阅读说明：本技术 包含电荷修饰的珠蛋白的抗肿瘤的细胞 (Antitumor cells comprising charge-modified globin ) 是由 亚当·威廉斯·佩里曼 本杰明·迈克尔·卡特 托马斯·莱恩·菲利普·格林 大卫·科埃 W·H· 于 2020-03-06 设计创作，主要内容包括：本发明提供一种抗肿瘤的细胞、脂质体或微团,其包含至少一种与所述细胞、脂质体或微团的膜相关联的电荷修饰的珠蛋白,以及其制备和使用的方法。(The present invention provides an anti-tumor cell, liposome or micelle comprising at least one charge-modified globin associated with the membrane of said cell, liposome or micelle, and methods of making and using the same.)

1. An anti-tumor cell, liposome or micelle comprising at least one charge-modified globin associated with a membrane of said cell, liposome or micelle.

2. The cell according to claim 1, wherein the cell is an immune cell, preferably a tumor infiltrating immune cell, more preferably a lymphocyte, a neutrophil, a dendritic cell or a macrophage.

3. The cell of claim 1 or 2, wherein the cell is a cytotoxic T cell, a natural killer T cell, or a natural killer cell.

4. The cell of any preceding claim, wherein the cell is a T cell.

5. The liposome or micelle of claim 1, wherein the liposome or micelle comprises a therapeutic agent; preferably wherein the therapeutic agent is a checkpoint inhibitor, an immunotherapeutic agent or a chemotherapeutic agent.

6. The cell, liposome or micelle of any preceding claim, wherein the globin is hemoglobin, myoglobin, neuroglobin or cytoglobin; myoglobin is preferred.

7. The cell, liposome or micelle of any preceding claim, wherein the globin is attached to a secondary anti-tumor molecule or a reactive functional group for attachment to a secondary anti-tumor molecule;

preferably wherein the secondary anti-tumour molecule is any one of an antibody, a lectin, an integrin or an adhesion molecule;

and/or, preferably, wherein said secondary anti-tumor molecule is: (1) a tumor cell binding molecule; (2) (ii) a checkpoint inhibitor; (3) enzymes that recombine tumor extracellular matrix; or (4) an enzyme that metabolizes the tumor-associated compound.

8. The cell, liposome, or micelle of claim 7, which contains a fusion protein comprising the globin and the secondary anti-cancer molecule.

9. The cell, liposome, or micelle of any preceding claim, wherein the globin is a cationized globin or an anionized globin.

10. The cell, liposome, or micelle of any preceding claim wherein the globin comprises a polymeric surfactant coating.

11. A pharmaceutical composition comprising the anti-tumor cell, liposome or micelle of any preceding claim, further comprising a pharmaceutically acceptable carrier, diluent or vehicle.

12. A cell, liposome or micelle according to any of claims 1-10, or a pharmaceutical composition according to claim 11, for use in the treatment of cancer.

13. A method of preparing the antitumor cell, liposome or micelle of any of claims 1-10, comprising:

a) providing a charge-modified globin; and

b) contacting said anti-tumor cell, liposome or micelle with said globin.

14. The method of claim 13, wherein step (a) comprises providing a charge-modified globin and a polymeric surfactant under conditions that enable electrostatic coupling of said polymeric surfactant to said globin.

15. The method of claim 13 or 14, wherein the globin is converted to charge-modified globin by a method comprising the steps of:

i) mixing the globin solution with a pH neutralized solution of N, N' -dimethyl-1,3-propanediamine (DMPA) or an analog thereof, and optionally adjusting the pH of the mixture to 5-7;

ii) subsequently or simultaneously adding a carbodiimide, such as N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC), and adjusting the pH of the mixture to a pH of 4-7;

iii) stirring said mixture in (ii) at a temperature of 0-25 ℃ and a pH of 4-7 for 1-30 hours;

iv) separating the proteins in the mixture of (iii) from water or buffer at a pH of 6.5 to 8.5 for at least 4 hours;

v) if necessary, adjusting the pH of the mixture in (iv) to 6.5-8.5.

16. The method of claim 13 or 14, wherein said charge-modified globin protein is obtained by a method comprising expressing a recombinant DNA sequence encoding said charge-modified globin protein.

17. A method of treating cancer comprising administering the cell, liposome or micelle of any of claims 1-10, or the pharmaceutical composition of claim 11 to a patient in need thereof.

18. The use of claim 12, or the method of claim 17, wherein the cancer is a solid tumor cancer.

19. The use of claim 12 or 18, or the method of claim 17 or 18, wherein the cancer is selected from: breast cancer, colorectal cancer, prostate cancer, lung cancer, stomach cancer, liver cancer, esophageal cancer, cervical cancer or pancreatic cancer.

20. A polypeptide comprising any one of the charge-modified globin sequences shown in SEQ ID NOs 1-14 or any one functional variant having at least about 60% sequence identity to a non-variant globin sequence.

Technical Field

The present invention relates to anti-tumor cells, liposomes and micelles comprising a membrane associated with said cells, liposomes or micelles, which enhance the anti-tumor properties of the cells, liposomes or micelles. The invention also provides a preparation method and a using method.

Background

Many anti-tumor therapies involve the use of cells, liposomes and micelles. Recent examples include treatment with engineered immune cells, such as CAR-T cells, which are cytotoxic T cells that are genetically engineered to express a Chimeric Antigen Receptor (CAR) with tumor specificity. The CAR binds T cells to tumor cells, which can then kill the tumor cells. Liposomes and micelles can also be used for therapy, which bind to tumors and deliver anti-tumor compositions in a manner similar to cytotoxic T cells.

However, these attempts have been frustrated by the ability of tumors to evade the immune system. The solid tumor microenvironment has a high degree of immunosuppressive effects: tumor cells express checkpoint inhibitors and recruit suppressive cell populations, and local hypoxia results in a cascade of immunosuppressive gene expression. As a result of the above-mentioned effects, tumor-killing cells such as cytotoxic T cells are "inactivated", i.e., they lose their ability to recognize and kill tumor cells.

