100173
Attorney, Agent or Firm:
SPOOR & FISHER et al. (11 Byls Bridge Boulevard, Building No. 14Highveld Ext 73, 0157 Centurion, 0157, ZA)
Download PDF:
Claims:
CLAIMS

1 . A recombinant polypeptide precursor, comprising the following formula:

Xi - X2 - X3 - X4 - X5 - X6 wherein,

X1 is a secretion leader sequence;

X2 is a purification tag;

X3 is a protease cleavage site;

X4 is a phosphatidylserine binding ligand;

X5 is a peptide linker comprising the amino acid sequence AAALESR; and X6 is a modified C^-alkylguanine-DNA alkyltransferase; wherein the protease cleavage site facilitates the cleavage of X4 - X5 - X6 from the recombinant polypeptide precursor to produce a mature recombinant polypeptide capable of binding to phosphatidylserine; and

further wherein X6 is capable of autoconjugation to an C^-benzylguanine- modified label.

2. The recombinant polypeptide precursor of claim 1 , wherein the phosphatidylserine binding ligand is annexin A5.

3. The recombinant polypeptide precursor of claim 1 or 2, wherein the secretion leader sequence is an IgK leader sequence.

4. The recombinant polypeptide precursor of any one of claims 1 to 3, wherein the purification tag is a poly histidine tag.

5. The recombinant polypeptide precursor of any one of claims 1 to 4, wherein the protease cleavage site is an enterokinase cleavage site.

6. The recombinant polypeptide precursor of any one of claims 1 to 5, wherein the modified 0®-alkylguanine-DNA alkyltransferase is C^-methylguanine-DNA methyltransferase.

7. The recombinant polypeptide precursor of any one of claims 1 to 6, wherein the C^-benzylguanine-modified label is selected from the group consisting of Alexa Fluor 488, Alexa Fluor 633, Allophycocyanin (APC), APC-Cy7, Carboxyfluorescein Diacetate (CFSE), Cy3, Cy5, Fluorescein isothiocyanate (FITC), Peridinin chlorophyll protein (PerCP), Phycoerythrin (PE or R-PE), Phycoerythrin-Cy5, Phycoerythrin- Texas Red (PE-Texas Red) and Texas Red.

8. The recombinant polypeptide precursor of any one of claims 1 to 7, comprising the sequence of SEQ ID NO:14.

9. A nucleic acid molecule encoding a recombinant polypeptide precursor of SEQ ID NO:14.

10. An expression vector comprising the nucleic acid molecule of claim 9.

1 1. A host cell transformed with the vector of claim 10.

12. A method for producing a mature recombinant polypeptide capable of binding to phosphatidylserine, the method comprising the steps of:

(i) providing an expression vector comprising a nucleic acid molecule encoding a recombinant polypeptide precursor having the formula:

Ci - X2 - X3 - X4 - X5- X6 wherein,

X1 is a secretion leader sequence;

X2 is a purification tag;

X3 is protease cleavage site;

X4 is a phosphatidylserine binding ligand;

X5 is a peptide linker comprising the amino acid sequence AAALESR; and

X6 is a modified C^-alkylguanine-DNA alkyltransferase; (ii) transfecting a host cell with the expression vector of step (i);

(iii) expressing the recombinant polypeptide precursor by the host cell in a cell culture medium;

(iv) secreting the recombinant polypeptide precursor from the host cell into the supernatant of the cell culture medium using the secretion leader sequence;

(v) recovering the recombinant polypeptide precursor from the supernatant using the purification tag;

(vi) cleaving the recombinant polypeptide precursor with a protease to produce the mature recombinant polypeptide, wherein the mature polypeptide comprises X4 - X5 - X6 of the recombinant polypeptide precursor; and

(vii) autoconjugating an C^-benzylguanine-modified label to X6.

13. The method of claim 12, wherein the phosphatidylserine binding ligand is annexin A5.

14. The recombinant polypeptide precursor of claim 12 or 13, wherein the secretion leader sequence is an IgK leader sequence.

15. The recombinant polypeptide precursor of any one of claims 12 to 14, wherein the purification tag is a 6x histidine tag.

16. The recombinant polypeptide precursor of any one of claims 12 to 15, wherein the protease cleavage site is an enterokinase cleavage site.

17. The method of any one of claims 12 to 16, wherein the modified O6- alkylguanine-DNA alkyltransferase is C^-methylguanine-DNA methyltransferase.

18. The method of any one of claims 12 to 17, wherein the C^-benzylguanine- modified label is selected from the group consisting of Alexa Fluor 488, Alexa Fluor 633, Allophycocyanin (APC), APC-Cy7, Carboxyfluorescein Diacetate (CFSE), Cy3, Cy5, Fluorescein isothiocyanate (FITC), Peridinin chlorophyll protein (PerCP), Phycoerythrin (PE or R-PE), Phycoerythrin-Cy5, Phycoerythrin-Texas Red (PE- Texas Red) and Texas Red.

19. The method of any one of claims 12 to 18, wherein the recombinant polypeptide precursor comprises the sequence of SEQ ID NO:14.

20. The method of any one of claims 12 to 19, wherein the host cell is a HEK293T cell.

21. The method of any one of claims 12 to 20, wherein the recombinant polypeptide precursor is recovered from the supernatant by affinity chromatography.

22. A mature recombinant polypeptide capable of binding to phosphatidylserine, produced according to the method of any one of claims 12 to 21.

23. A kit comprising the mature recombinant polypeptide of claim 22 and instructions for use.

24. An assay for detecting apoptosis in a cell population from a subject, comprising:

(i) contacting the cell population with the mature recombinant polypeptide of claim 22; and

(ii) detecting the binding of the phosphatidylserine binding ligand to phosphatidylserine on the cell surface, wherein binding of the ligand is indicative of apoptosis in the cell population.

Description:
FUSION PROTEINS FOR THE DETECTION OF APOPTOSIS

BACKGROUND OF THE INVENTION

The present invention relates to fusion proteins which are capable of binding to phosphatidylserine, comprising a modified C^-alkylguanine-DNA alkyltransferase which is capable of autoconjugation to an C^-benzylguanine-modified label, the fusion proteins being capable of detecting apoptosis in a cell population. The invention also relates to recombinant polypeptide precursors which comprise a secretion leader sequence, purification tag, protease cleavage site and the fusion protein. Also included in the scope of the invention are nucleic acids encoding the recombinant polypeptide precursor, vectors comprising the nucleic acids, host cells comprising the vectors, methods of production of the fusion proteins, kits and assays for detecting apoptosis.

Apoptosis, also termed programmed cell death, is a natural process used by cells to self-destruct and control aberrant growth. To date, scientists have continued to seek more insight in order to understand the complexity of the cellular events that occur when cells undergo apoptosis. The field of drug discovery has also witnessed development of therapies that seek to eliminate diseased cell populations by selectively driving target cells into apoptosis. In both cases, availability of tools that allow for the specific detection and study of apoptotic cell populations are important.

Annexin A5 (or annexin V) is a cellular protein in the annexin group and is commonly used in flow cytometry to detect apoptotic cells by its ability to bind to phosphatidylserine. Phosphatidylserine acts as a marker of apoptosis when it occurs on the outer surface of the plasma membrane of a cell. Annexin A5 has been proposed to play a role in the inhibition of blood coagulation by competing for phosphatidylserine binding sites with prothrombin and also to inhibit the activity of phospholipase A1 . These properties have been found by in vitro experiments.

The use of Annexin A5 (36-kDa) as a ligand that specifically binds phosphatidylserine residues on the surface of cells undergoing apoptosis remains one of the most popular methods of detection of apoptosis. This is possible because in normal cells phosphatidylserine residues are restricted to the inner layer of the cell membrane where they contribute to intracellular signalling. On the other hand, when cells undergo apoptosis, phosphatidylserine is flipped and exposed to the outer surface of the cell membrane where it plays a role in the recognition of apoptotic cells by phagocytes, such as macrophages and dendritic cells. The presence of phosphatidylserine on the cell surface is a clear indication of apoptosis and the ability of Annexin A5 to bind to phosphatidylserine is useful for the in vitro or ex vivo detection of apoptosis. Because, annexin A5 binds to phosphatidylserine with high affinity; fluorescently labelled Annexin V can be used to detect phosphatidylserine that is exposed on the outside of apoptotic cells.

Previously several types of Annexin A5-fluorescent probes have been developed and are readily available commercially from different vendors. Most of these probes have been optimized for molecular imaging and are delivered in the form of an easy to use kit. Most of the commercially available annexin A5-fluorescent probes are prepared by chemical modification and conjugation strategies, which have several limitations. For example, one method relies on the coupling of fluorophores to annexin A5 via the lysine side chains of the annexin A5 protein using amine-reactive fluorophores. This approach has several drawbacks which includes heterogeneous annexin A5 conjugated products as a result of inability to control the number of bound fluorophores. In addition, in some instances fluorescence quenching may occur as a result of multiple fluorophores binding to annexin A5 in close proximity to each other. A further, drawback of the chemical conjugation approach is that the conjugation of fluorophores to freely accessible lysine residues may occur in the active binding site of the ligand which inhibits the ability of annexin A5 to bind to phosphatidylserine.

Other approaches which have sought to solve the abovementioned shortcomings of chemical conjugation include the genetic fusion of annexin A5 to fluorescent proteins such as eGFP. This approach has resulted in functional proteins being produced but the protein products also suffer from several limitations, including high background noise, irradiation with other dyes and the high cost of production of each different annexin A5-fluorophore colour or wavelength.

The present invention is aimed at overcoming the difficulties experienced with chemically conjugating dyes to annexin A5 and/or using non-specific fluorescent proteins.

SUMMARY OF THE INVENTION

The present invention relates to fusion proteins which are capable of binding to phosphatidylserine comprising a modified C^-alkylguanine-DNA alkyltransferase which is capable of autoconjugation to an C^-benzylguanine-modified label, the fusion proteins being capable of detecting apoptosis in a cell population.