Current approaches to overcoming this immunosuppression include systemic administration of antibodies to block proteins that inactivate cytotoxic T cells (e.g., PD-1, PD-L1, and CTLA4), depletion of immunosuppressive regulatory T cells (an immunosuppressive T cell), and use of oxygen-enriched gas or erythropoietin to increase oxygen levels. It has also been reported that administration of globin reduces hypoxia in cancer cells and so it can be attempted to enhance the effectiveness of chemotherapeutic drugs (e.g. as described in US 2015/0374796).

The present invention provides anti-tumor cells, liposomes and micelles, wherein the anti-tumor activity of the cells, liposomes and micelles is enhanced, in particular by reducing the ability of a tumor to evade the anti-tumor effect of the anti-tumor cells, liposomes and micelles, and reducing the ability of a tumor to evade immune responses.

Summary of The Invention

According to a first aspect, the present invention provides an anti-neoplastic cell, an anti-neoplastic liposome or an anti-neoplastic micelle comprising at least one charge-modified globin associated with the membrane of said cell, liposome or micelle.

The inventors have identified an improved therapeutic composition for the treatment of tumours, in particular for solid tumours. Surprisingly, the inventors have found that charge-modified globin proteins can be successfully associated with the membranes of cells, liposomes and micelles, said globin proteins, although charge-modified and associated with cells, liposomes or micelles, successfully retain their oxygen transport and delivery functions. Furthermore, the inventors have found that such binding can be achieved without losing key properties of the cells, liposomes or micelles, such as stability, viability and activity. In particular, experiments with T cells have shown that their viability, proliferation and key activities are not lost. An important aspect of binding charge-modified globin to the membrane of an anti-tumor cell, liposome or micelle is that the charge-modified globin can provide multiple beneficial effects to the cell, liposome or micelle and tumor cell simultaneously. For example, as discussed in detail below, the charge-modified globin can modulate and/or enhance the activity of anti-tumor cells on hypoxic solid tumor cells. In addition, the charge-modified globin can also reduce hypoxia in hypoxic solid tumors, thereby alleviating the effects of hypoxic conditions. For example, reducing hypoxia may sensitize tumor cells to the effects of anti-tumor cells and/or chemotherapy, possibly delivered by liposomes or micelles. Thus, the cells, liposomes or micelles of the invention have the inherent ability to overcome the ability of a tumor to evade the anti-tumor effects of the cells, liposomes or micelles, as well as the ability to reduce the tumor's ability to evade any immune response that occurs simultaneously with the effects of the anti-tumor cells, liposomes or micelles of the invention. The association of the charge-modified bead protein with the membrane of the cell, liposome or micelle means that the local concentration of the globin in the region of the cell, liposome or micelle can only be matched in a non-binding system by systemically administering a large excess of globin. This also applies to the local concentration of globin in the area of hypoxic solid tumors, when the cells, liposomes or micelles are targeted to hypoxic solid tumors. Furthermore, by conjugating the charge-modified globin to an anti-tumor cell, liposome or micelle, the anti-tumor effect of the cell, liposome or micelle on a solid tumor inherently occurs while the globin acts on the solid tumor. Furthermore, when an anti-tumor cell, liposome or micelle binds to and acts on a specific moiety within a solid tumor, the protein of the relevant charge-modified bead itself acts on the same moiety within the solid tumor.

Globin is charge modified. This means that the net surface charge of the globin is modified relative to the native (or "unmodified" or "wild-type") globin. In other words, at least one residue that is negatively charged or neutral in the native protein has been modified to be positively charged, or at least one residue that is positively charged or neutral in the native protein has been modified to be negatively charged. "charged residues" are understood to include inherently charged residues (e.g., the proteinogenic amino acids glutamic acid, aspartic acid, arginine, lysine and histidine) as well as charged residues modified to be inserted into at least one functional group with one or more charges.

Typically, the charge is assessed at physiological pH, e.g., pH of about 6-9, e.g., pH of about 6, 6.5, 7, 7.5, 8, 8.5, or about 9.

Charge modification of globin can be achieved in various ways. For example, native globin can be provided and then chemically modified to change the charge state of one or more residues. Alternatively, the charge-modified globin can be generated by recombinant expression of a sequence encoding the charge-modified globin. Charge modified globin can also be produced by a combination of expression and chemical modification of charge modified globin.

As described above, the charge-modified globin proteins have one or more charge modifications relative to the native protein. For example, the number of positively charged residue or residues modified to carry may be 1 to 100, e.g., 1 to 80, 10 to 70, 20 to 60, or 30 to 50, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 51, 52, 53, 54, or 55. Additionally, or alternatively, the number of one or more negatively charged residues modified to carry may be 1 to 100, e.g., 1 to 80, 10 to 70, 20 to 60, or 30 to 50, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 51, 52, 53, 54, or 55.

The charge-modified globin can be referred to as a cationic globin in the case of an increase in the total surface positive charge after modification, or as an anionic globin in the case of an increase in the total surface negative charge after modification. For the cationized globin, the overall change in surface positive charge may be +1 to +100, for example +1 to +80, +10 to +70, +20 to +60 or +30 to +50, for example about +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25, +26, +27, +28, +29, +30, +31, +32, +33, +34, +35, +36, +37, +38, +39, +40, +41, +42, +43, +44, +45, +46, +47, +48, +49, +50, +51, +52, +53 or + 55. The total surface positive charge of the cationized globin may be +1 to +100, such as +1 to +80, +10 to +70, +20 to +60 or +30 to +50, such as at least about +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25, +26, +27, +28, +29, +30, +31, +32, +33, +34, +35, +36, +37, +38, +39, +40, +41, +42, +43, +44, +45, +46, +47, +48, +49, +50, +51, +52, +53, +54 or + 55.

For the anionized globin proteins, the overall change in surface negative charge may be-1 to-100, e.g., -1 to-80, -10 to-70, -20 to-60, or-30 to-50, e.g., about-5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20, -21, +23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, -40, -41, or-30, -42, -43, -44, -45, -46, -47, -48, -49, -50, -51, -52, -53, -54, or-55. The total surface negative charge of the anionized globin can be-1 to-100, e.g., -1 to-80, -10 to-70, -20 to-60, or-30 to-50, e.g., at least about-5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, -40, or, -41, -42, -43, -44, -45, -46, -47, -48, -49, -50, -51, -52, -53, -54, or-55.