According to a first aspect of the invention there is provided for a recombinant polypeptide precursor, comprising the following formula:

X 1 - X 2 - X 3 - X 4 - X 5 - X6 wherein, is a secretion leader sequence, X 2 is a purification tag, X 3 is a protease cleavage site, X 4 is a phosphatidylserine binding ligand, X 5 is a peptide linker comprising the amino acid sequence AAALESR; and X 6 is a modified O 6 - alkylguanine-DNA alkyltransferase, wherein the enterokinase cleavage site facilitates the cleavage of X 4 - X 5 - X 6 from the recombinant polypeptide precursor to produce a mature recombinant polypeptide capable of binding to phosphatidylserine; and further wherein X 6 is capable of autoconjugation to an C^-benzylguanine-modified label.

In one embodiment of the invention the phosphatidylserine binding ligand may be any amino acid sequence that facilitates binding of the fusion protein to a cell which presents phosphatidylserine on its surface. Preferably, the phosphatidylserine binding ligand is Annexin A5.

In one embodiment of the invention the secretion leader sequence may be an amino acid sequence that facilitates the secretion of a polypeptide of interest from a cell. Those of skill in the art will appreciate that non-limiting examples of typical secretion leader sequences may include mammalian, murine and prokaryotic signal peptide, human placental secretory alkaline phosphatase (SEAP), interleukin-2, CD5, Immunoglobulin Kappa light chain, trypsinogen, serum albumin, prolactin or CD33 leader sequence. Preferably, the secretion leader sequence is an IgK leader sequence.

In another embodiment of the invention the purification tag may be any amino acid sequence that facilitates the purification of a polypeptide of interest. Those of skill in the art will appreciate that non-limiting examples of typical purification tags may include poly-histidine, glutathione S-transferase, maltose binding protein, calmodulin binding peptide, streptavidin or biotin-based tags, HALO tag, FLAG tag thioredoxin-tag, AviTag, Myc-tag, NE-tag, green fluorescent protein-tag, polyglutamate tag etc. Preferably, the purification tag is a poly histidine sequence and most preferably the purification tag is a 6x-His sequence. In yet a further embodiment of the invention the protease cleavage site may be any amino acid sequence that is capable of being cleaved by a proteolytic enzyme. Those of skill in the art will appreciate that non-limiting examples of typical protease enzymes may include an enterokinase, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, factor Xa, granzyme B, HRV3C protease, TEV protease and thrombin. Preferably, the protease cleavage site is an enterokinase cleavage site.

In a preferred embodiment of the invention the C^-alkylguanine-DNA alkyltransferase is an C^-methylguanine-DNA methyltransferase.ln yet a further embodiment of the invention the C^-benzylguanine-modified label is selected from the group consisting of 0 ® -benzylguanine modified with an label such as Alexa Fluor 488, Alexa Fluor 633, Allophycocyanin (APC), APC-Cy7, Carboxyfluorescein Diacetate (CFSE), Cy3, Cy5, Fluorescein isothiocyanate (FITC), Peridinin chlorophyll protein (PerCP), Phycoerythrin (PE or R-PE), Phycoerythrin-Cy5, Phycoerythrin- Texas Red (PE-Texas Red) and Texas Red. Those of skill in the art will however appreciate that this is a non-exhaustive list of possible labels.

In a most preferred embodiment of the invention the recombinant polypeptide precursor comprises a sequence of SEQ ID NO:14.

According to a second aspect of the invention provides for nucleic acid molecules encoding the recombinant polypeptide precursor of the invention. In some embodiments, the nucleic acid molecules of the invention may be operably linked to regulatory sequences in such a way as to permit gene expression thereof when the appropriate molecules are bound to the regulatory sequences. Such operably linked sequences may be in the form of vectors or expression constructs that can be transformed or transfected into host cells for expression. It will be appreciated by those of skill in the art that any suitable vector can be used for this purpose. Further it will be appreciated that any suitable expression system may be used, expression systems include host cells transformed with the expression vectors and may include bacterial cells, yeast cells, insect cells, plant cells and/or mammalian cells.

In a preferred embodiment of this aspect of the invention the nucleic acid molecule encodes a recombinant polypeptide precursor comprising a sequence of SEQ ID NO:14.

It will also be appreciated that expression vectors which comprise the nucleic acid molecules encoding the recombinant polypeptide precursors and expression cassettes comprising the nucleic acid molecules encoding the recombinant polypeptide precursors also fall within the scope of an embodiment of the invention.

In a third aspect of the invention there is provided for a method for producing a mature recombinant polypeptide capable of binding to phosphatidylserine, the method comprising the steps of:

(i) providing an expression vector comprising a nucleic acid molecule encoding a recombinant polypeptide precursor having the formula X 1 - X 2 - X 3 - X 4 - X 5 - X 6 , wherein X 1 is a secretion leader sequence, X 2 is a purification tag, X 3 is protease cleavage site, X 4 is a phosphatidylserine binding ligand, X 5 is a peptide linker comprising the amino acid sequence AAALESR; and X 6 is a modified O 6 - alkylguanine-DNA alkyltransferase;

(ii) transfecting a host cell with the expression vector of step (i);

(iii) expressing the recombinant polypeptide precursor by the host cell in a cell culture medium;

(iv) secreting the recombinant polypeptide precursor from the host cell into the supernatant of the cell culture medium using the secretion leader sequence;

(v) recovering the recombinant polypeptide precursor from the supernatant using the purification tag;

(vi) cleaving the recombinant polypeptide precursor with a protease to produce the mature recombinant polypeptide, wherein the mature polypeptide comprises X 4 - X 5 - X 6 of the recombinant polypeptide precursor; and

(vii) autoconjugating an C^-benzylguanine-modified label to X 6 .

In a preferred embodiment of the invention the invention the host cell is a HEK293T cell.

In one embodiment of the invention the recombinant polypeptide precursor is recovered from the supernatant by affinity chromatography.

In a fourth aspect of the invention there is provided for a mature recombinant polypeptide capable of binding to phosphatidylserine, produced according to the methods set out herein.

A fifth embodiment of the invention provides for a kit comprising the mature recombinant polypeptide of the invention and instructions for its use.

In a sixth embodiment of the invention there is provided for an assay for detecting apoptosis in a cell population from a subject, the assay comprising the steps of: (i) contacting the cell population with the mature recombinant polypeptide of the invention; and

(ii) detecting the binding of the phosphatidylserine binding ligand to phosphatidylserine on the cell surface, wherein binding of the ligand is indicative of apoptosis in the cell population. It will be appreciated by those of skill in the art that the step of detection of binding of the mature recombinant polypeptide to phosphatidylserine in the cell population is performed by detecting the presence of the C^-benzylguanine-modified label

BRIEF DESCRIPTION OF THE FIGURES

Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

Figure 1 : Plasmid map of constructed Annexin A5-SNAP pMS Vector.

The eGFP protein is expressed as a reporter protein under the control of an IRES sequence.

Figure 2: Amino acid sequence of a first generation construct of the invention (SEQ ID NO:1 ) comprising IgKappa, poly-His tag, enterokinase cleavage site, Annexin A V and SNAP-tag. IgK is a leader sequence helping this protein to be excreted from the producing cells, poly-His tag allows for purification of the protein via immobilized metal affinity chromatography, the enterokinase cleavage site allows removal of the N-terminal tag.

Figure 3: Flow cytometry analysis of selective elimination of M1 - polarized macrophages in vitro.

Figure 4: Nucleic acid sequence encoding the first generation recombinant polypeptide precursor (SEQ ID NO:2).

Figure 5: Amino acid sequence of the mature recombinant polypeptide

(SEQ ID NO:3).

Figure 6: Nucleic acid sequence of the annexin A5 ORF modified with

Sfil and Notl restriction sites (SEQ ID NO:4).

Figure 7: Amino acid sequence of the annexin A5 protein (SEQ ID

NO:5).

Figure 8: Nucleic acid sequence of the SNAP-tag (SEQ ID NO:6).

Figure 9: Amino acid sequence of the SNAP-tag (SEQ ID NO:7). Figure 10: Amino acid sequence of the construct of the invention (SEQ ID NO:14) comprising IgKappa, poly-His tag, enterokinase cleavage site, Annexin A V and SNAP-tag. IgK is a leader sequence helping this protein to be excreted from the producing cells, poly-His tag allows for purification of the protein via immobilized metal affinity chromatography, the enterokinase cleavage site allows removal of the N-terminal tag.

Figure 11 : Nucleic acid sequence encoding the recombinant polypeptide precursor (SEQ ID NO:15).

SEQUENCE LISTING

The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and the standard three letter abbreviations for amino acids. It will be understood by those of skill in the art that only one strand of each nucleic acid sequence is shown, but that the complementary strand is included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO:1 - Amino acid sequence of a first generation construct of the recombinant polypeptide precursor comprising Ig-Kappa, poly-His tag, enterokinase cleavage site, Annexin A V and SNAP-tag fusion protein.

SEQ ID NO:2 - Nucleic acid sequence encoding the first generation construct of the recombinant polypeptide precursor comprising Ig-Kappa, 6x His-tag, enterokinase cleavage site, Annexin A V and SNAP-tag construct.

SEQ ID NO:3 - Amino acid sequence of the mature first generation recombinant polypeptide comprising the Annexin A V - SNAP-tag cleavage product.

SEQ ID NO:4 - Nucleic acid sequence of the annexin A5 ORF modified with Sfi\ and Not\ restriction sites.

SEQ ID NO:5 - Amino acid sequence of the annexin A5 protein.

SEQ ID NO:6 - Nucleic acid sequence of the SNAP-tag.

SEQ ID NO:7 - Amino acid sequence of the SNAP-tag.

SEQ ID NO:8 - Nucleic acid sequence of the Ig-Kappa leader sequence.

SEQ ID NO:9 - Amino acid sequence of the Ig-Kappa leader sequence.

SEQ ID NO:10 - Nucleic acid sequence of the 6xHis tag.

SEQ ID NO:1 1 - Amino acid sequence of the 6xHis tag.

SEQ ID NO:12 - Nucleic acid sequence of the enterokinase cleavage site.

SEQ ID NO:13 - Amino acid sequence of the enterokinase cleavage site. SEQ ID NO:14 - Amino acid sequence of the second generation recombinant polypeptide precursor comprising Ig-Kappa, poly-His tag, enterokinase cleavage site, Annexin A V, AAASLR peptide linker and SNAP-tag fusion protein.