Typically, the charge-modified globin comprises a percentage of positively or negatively charged residues as a percentage of its total number of amino acid residues in the protein. The percentage of positive or negative charges is greater than the percentage of positive or negative charges in the corresponding native globin. For example, 5.0-40% of the total amino acid residues of native globin are positively charged residues, while charge-modified globin may have a higher percentage than in the corresponding native globin. For example, native human myoglobin has 14% of the amino acid residues that are positively charged residues. Similarly, human hemoglobin has 10% of its amino acid residues as positively charged residues, while horse cardiac myoglobin and chimpanzee myoglobin have 14% of its amino acid residues as positively charged residues. The charge-modified globin can have at least about 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or at least about 30% of the total number of amino acid residues as positively charged residues. The same principles apply to negatively charged residues.

The charge-modified globin may also be referred to as superglobin. Thus, the term "supercharged globin" as used herein can refer to any globin protein having one or more charge modifications thereon relative to the native protein, as described above. Production of supercharged proteins is known in the art. Examples of the production of such supercharged proteins in the context of Green Fluorescent Protein (GFP) are disclosed in Lawrence et al (j.am. chem.soc. (2007) vol.129p.10110-10112). This so-called "overloaded" protein has previously been used to facilitate the transport of molecules across phospholipid bilayer membranes into the interior of cells (Zang et al (2017) PLoS One 12(6): e 0180138; WO 2009/134808; WO 2010/129023; WO 2016/069910; Thompson et al (2012) Methods enzymol. vol.503p.293-319; McNaugton et al (2009) Proc. Natl.Acad.Sci.U.S.vol.A.106p.6111-6116). Thus, it is entirely surprising that when the above-described method is used to produce charge-modified globin and charge-modified globin comprising a polymeric surfactant coating as described herein, an anti-tumor cell, liposome or micelle comprising at least one charge-modified globin associated with the membrane of said cell, liposome or micelle can be obtained.

The charge-modified globin is associated with a membrane of a cell, liposome or micelle. In one embodiment, the protein of the charge-modified bead is associated with the membrane by binding to the membrane. Such binding may be mediated by one or more covalent bonds and/or one or more intermolecular forces (e.g., electrostatic forces, hydrogen bonding, and/or hydrophobic interactions). The charge-modified globin may also, or alternatively, be associated with the membrane by being sterically locked in position. One advantage associated with membranes is that globin can readily deliver oxygen to target tumor cells and anti-tumor cells, liposomes or micelles. Another advantage is that membrane-associated globin can also serve as an anchor for secondary anti-tumor molecules.

By "membrane" we mean a structure that separates the interior of a cell, liposome or micelle from the external environment. In cells produced by natural organisms, the membrane is a phospholipid bilayer, also known as the cell membrane, plasma membrane or cytoplasmic membrane. The charge-modified globin can be associated with the membrane by intercalation into the phospholipid bilayer (i.e., the hydrophobic lipid region of the membrane), or can be associated with the outer surface of the phospholipid bilayer that is exposed to the solvent. This association with the solvent-exposed surface of the phospholipid bilayer can be mediated by direct binding to hydrophilic phospholipid heads and/or by binding to cell surface proteins. In liposomes, the membrane is a structure resembling a cell membrane. In other words, the membrane is the outer membrane of the liposome. The liposome membrane may comprise a phospholipid bilayer. It is noted, however, that the liposome membrane may comprise bilayers formed of different amphiphilic molecules, as is known in the art. In micelles, the membrane is the periphery of the micelle, which consists of hydrophobic heads of amphiphilic molecules (such as phospholipids).

In preferred embodiments, the charge-modified globin is embedded in the membrane or bound (e.g., bound) to the outer surface of the membrane (i.e., exposed to solvent).

The cell membrane preferably comprises a phospholipid bilayer. The liposome membrane may comprise a phospholipid bilayer. The micelles may comprise a phospholipid membrane. In these embodiments, the charge-modified globin is bound to the phospholipid membrane by binding to the phospholipid membrane. The membrane may comprise lipids other than phospholipids, such as cholesterol. The membrane may also comprise other components, such as integral membrane proteins. This may be particularly applicable where the membrane is a cell membrane.

In one embodiment, the charge-modified globin is bound to the outer surface of the phospholipid membrane exposed to the solvent. This binding is preferably mediated by electrostatic forces. Typically, the outer surface of the phospholipid membrane exposed to the solvent comprises a negative charge, particularly in the case of cells. However, the membrane may also comprise a solvent-exposed surface containing a positive charge. To modulate electrostatic interactions with such membranes, charge-modified globin proteins may comprise an increased total surface positive charge (i.e. cationized globin) or an increased total surface negative charge (i.e. anionized globin).

The cell, liposome or micelle may have a membrane that further comprises a molecule that can act as a charge-modified globin binding site. For example, the outer surface of cells displays various proteins, lipids and glycans. Liposomes and micelles can display various labels on their surface. In one embodiment, the charge-modified globin is associated with a molecule, liposome or micelle displayed on the outer surface of the cell.

In one embodiment, the charge-modified globin is embedded in the membrane. Cell membranes, liposomes and liposomes typically comprise at least one lipid bilayer having a hydrophobic interior, defined on each surface by hydrophilic functional groups. The charge-modified globin can be coated with a hydrophobic coating, and the hydrophobic charge-modified globin that can be embedded within the lipid bilayer is provided as discussed in more detail below. The term "intercalator" means that the hydrophobically charge-modified globin is at least partially located within the phospholipid bilayer or within the micellar layer. That is, the hydrophobically charge-modified globin protein at least partially intersects a phospholipid bilayer or a phospholipid layer, and does not interact only with the surface of a phospholipid bilayer molecule or a phospholipid layer.

In a preferred embodiment, the charge-modified globin is not internalized. In other words, the charge-modified globin remains in contact with the membrane and is not released into the cell or liposome interior.