SEQ ID NO:15 - Nucleic acid sequence encoding the second generation recombinant polypeptide precursor comprising Ig-Kappa, 6x His-tag, enterokinase cleavage site, Annexin A V, AAASLR peptide linker and SNAP-tag construct.

SEQ ID NO:16 - Amino acid sequence of the peptide linker.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

As used throughout this specification and in the claims which follow, the singular forms“a”,“an” and“the” include the plural form, unless the context clearly indicates otherwise.

The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms “comprising”, “containing”, “having” and “including” and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Once a cell commences with the process of apoptosis it exposes phosphatidylserine (PS) on its surface. Annexin A5 is a ligand which specifically binds to phosphatidylserine on the surface of a cell. The present inventors have developed a SNAP tag based fusion protein for the selective detection of phosphatidylserine on the surface of a cell. The present invention has a distinct advantage in comparison to the previously described state of the art annexin A5 detection molecules.

The present invention has several major differences between the previously described chemically modified annexin A5-fluorescent probes and/or annexin A5- eGFP proteins, these include: 1 . The use of a SNAP-tag as a labelling tag which allows for simple site- specific conjugation of any BG-modified fluorophore and a 1 :1 stoichiometric conjugation ratio of the BG-modified fluorophore to annexin A5. The ability of being able to conjugate any BG-modified fluorophore to the fusion protein of the invention will result in a substantially lower cost for the preparation of a panel of annexin A5 detection fluorochromes, together with the ability to produce reproducible results.

2. The covalent SNAP-tag labelling reaction is an autoconjugation event which is occurs under physiological conditions and which does not require enzymatic attachment or acidic or alkaline media to facilitate attachment of the fluorophore which can be deleterious to the functional properties of either the protein or fluorophore.

3. The currently commercially available annexin A5-fluorophore products are not recommended for use in the detection of apoptotic adherent cells. The present inventors have however shown that optimal results are obtained for the use of Annexin A5-SNAP conjugated to different fluorophores including Alexa 488, Alexa 449, and Alexa 649 in the detection of apoptotic macrophages. This shows the versatility of the annexin A5-SNAP protein of the invention as tool for the detection of apoptosis across all cell types.

The present invention relates to the development of an Annexin A5-SNAP fusion protein with robust function in targeting phosphatidylserine on the surface of cells undergoing apoptosis. The inventors have demonstrated the use and ability of annexin A5-SNAP coupled to a detectable label to monitor induction of apoptosis on cells by identifying the selective killing of M1 macrophages by Pseudomonas exotoxin A-based recombinant immunotoxins and Angiogenin based human cytolytic fusion proteins.

These detectable labels may include one of the following SNAP-Cell® Oregon Green®, SNAP-Ce!i® TMR-Star. SNAP-Cell® 430, SNAP-Cei® 647-SIR, SNAP- Surface® 488, SNAP-Surface® 549, SNAP-Surface® 594, SNAP-Surface® 649, SNAP-Surface® Alexa Fluor® 488, SNAP-Surface® Alexa Fluor® 546, SNAP- Surface® Alexa Fluor® 647, SNAP- Vista® Green.

Preferably the detectable label may be an C^-benzylguanine-modified label selected from the group consisting of Alexa Fluor 488, Alexa Fluor 633, Allophycocyanin (APC), APC-Cy7, Carboxyfluorescein Diacetate (CFSE), Cy3, Cy5, Fluorescein isothiocyanate (FITC), Peridinin chlorophyll protein (PerCP), Phycoerythrin (PE or R-PE), Phycoerythrin-Cy5, Phycoerythrin-Texas Red (PE- Texas Red, also written Texas Red-PE) and Texas Red.

The present invention specifically relates to a phosphatidylserine-binding ligand, preferably annexin A5 fusion protein recombinantly fused to a SNAP tag and separated by an amino acid linker. In a preferably embodiment the fusion protein comprises an IgK leader sequence, a poly-His tag purification tag, and a protease cleavage site, preferably the protease cleavage site is an enterokinase cleavage site (SEQ ID NO:14), hereinafter referred to as the fusion protein of the invention.

A“protein,”“peptide” or“polypeptide” is any chain of two or more amino acids, including naturally occurring or non-naturally occurring amino acids or amino acid analogues, irrespective of post-translational modification (e.g., glycosylation or phosphorylation).

The terms “nucleic acid” or “nucleic acid molecule” encompass both ribonucelotides (RNA) and deoxyribonucleotides (DNA), including cDNA, genomic DNA, and synthetic DNA. The nucleic acid may be double-stranded or single- stranded. Where the nucleic acid is single-stranded, the nucleic acid may be the sense strand or the antisense strand. A nucleic acid molecule may be any chain of two or more covalently bonded nucleotides, including naturally occurring or non- naturally occurring nucleotides, or nucleotide analogs or derivatives. By“RNA” is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides. The term “DNA” refers to a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides. By“cDNA” is meant a complementary or copy DNA produced from an RNA template by the action of RNA- dependent DNA polymerase (reverse transcriptase).

Accordingly, a “cDNA clone” refers to a duplex DNA sequence which is complementary to an RNA molecule of interest, and which is carried in a cloning vector. The term“complementary” refers to two nucleic acids molecules, e.g., DNA or RNA, which are capable of forming Watson-Crick base pairs to produce a region of double-strandedness between the two nucleic acid molecules. It will be appreciated by those of skill in the art that each nucleotide in a nucleic acid molecule need not form a matched Watson-Crick base pair with a nucleotide in an opposing complementary strand to form a duplex. One nucleic acid molecule is thus “complementary” to a second nucleic acid molecule if it hybridizes, under conditions of high stringency, with the second nucleic acid molecule. A nucleic acid molecule according to the invention includes both complementary molecules. In some embodiments, a fusion protein of the invention may include, without limitation, a fusion polypeptide including an amino acid sequence substantially identical to the amino acid sequence a fusion protein comprising a secretion leader sequence, a purification tag, a protease cleavage site, a phosphatidylserine-binding ligand and a SNAP tag or a derivative thereof. In a preferred embodiment of the invention the fusion protein comprises a secretion leader sequence which is an IgK or a derivative thereof, the purification tag is a poly-His tag, the protease cleavage site is an enterokinase cleavage site or a derivative thereof, the phosphatidylserine- binding ligand is annexin A5 or a derivative thereof and a SNAP tag or a derivative thereof. Preferably, the fusion protein of the invention comprises SEQ ID NO:14. Another embodiment of the invention includes, without limitation, nucleic acid molecules encoding the aforementioned fusion protein. A further embodiment of the invention comprises a derivative of the fusion protein of the invention comprising annexin A5 fused to a SNAP-tag after the IgK leader sequence which facilitates excretion of the fusion protein from the cells in which it is produced, the 6xHis tag which facilitates purification of the fusion protein via immobilized metal affinity chromatography and the enterokinase cleavage site which allows removal of the N- terminal tag have been cleaved from each other at the enterokinase cleavage site.

The term“isolated”, is used herein and means having been removed from its natural environment.

The term “poly-His tag”, as used herein refers to a linear sequence of histidine residues allowing for the purification of a recombinant protein by metal chelate affinity chromatography. Alternatively, a poly-His tag may be used to detect a recombinant polypeptide using an anti-poly-His tag antibody. A poly-His tag may comprise 6, 7, 8, 9 or 10 consecutive Histidine residues.

The term“purified”, relates to the isolation of a molecule or compound in a form that is substantially free of contamination or contaminants. Contaminants are normally associated with the molecule or compound in a natural environment, purified thus means having an increase in purity as a result of being separated from the other components of an original composition. The term "purified nucleic acid" describes a nucleic acid sequence that has been separated from other compounds including, but not limited to polypeptides, lipids and carbohydrates which it is ordinarily associated with in its natural state.

The term“complementary” refers to two nucleic acid molecules, e.g., DNA or RNA, which are capable of forming Watson-Crick base pairs to produce a region of double-strandedness between the two nucleic acid molecules. It will be appreciated by those of skill in the art that each nucleotide in a nucleic acid molecule need not form a matched Watson-Crick base pair with a nucleotide in an opposing complementary strand to form a duplex. One nucleic acid molecule is thus “complementary” to a second nucleic acid molecule if it hybridizes, under conditions of high stringency, with the second nucleic acid molecule. A nucleic acid molecule according to the invention includes both complementary molecules.

As used herein a “substantially identical” sequence is an amino acid or nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, or by one or more non-conservative substitutions, deletions, or insertions located at positions of the sequence that do not destroy or substantially reduce the antigenicity of the expressed fusion protein or of the polypeptide encoded by the nucleic acid molecule. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the knowledge of those with skill in the art. These include using, for instance, computer software such as ALIGN, Megalign (DNASTAR), CLUSTALW or BLAST software. Those skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In one embodiment of the invention there is provided for a polypeptide or polynucleotide sequence that has at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the sequences described herein.

Alternatively, or additionally, two nucleic acid sequences may be “substantially identical” if they hybridize under high stringency conditions. The “stringency" of a hybridisation reaction is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation which depends upon probe length, washing temperature, and salt concentration. In general, longer probes required higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridisation generally depends on the ability of denatured DNA to re anneal when complementary strands are present in an environment below their melting temperature. A typical example of such“stringent” hybridisation conditions would be hybridisation carried out for 18 hours at 65 °C with gentle shaking, a first wash for 12 min at 65 °C in Wash Buffer A (0.5% SDS; 2XSSC), and a second wash for 10 min at 65 °C in Wash Buffer B (0.1% SDS; 0.5% SSC). In an alternative embodiment of the invention, the fusion proteins of the invention may be prepared by, for instance, inserting, deleting or replacing amino acid residues at any position of the polypeptide sequences and/or, for instance inserting, deleting or replacing nucleic acids at any position of the nucleic acid molecule encoding the fusion protein of the invention.

Those skilled in the art will appreciate that polypeptides, peptides or peptide analogues can be synthesised using standard chemical techniques, for instance, by automated synthesis using solution or solid phase synthesis methodology. Automated peptide synthesisers are commercially available and use techniques known in the art. Polypeptides, peptides and peptide analogues can also be prepared from their corresponding nucleic acid molecules using recombinant DNA technology.