In a preferred embodiment, the anti-tumor cell, anti-tumor liposome or anti-tumor micelle is an anti-tumor cell. "anti-tumor cell" can refer to any cell having anti-tumor properties. As such, the cell may be a natural cell, an artificial cell, a modified cell, or an organelle. The term "modified cell" includes cells that have been modified in vitro and cells that have been modified in vivo (e.g., by in vivo gene editing). In the present specification, the term "cell" includes protoplasts or spheroplasts, i.e. cells which typically comprise a cell membrane but at least part of which is removed or destroyed (e.g. by mechanical or enzymatic treatment). This may include, for example, a kit comprising cells, wherein at least a portion of the membrane has been removed or disrupted to facilitate further modification of the cells, for example by cell transformation.

Typically, the cell is an animal cell, such as a mammalian cell. The mammalian cell may be a human, mouse, dog, cat or horse cell, or a bovine, porcine or ovine cell. In a preferred embodiment, the cell is a human cell. Alternatively, the cell may be from a humanized animal, such as a humanized mouse. Human or humanized cells are particularly preferred because they should be less immunogenic and therefore more preferred.

Typically, the cell is an immune cell, has cytotoxic properties, and can kill tumor cells. Preferably, the immune cell is a tumor infiltrating cell, such as a lymphocyte, a neutrophil, a dendritic cell, or a macrophage. More preferably, the cell is a lymphocyte, such as a cytotoxic T cell, a natural killer T cell, or a natural killer cell. It is particularly preferred that the cells are T cells, for example CD3+ T cells. The CD3+ T cells are preferably of the CD4+ or CD8+ subtype, preferably of the CD8+ subtype. In one embodiment, the T cell is a chimeric antigen receptor T (CAR-T) cell.

Importantly, binding of certain proteins (i.e., charge-modified GFP) to T cell membranes has been shown to have toxic effects on T cells (fig. 2). In contrast, a representative charge-modified globin (myoglobin) was associated with mouse T cell membranes with an unexpected decrease in toxicity compared to GFP (fig. 2b), and more surprisingly, no apparent toxicity to human Jurkat T cells (fig. 2 a).

Furthermore, the activity of T cells depends on the structure and composition of the outer surface of the T cell membrane exposed to the solvent, where a set of proteins is required to recognize ligands that mediate the immune response. The globin of the invention is associated with the T cell membrane but the association must be such that it does not interfere with T cell function. A very important and surprising result is that the globin can bind to human Jurkat T cell membranes without any significant loss of T cell activity (figure 3). This indicates that the globin does not present a spatial or other interference with the interaction between the T cell receptor and its ligand.

More surprising effects were observed on T cell behavior in an normoxic environment compared to behavior in a hypoxic environment as a representative model of a solid tumor environment. Mouse T cells were tested for CD4+ and CD8+ subtypes. Upon binding of myoglobin to the T cell membrane, T cell proliferation decreased when exposed to normoxic environment, but proliferation recovered in hypoxic environment (fig. 4a and b). Similar patterns of activity are seen for the CD4+ subtype. In the normoxic environment, the activity of T cells in the T cell/myoglobin complex decreased, but in the hypoxic environment, the activity of the T cell/myoglobin complex recovered (fig. 4 c). This unexpected result suggests that these T cell/myoglobin complexes are particularly suitable for hypoxic solid tumor environments. That is, reducing proliferation of the T cell/myoglobin complex under normoxic conditions helps to ensure that globin is not diluted each time the T cell proliferates (i.e., by dilution between T cell progeny during proliferation), thereby producing T cell progeny with reduced globin concentrations. Also, the activity of the T cell/myoglobin complex in fig. 4c is inhibited until the cells are in a hypoxic environment, which makes these cells particularly suitable for targeting and acting on hypoxic solid tumors. A more surprising effect was observed for the activity of the CD8+ cell/myoglobin complex. In an oxygen deficient environment, T cell activity is in fact significantly enhanced, which makes it a particularly exciting complex in antitumor therapy.

The charge modified globin can be associated with a cell by contacting the cell with the charge modified globin, which association can occur 1-30 days or 1-15 days or 1-10 days or 1-5 days after the formation of the cell according to the invention. For example, the charge-modified globin can be associated for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 days.

In one embodiment, the anti-tumor cell, liposome or micelle is an anti-tumor liposome or micelle. The liposomes or micelles are typically water-soluble liposomes or micelles. The liposomes or micelles will comprise a component that imparts tumor killing properties to the liposomes or micelles. In a preferred embodiment, the anti-tumor cell, liposome or micelle comprises a therapeutic agent, such as a checkpoint inhibitor, an immunotherapeutic agent or a chemotherapeutic agent. Checkpoint inhibitors include, but are not limited to, peptides or proteins that bind to and block PD-1, PDL-1 and/or CTLA-4, such as peptides generated by phage display technology that have high affinity and inhibitory properties for PD-1, PDL-1 and/or CTLA-4, or antibodies or antibody fragments that recruit or design antibodies or antibody fragments that have high affinity and inhibitory properties for PD-1, PDL-1 and/or CTLA-4. Many checkpoint inhibitors are known in the art.

The liposomes or micelles may also comprise a targeting component, such as an antibody or other targeting protein, which specifically targets the liposomes or micelles to tumor cells.

The globin can be hemoglobin, myoglobin, neuroglobin or cytoglobin. In a preferred embodiment, the globin is myoglobin. The globin reversibly binds and transports oxygen. Thus, the globin binds and carries oxygen until it is aerobic to release oxygen. Especially myoglobin, is also considered to be a weak peroxidase and free radical scavenger.

The globin may be linked to a secondary anti-cancer molecule. Alternatively, the globin may be linked to a reactive functional group for attachment to a secondary anti-tumour molecule. For example, the reactive functional group may be one half of a biological binding system such as the SpyCatcher/SpyTag system (Reddington & Howarth (2015) curr. op. chem. biol. vol.29p94-99; WO2014/176311) or streptavidin/biotin.

Thus, globin acts as an "ankyrin" anchoring the secondary anti-tumor molecule to the cell, liposome or micelle. This means that charge-modified globin bound to the membrane of a cell, liposome or micelle provides at least two important advantages. The first advantage is the ability to readily deliver oxygen to tumor cells and anti-tumor cells, liposomes or micelles. A second advantage is the ability to anchor secondary anti-tumor molecules to the membrane. As the charge-modified globin is bound to the membrane, the secondary molecule is advantageously present on the outer surface of the antitumor cell, liposome or micelle.

The secondary molecule may be a protein that is not a cationized protein or a non-anionized protein. In the case where the charge-modified globin is embedded in the membrane, the secondary anti-tumour molecule may not be embedded in the membrane whilst localised, as described in PCT/GB 2018/052534.

The secondary anti-tumor molecule may be selected from any anti-tumor molecule known in the art. For example, the secondary anti-tumor molecule may be any of an antibody, a lectin, an integrin, or an adhesion molecule. Specifically, the anti-tumor molecule may be any one of the following:

(1) a tumor cell binding molecule. The tumor cell binding molecules facilitate targeting and sustained binding of tumor cells. The sustained binding helps to ensure that the anti-tumor cells, liposomes or micelles remain in the tumor cell region for a period of time, thereby allowing the cells, liposomes or micelles and globin to have a stronger effect on the tumor cells.

(2) A checkpoint inhibitor. Checkpoint inhibitors help to reduce the ability of tumors to evade the immune system, and complement the effects of globin to reduce tumor hypoxia, which also reduces the ability of tumors to evade anti-tumor therapies (e.g., anti-tumor cells, liposomes, and micelles).

(3) Recombining the enzymes of the tumor extracellular matrix to enhance the penetration of immune cells into the tumor mass.

(4) An enzyme that metabolizes a tumor-associated compound. For example, the tumor's rate of adenosine triphosphate metabolism is increased, resulting in an excess of adenosine in the tumor microenvironment. This stimulates the adenosylergic receptors which lead to immunosuppression. The presence of an enzyme, such as adenosine deaminase, on the surface of immune cells will reduce this immunosuppressive signal.

In one embodiment, said globin and said secondary anti-cancer molecule are comprised within a fusion protein.

The globin is a charge modified globin. The globin may be a cationic globin or an anionic globin.

Cationized globin can be obtained by covalent bonding of a cationic or polycationic linker to the acidic amino acid side chain on the parent globin. For example, this can be accomplished by mixing the protein with N, N ' -dimethyl-1,3-propanediamine (N, N ' -dimethyl-1,3-propanediamine, DMPA) or the like in the presence of a carbodiimide such as N- (3-dimethylaminopropyl) -N ' -ethylcarbodiimide hydrochloride (EDC) or Dicyclohexylcarbodiimide (DCC). The reaction is as followsThis shows that the acidic residue (1) is activated for nucleophilic attack by the addition of the zero-length crosslinker EDC (2) to form the activated o-acylureido group (3). The nucleophilic DMPA (4) then attacks the active carbonyl and eliminates the isourea to form a cationized residue (5). Thus, the cationized globin can comprise the linker-CH₂C(O)NCH₃(CH₂)₃N(CH₃)₂H⁺. DMPA or an analogue thereof may be added to the protein prior to mixing with EDC to ensure that there is an excess of DMPA or an analogue thereof, thereby avoiding cross-linking of the proteins with each other.

The covalent bonding step of the cationic linker to the acidic amino acid side chain on the protein can be performed in the presence of N-hydroxysuccinimide (NHS) or its water-soluble analogue, sulfo NHS, to improve the stability of the electrostatic coupling.

In the present invention, mixing of the protein with DMPA or an analogue thereof in the presence of carbodiimide may be allowed for a limited time to avoid denaturation and/or polymerization of the protein. Such limited time may be, for example, up to or about 2 hours, or up to or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or about 90 minutes. Alternatively or additionally, the product mixed with DMPA in the presence of carbodiimide may then be subjected to size exclusion chromatography, the chromatography product being used as a cationized protein. The skilled person will be able to determine the theoretical size of the desired charge modified globin for determining a suitable chromatographic eluate for collection, for example by using a calibration chromatographic column.

The analogs of DMPA may be N, N '-dimethylhexane-1, 6-Diamine (DMHA), N' -Dimethylethylenediamine (DMEA), 3-Dimethylaminopropylamine (DMAPA), Ethylenediamine (EN), 1,3-diaminopropane (1,3-diaminopropane, DAP), 1,4-diaminobutane (1,4-diaminobutane, DAB), 1, 5-diaminopropane (1, 5-diaminopropane, DAP), 1,6-diaminohexane (1,6-diaminohexane, DAH), hexamethylenediamine (HMA ), 1, 7-diaminoheptane (1, 7-diaminoheptane), 1, 8-diaminoheptane (1, 8-diaminoheptane), 1, 8-diaminoethylamine (2, 8-Diaminoethylamine) (DAO) and 2- (DAO-8-diaminoethylamine), AEG). Other suitable nucleophiles, for example, charged nucleophiles, are contemplated by those skilled in the art. For example, the nucleophile may also include other primary, secondary, and tertiary amines, as well as alkyldiamines capped with quaternary amines if the opposite terminus contains a primary, secondary, or tertiary amine. Polyalkylamines are also contemplated, for example, polyethyleneimine as a linear chain or branched structure.

Alternatively, electrostatically modified proteins can be obtained by anionization of the protein. This can be achieved, for example, by nucleophilic addition of a dicarboxylic acid (HOOC-R-COOH) to the lysine side chain of the native protein.

In another alternative, the charge-modified globin proteins can be obtained by recombinant expression of sequences with altered charge relative to the native globin protein, in particular sequences with a more positive or more negative overall charge relative to the native globin protein. The recombinant modification may comprise recombinant expression of a charge-modified globin, which is a variant comprising one or more amino acid substitutions in its entire amino acid sequence compared to the sequence of a non-variant globin. The amino acid substitutions introduce different surface charge distributions to the charge-modified globin protein by providing different amino acid charges to the natural amino acid at the or each substitution position relative to the natural globin protein.

For example, an amino acid with an uncharged side group can be replaced with an amino acid with a positively or negatively charged side chain group (to give a total charge change of +1 or-1, respectively), or an amino acid with a negatively charged side chain group can be replaced with an amino acid with a positively charged side chain group (to change the total charge to +2), or an amino acid with a positively charged side chain group can be replaced with an amino acid with a negatively charged side chain group (to change the total charge to-2), without significantly changing the tertiary structure and/or biological activity of the protein. This rational design approach may be advantageous if the function/activity of the protein is dependent on the involvement of specific amino acids, such as amino acids with charged side chain groups, since the user can make globin surface charge changes at non-critical amino acid positions; this is not always possible in the chemical modification methods described elsewhere herein.