In some embodiments, the nucleic acid molecules of the invention may be operably linked to other sequences. By“operably linked” is meant that the nucleic acid molecules encoding the fusion proteins of the invention and regulatory sequences are connected in such a way as to permit expression of the fusion proteins when the appropriate molecules are bound to the regulatory sequences. Such operably linked sequences may be contained in vectors or expression constructs which can be transformed or transfected into host cells for expression. It will be appreciated that any vector can be used for the purposes of expressing the fusion proteins of the invention.

As used herein, the term “gene” refers to a nucleic acid that encodes a functional product, for instance a RNA, polypeptide or protein. A gene may include regulatory sequences upstream or downstream of the sequence encoding the functional product.

As used herein, the term“coding sequence” refers to a nucleic acid sequence that encodes a specific amino acid sequence. On the other hand a “regulatory sequence” refers to a nucleotide sequence located either upstream, downstream or within a coding sequence. Generally regulatory sequences influence the transcription, RNA processing or stability, or translation of an associated coding sequence. Regulatory sequences include but are not limited to: effector binding sites, enhancers, introns, polyadenylation recognition sequences, promoters, RNA processing sites, stem-loop structures, translation leader sequences and the like.

The term“promoter” refers to a DNA sequence that is capable of controlling the expression of a nucleic acid coding sequence or functional RNA. A promoter may be based entirely on a native gene or it may be comprised of different elements from different promoters found in nature or a promoter could be an entirely synthetic construct. Different promoters are capable of directing the expression of a gene in different cell types, or at different stages of development, or in response to different environmental or physiological conditions. A“constitutive promoter” is a promoter that directs the expression of a gene of interest in most host cell types most of the time.

The term“recombinant” means that something has been recombined. When used with reference to a nucleic acid construct the term refers to a molecule that comprises nucleic acid sequences that are joined together or produced by means of molecular biological techniques. The term“recombinant” when used in reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed from a recombinant nucleic acid construct created by means of molecular biological techniques. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Accordingly, a recombinant nucleic acid construct indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention. Recombinant nucleic acid constructs may be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species.

As used herein, the term“chimaeric”, means that a sequence comprises of sequences that have been “recombined”. By way of example sequences are recombined and are not found together in nature. The term “recombine” or “recombination” refers to any method of joining two or more polynucleotides. The term includes end to end joining, and insertion of one sequence into another. The term is intended to include physical joining techniques, for instance, sticky-end ligation, blunt-end ligation, as well as PCR-mediated fusion by overlap extension PCR. Sequences may also be artificially synthesized to contain a recombined sequence. The term may also encompass the integration of one sequence into a second sequence by way of, for example, homologous recombination.

The term “vector” refers to a means by which polynucleotides or gene sequences can be introduced into a cell. There are various types of vectors known in the art including plasmids, viruses, bacteriophages and cosmids. Generally polynucleotides or gene sequences are introduced into a vector by means of a cassette. The term“cassette” refers to a polynucleotide or gene sequence that is expressed from a vector, for example, the polynucleotide or gene sequences encoding the fusion proteins of the invention. A cassette generally comprises a gene sequence inserted into a vector, which in some embodiments, provides regulatory sequences for expressing the polynucleotide or gene sequences. In other embodiments, the vector provides the regulatory sequences for the expression of the fusion protein. In further embodiments, the vector provides some regulatory sequences and the nucleotide or gene sequence provides other regulatory sequences. “Regulatory sequences” include but are not limited to promoters, transcription termination sequences, enhancers, splice acceptors, donor sequences, introns, ribosome binding sequences, poly(A) addition sequences, and/or origins of replication.

In some embodiments, the fusion proteins or compositions according to the invention may be provided in a kit, together with instructions for use.

A major advantage of the present invention is the autoconjugation of any label under physiological conditions needed for specific detection of an interaction of the fusion protein of the invention with apoptotic cells. The biology can be adjusted and is not restricted to a fluorophore of a precast wavelength being attached to annexin A5.

The following example is offered by way of illustration and not by way of limitation.

EXAMPLE 1

Generation of Annexin A5-SNAP

The open reading frame (ORF) for annexin A5 (SEQ ID NO:5) was modified with 5'-S//l and 3'-Not\ restriction sites in-silico and synthesized by Genescript (NJ, USA) (SEQ ID NO:4). The synthesized ORF was ligated into a S/zl/A/ofl-linearized pMS vector already containing the SNAP-tag sequence (Figure 1 ) (SEQ ID NO:6). An Ig-Kappa leader sequence ((METDTLLLWVLLLWVPGSTGD (SEQ ID NO:9) encoded by the nucleic acid sequence ATGGAGACAG ACACACTCCT GCTATGGGTA CTGCTGCTCT GGGTTCCAGG TTCCACTGGT GAC (SEQ ID NO:8)) was introduced at the N-terminal of the fusion construct to allow secretion of the protein into the culture media. A 6x Histidine tag (HHHHHH (SEQ ID NO:1 1 ) encoded by the nucleic acid sequence CATCATCATC ATCATCAT (SEQ ID NO:10)) was also introduced to allow purification of the protein by immobilized metal ion affinity chromatography. An enterokinase cleavage site (DDDDK (SEQ ID NO:13) encoded by the nucleic acid sequence GACGACGACG ACAAG (SEQ ID NO:12)) was also introduced to allow separation of Annexin A5-SNAP from its N-terminal tags (SEQ ID NO:12) and a peptide linker (AAALESR (SEQ ID NO:16) was introduced in order to functionalise the mature recombinant polypeptide. Successful cloning was confirmed by sequencing.