Typically, the determined amino acid sequence identity (also referred to as "overall sequence identity") at an overall level is at least about 60%, e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, between native globin and recombinantly modified charge-modified globin. Sequence identity can be determined at a global level using, for example, the Needleman-Wunsch global sequence alignment tool, which can be passed through the NCBIWebsites (blast. ncbi. nlm. nih. gov/blast. cgi) are available on the internet. The tool allows the user to compare two sequences over the full length. If the globin is part of a fusion protein, the comparison is made in the globin portion of the fusion protein.

Typically, native globin proteins consist of naturally occurring amino acids, such as amino acids selected from proteinogenic amino acids (including standard amino acids). Proteinogenic amino acids are amino acids that are incorporated into proteins by natural translation processes. Non-limiting examples of amino acids that may be included in the charge-modified globin are provided in tables 1 and 2 below:

alanine	Phenylalanine (phenylalanine)	Glutamine	Arginine	Selenocysteine
					Isoleucine	Tryptophan	Serine	Histidine	Pyrrolysine
Leucine	Tyrosine	Threonine	Lysine
					Methionine	Asparagine	Aspartic acid	Glycine
Valine	Cysteine	Glutamic acid	Proline

Table 1: examples of proteinogenic amino acids; bold indicates positively charged amino acids and italics indicates negatively charged amino acids.

Modifications involving non-proteinogenic amino acids are also contemplated. Non-naturally occurring amino acids (e.g., amino acids that can be introduced into a protein by using a unique codon and corresponding aminoacyl-tRNA system) can also be included in the invention.

Table 2: examples of amino acids of non-protein origin

In one embodiment, the recombinantly expressed charge-modified globin can be chemically cationized or anionized to further modify the charge.

When recombinant techniques are used, as described above, the recombinant DNA sequence may encode a fusion protein comprising a charge-modified globin and a proteinogenic secondary anti-tumor molecule.

The charge modified globin can comprise a polymeric surfactant coating to provide a polymer coated charge modified globin. The construct may comprise charge-modified globin proteins having one or more surfactant molecules electrostatically complexed to charged amino acid residues on the surface of the protein. For example, Perriman et al (2010; Nature chem.vol.2622-626), Brogan et al (2013; J.Phys.chem.B vol.1178400-8407) and Sharma et al (2013; adv.Mate.vol.252005-2010) describe the preparation of similar constructs. Conjugates proteins having an amphiphilic surfactant crown around at least a portion of the entire structure, as described herein. The presence of such a corona can be confirmed by comparing the conjugate to the corresponding native globin to detect changes in charge and/or size. Techniques such as mass spectrometry, zeta potentiometry, small angle X-ray scattering and/or dynamic light scattering, among others, combinations of two or more of these may be used to detect such changes.

The polymer-coated charge-modified globin can comprise a surfactant comprising polyethylene glycol (PEG). For example, the surfactant may have the general structure of formula I below:

in formula I, n can be any integer including or between 5 and 150, for example, any integer including or between 8 and 110. For example, n can be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110.

R₁Can be as follows:

R₂can be C_xH_(2x+1)Wherein x is any integer including or between 8-18; for example, x may be 11, 12, or 13. R₂Unsaturated hydrocarbons having from 8 to 18 carbon atoms, for example 11, 12 or 13 carbon atoms, are also possible. In another alternative, R₂Can be as follows:

the surfactant may be one of those described herein, such as S621(Sigma-Aldrich catalog No. 463221), S907(Sigma-Aldrich catalog No. 463256), S1198(Sigma-Aldrich catalog No. 473197), or S1783 (oxidized form of glycolic acid ethoxylate 4-nonylphenyl ether, Sigma-Aldrich catalog No. 238678).

These anionic surfactants have the following structure:

for S621 and S907, x is 11-13

For S621, y is 7-9

For S907, y is 14-15

The molecular weight and polydispersity were measured by mass spectrometry with the results shown below:

TABLE 3 molecular weight and polydispersity of the surfactants

"polydispersity" reflects the fact that a synthetic polymer produced by a chemical reaction has a molecular mass distribution that results from the intrinsic entropy process resulting from polymerization. The degree of change depends on the reaction mechanism and the reaction conditions. The degree of variation is determined by the degree of dispersionBy definition, it has not until recently been referred to as "polydispersity". It is defined by the following equation:

in the formula, M_wIs the weight average molar mass, M_nIs the number average molar mass.

The polymer dispersibility can be estimated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).

The protein polymer surfactant conjugate may comprise a surfactant having a molecular weight of at least about 500Da, for example, at least about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, or at least about 4000 Da.

The protein-polymer surfactant conjugate may comprise S1783 (i.e., glycolic acid ethoxy 4-nonylphenyl ether oxide) as a surfactant. Alternatively or additionally, the protein-polymer surfactant conjugate may comprise a cationic surfactant, such as PEG-15 hydrogenated tallow methyl ammonium chloride(bySold as HT 25).

The cells, liposomes or micelles according to the invention may be present in a complex composition further comprising at least one additional component, such as water, buffer solution, one or more components required to form a pharmaceutical composition as described below.

According to a second aspect, the present invention provides a pharmaceutical composition comprising the anti-tumor cell, liposome or micelle of the first aspect, further comprising a pharmaceutically acceptable carrier, diluent or vehicle.

According to a third aspect, the present invention provides a cell, liposome or micelle according to the first aspect, or a pharmaceutical composition according to the second aspect, for use in the treatment of cancer.

The term "pharmaceutical composition" referred to throughout this specification may be a composition comprising a pharmaceutically acceptable carrier, diluent or vehicle. For example, the pharmaceutical compositions described herein may be in the form of a sterile injectable preparation which may be in the form of an aqueous or oleaginous suspension or suspension in a non-toxic parenterally-acceptable diluent or solvent. Aqueous suspensions may be prepared, for example, in mannitol, water, ringer's solution or isotonic sodium chloride solution. Alternatively, it can be prepared in phosphate buffered saline. The oily suspension may be prepared in artificial monoglyceride, artificial diglyceride, fatty acid or pharmaceutically acceptable natural oil. The fatty acid may be an oleic acid or a glyceryl oleate derivative. The pharmaceutically acceptable natural oil may be olive oil, castor oil or polyoxyethylenated olive oil or castor oil. The oily suspension may contain a long chain ethanol diluent or dispersant, for example, in accordance with the european pharmacopoeia and/or the Helv pharmacopoeia. In addition to the phospholipid composition of the present invention, the pharmaceutical composition may also comprise one or more pharmaceutically or other biologically active agents. For example, the composition may include a therapeutic agent, such as a conventional drug, antibody, or other protein component

According to a fourth aspect, the present invention provides a method of preparing an anti-tumor cell, liposome or micelle according to the first aspect, comprising a) providing a charge-modified globin; and b) contacting an anti-tumor cell, liposome or micelle with said globin.

In the method according to the fourth aspect of the invention, when cells are used, step (b) may comprise incubation at a temperature of at least about 1 ℃ for at least about 2 minutes, for example at a temperature of at least about 10 ℃ for at least about 2 minutes. The temperature may typically be about 30-40 ℃, e.g., about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or about 40 ℃, e.g., about 37 ℃ ± about 1 ℃. Alternatively, certain cells, liposomes or micelles benefit from treatment at lower temperatures, e.g., 1-8 ℃, 2-7 ℃, or 3-6 ℃, e.g., about 1, 2, 3, 4, 5, 6, 7, or about 8 ℃. The time period is typically from 2 to 60 minutes, for example about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50 or about 60 minutes, for example about 15, about 20 or about 30 minutes. This step may be performed at about 0-10% CO₂(e.g., about 5% CO)₂) Is carried out in an atmosphere of (2). When liposomes or micelles are used, step (b) may be carried out at CO₂At room temperature (e.g., between about 15 ℃ to about 25 ℃), e.g., in air, in an amount of less than 1%.

Step (b) of the method according to the fourth aspect of the invention may optionally be followed by step (c) by washing the cells, liposomes or micelles, for example using a buffer such as Phosphate Buffered Saline (PBS), for example using two or more washing steps. One skilled in the art can readily adjust these steps as needed and determine when a washing step is required.

Step (a) may comprise providing charge modified globin and a polymeric surfactant under conditions that enable electrostatic coupling of the polymeric surfactant and the globin. The surfactant may be added to the globin solution in solid or liquid form. The surfactant may be added in an amount corresponding to 0.5 to 5 moles of surfactant per cationic site on the protein, for example about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.1, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or about 3 moles of surfactant per cationic site on the proteinAmount of the compound (A). The protein may be present in solution with a suitable buffer, e.g., HEPES buffer, with or without CoCl₂Or in Tris-HCl buffer. The selection of an appropriate buffer is within the routine ability of those skilled in the art. The conditions may include a pH between 5 and 8, such as about 5, 6, 7, or about 8 (including any intermediate pH between 5.1 and 5.9, between 6.1 and 6.9, and between 7.1 and 7.9), and may include stirring the mixture for 0 to 30 hours, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or about 12 hours, and may also include a temperature of 0 to 25 ℃, such as at about 4 ℃ or at about room temperature. For example, the coupling conditions described by Armstrong et al (nat. Commun. (2015) Jun 17; 6:7405) may be suitable.

A "cationic site" is a site in the amino acid sequence of a protein that has an amino acid with a positively charged side chain or a linker that contains a cation (i.e., positively charged). One skilled in the art can determine the number of cationic sites within a globin without using the techniques of the present invention.

The surfactant may comprise a polyethylene glycol, which may, for example, have a molecular weight of at least about 500Da, such as at least about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, or at least about 4000 Da. The surfactant may be in a buffered solution at a concentration of 5-50mg/mL, for example, about 10, 15, 20, 25, or about 30 mg/mL.

The surfactant may be S1783 (i.e., glycolic acid ethoxy 4-nonylphenyl ether oxide). Alternatively, the surfactant conjugate may comprise a cationic surfactant, such as PEG-15 hydrogenated tallow ammonium chloride (fromSold as HT 25).

As described above, the charge-modified globin can be attached to a secondary anti-tumor molecule prior to contact with the surfactant.

Step (a) may further comprise a buffer exchange step prior to contacting the cell, liposome or micelle with the globin. The buffer exchange step may comprise rotary concentration of the product of the step of contacting the cationized or anionized protein with a surfactant. Alternatively, the buffer exchange step may comprise a dialysis step. Such methods are within the routine ability of those skilled in the art.

The charge-modified globin can be generated by chemically modifying the charge on the native globin. For example, at least one acidic amino acid side chain may comprise-CH₂C(O)NCH₃(CH₂)₃N(CH₃)₂H⁺A linker. This can be achieved by: a solution of N, N '-dimethyl-1, 3-propanediamine (DMPA) or analog thereof is mixed with native globin (as described above) in the presence of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC). The analogs of DMPA may be N, N '-dimethylhexane-1, 6-Diamine (DMHA), N' -Dimethylethylenediamine (DMEA), 3-Dimethylaminopropylamine (DMAPA), Ethylenediamine (EN), 1,3-Diaminopropane (DAP), 1,4-Diaminobutane (DAB), 1, 5-Diaminopropane (DAP), 1,6-Diaminohexane (DAH), Hexamethylenediamine (HMA), 1, 7-Diaminoheptane (DAH), 1, 8-Diaminooctylamine (DAO), and 2- (2-aminoethyl) guanidine (AEG). Other suitable nucleophiles, such as charged nucleophiles, are contemplated by those skilled in the art. For example, the nucleophile may also include other primary, secondary, and tertiary amines, as well as alkyldiamines capped with quaternary amines if the opposite terminus contains a primary, secondary, or tertiary amine. Polyalkylamines are also contemplated, for example, polyethyleneimine as a linear chain or branched structure.