Expression and purification of Annexin A5-SNAP

Once molecular cloning was confirmed by sequencing, the pMS expression vector was used for the transfection of HEK293T cells (ATCC, Wesel, Germany, CRL-1 1268) using X-tremeGENE™ HP DNA Transfection Reagent (Roche, Germany) according to the manufacturer’s instructions. For selection of successfully transfected cells, the culture medium was supplemented with Zeocin (Invitrogen, Carlsbad) 100 ng/ml_. The recombinant proteins were purified from the cell culture supernatant by immobilized metal ion affinity chromatography using a 5ml_ Ni 2+ -NTA Superflow cartridge on an AKTA purifier system). Elution fractions were pooled and concentrated using 10 kDa Amicon Ultracentrifugation filters (Millipore, Germany). Protein concentration was estimated by nanodrop reading and structural integrity confirmed by SDS-PAGE and Coomassie Brilliant Blue staining. The inventors found that a first generation fusion protein comprising a linker sequence of AAASR only resulted in minor binding in the supernatant of the fusion protein. Additionally, it was found that the first generation fusion protein did not bind to the eluate. As a result the first generation fusion protein could not be used in a functional apoptosis assay. On the other hand a second generation fusion protein comprising a linker sequence of AAALESR (SEQ ID NO:16) worked surprisingly well and provided a mature fusion protein that could be used in functional apoptosis assays.

Labelling of Annexin A5-SNAP with BG-modified fluorophore

The labelling of purified annexin A5-SNAP with BG-Alexa 488 (Cat no: S9129S; New England Biolabs; NEB, Ipswich, MA, USA) was carried out using 1.5- fold molar excess of dye to protein. Coupling was performed for 30 mins at 37 °C. Successful coupling and fluorescence of labelled proteins was confirmed by SDS- PAGE and detection with a dark reader blue light transilluminator (Clare Chemical, Colorado, United States, MA, USA).

Preparation of and treatment of ex vivo differentiated macrophages with RIT and hCFPs Human PBMCs were isolated from buffy coats by gradient centrifugation with lymphroprep (Alere Technologies, Norway) and cultured for 3 hours at a density of 10 x 10 6 cells/well in serum free media to select for monocytes by adherence. After which each well was subsequently washed with 1 ml of PBS (Sigma-Aldrich, Germany) to remove non-adherent cells. Polarization was carried out for 72 hours using 100 U/ml human IFN-g (Sigma-Aldrich, Germany) and 1 pg/ml LPS (Sigma- Aldrich, Germany) for M1 and 20 ng/ml human IL-4 (Peprotech, Germany) for M2 macrophages. After 72 hours, the polarization was boosted with 50 U/ml human IFN- Y (Sigma-Aldrich, Germany) and 0.5 pg/ml LPS (Sigma-Aldrich, Germany) for M1 and 10 ng/ml human IL-4 (Peprotech, Germany) for M2 macrophage for additional 24 hours, after which macrophages were used for functional assays. The selective killing of M1 macrophages was demonstrated by treating cells with 200 nM concentration of H22(SCFV)-ANG GG RR and 100 nM OF H22(scFv)-ETA for 24 hours. Negative control cells were treated with 30 mL PBS.

Apoptosis assays

After 24 hours of treatment, selective induction of apoptosis in M1 IFN-Y/LPS polarized macrophages was measured by annexin A5/7-AAD staining. Briefly, cells were washed with 1 ML of PBS and lifted by incubating in 500 pi of Accutase (Sigma, Germany) for 10 mins. The cells were washed once with PBS and again with 1x annexin A5 binding buffer containing 10 mM HEPES, 140 mM NaCI, and 2.5 mM CaCI 2 (pH 7.4). Afterwards, cells were resuspended in 200 pi of 1x annexin A5 binding buffer and stained with 1.5 pg of annexin A5-SNAP-BG-Alexa 488 protein for 30 minutes. Cells were then washed with 1 ml 1 x binding buffer and resuspended in 400 pi of 1 x binding buffer including 7-AAD (1/1000 dilution).

Use of Annexin A5-SNAP-Alexa-488 for the measurement of apoptosis induction in M1 and M2 macrophages

Specific and selective induction of apoptosis by anti-CD64 based RIT and hCFP was analysed by flow cytometry after staining of phosphatidylserine residues on the surface of apoptotic cells with annexin V5-SNAP-Alexa 488. As depicted in Figure 3, treatment with H22(scFv)-ETA and H22-ANG(SCFV) GGRR shows selectively killing of M1 polarized macrophages after 24 hours.