Thus, globin (i.e. a charge-modified globin precursor) can be converted to charge-modified globin by a method comprising:

i) mixing the globin solution with a pH neutralized solution of N, N' -dimethyl-1,3-propanediamine (DMPA) or an analog thereof, and optionally adjusting the pH of the mixture to 5-7;

ii) subsequently or simultaneously adding a carbodiimide, such as N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC), and adjusting the pH of the mixture to a pH of 4-7;

iii) stirring said mixture in (ii) at a temperature of 0-25 ℃ and a pH of 4-7 for 1-30 hours;

iv) separating the proteins in the mixture of (iii) from water or buffer at a pH of 6.5 to 8.5 for at least 4 hours;

v) if necessary, adjusting the pH of the mixture in (iv) to 6.5-8.5.

In the method, any step (iii) lasts for no more than about 120 minutes, for example, no more than about 90 minutes; and/or the method further comprises the step (vi) of subjecting the mixture of step (iv) or step (v), if present, to size exclusion chromatography and obtaining an eluate comprising charge-modified globin of the desired molecular weight. One or both of these two constraints may ensure that the above process is controlled to reduce or prevent protein denaturation and/or aggregation.

The native globin solution used in step (i) may be prepared in any conventional buffer, for example HEPES. Natural globin, as DMPA moles number of anionic sites on the protein 100:1-400:1 mixed with DMPA, e.g., about 100:1, 150:1, 200:1, 250:1 or about 300: 1. EDC is added to the protein in a ratio of moles of EDC to the number of anionic sites on the protein from 30:1 to 60:1, e.g., about 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 40:1, 45:1, or about 50: 1.

An "anionic site" is a site in the globin amino acid sequence that has an amino acid with a negatively charged side chain. The number of anionic sites within the globin can be determined using routine capabilities of a person skilled in the art.

Step (ii) may be done simultaneously with step (i), i.e. the protein solution, DMPA and EDC may be mixed simultaneously. If step (ii) is completed after step (i), step (ii) may be a single step as defined above, followed by step (iii); or may be subdivided into two steps: (iia) wherein a portion of the EDC is added to the mixture in step (i) and the resulting mixture is stirred at a temperature of 0-25 ℃ for about 2, 3, 4, 5, 6, 7, or about 8 hours, followed by (iib) wherein EDC is further added to the mixture described in (iia) and stirring is continued; step (iib) is followed by step (iii).

The stirring required in step (iii) may be achieved by any conventional means, for example agitation, and the pH may be about 4, about 5, about 6 or about 7 (including any intermediate pH between 4.1 and 4.9, between 5.1 and 5.9 and between 6.1 and 6.9). When the time in step (iii) exceeds 120 minutes, it may last for about 20-30 hours, for example, about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or about 30 hours, for example, about 24 hours. For example, all steps may be performed at about room temperature (e.g., 18-23 ℃), or may be performed at about 4 ℃.

The appropriate length of time for step (iii) can be determined by one skilled in the art by determining the optimal length of time for step (iii) by testing the retention of globin activity over a range of time for step (iii), whether or not a subsequent size exclusion chromatography step is present. .

In an alternative general method, the polymeric surfactant coated charge-modified globin can be prepared by contacting charge-modified globin (e.g., the anionic globin) with a surfactant (e.g., a cationic surfactant). For example, globin can be anionized by nucleophilic addition of a dicarboxylic acid (HOOC-R-COOH) to the lysine side chain of the native protein.

Alternatively or in addition to the above modifications, the charge modified globin may be obtained by a method comprising expressing a recombinant DNA sequence encoding the charge modified globin. For example, the charge modified bead protein can be obtained by a method comprising expressing a recombinant DNA sequence encoding the charge modified bead. The resulting protein, i.e., the charge-modified globin, can then be isolated.

For example, the preparation of charge-modified globin proteins can involve replacing an amino acid having an uncharged side chain group with an amino acid having a charged side chain group, or replacing an amino acid having a charged side chain group with an oppositely charged side chain group, without significantly altering the tertiary structure and/or biological activity of the protein. This may be a particularly advantageous solution if the function/activity of the protein is dependent on the involvement of amino acids with charged side chain groups, since the user can direct changes in the protein surface charge to non-critical amino acid positions.

Typically, the determined amino acid sequence identity (also referred to as "overall sequence identity") at an overall level is at least about 60%, e.g., at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%, between the recombinant modified charge-modified globin (i.e., charge-modified globin) and the native globin. Sequence identity can be determined at a global level using, for example, the Needleman-Wunsch global sequence alignment tool, which can be passed through the NCBIWebsites (blast. ncbi. nlm. nih. gov/blast. cgi) are available on the internet. As described above, the sequence identity of the functionally important domains may be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% between the charge-modified globin and the native globin.

The recombinant DNA sequence may be expressed in any conventional manner, e.g.using any expression system, e.g.in E.coli, according to the routine abilities of a person skilled in the art. It is also within the routine ability of the person skilled in the art to isolate the expressed ankyrin from the expression system.

According to a fifth aspect, the present invention provides a method of treating cancer comprising administering a cell, liposome or micelle as described above in the first aspect or a pharmaceutical composition as described in the second aspect to a patient in need thereof.

In a preferred embodiment, the cancer according to the third or fifth aspect of the invention is a solid tumor cancer. In particular, the cancer may be selected from the following cancers: breast cancer, colorectal cancer, prostate cancer, lung cancer, stomach cancer, liver cancer, esophageal cancer, cervical cancer or pancreatic cancer. These are the most common solid tumor cancers. More specifically, the cancer may be selected from ICD-10 version: 2016 (International Classification of diseases, 10 th edition, published by the world health organization, ICD. who. int/brown 10/2016/en) code C00-C75.9.

According to a sixth aspect, the present invention provides a charge-modified globin protein comprising any one of the sequences shown in SEQ ID NO. 1-12 or any one of the functional variants having at least about 60% sequence identity to any one of SEQ ID NO. 1-12. For example, the charge-modified globin protein may have at least about 65% sequence identity, e.g., at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% sequence identity to any one of SEQ ID NOs 1-12. The charge-modified globin protein may optionally form part of a larger construct, such as a fusion protein.

Throughout the description and claims of this specification, the words "comprise" and variations of the words, for example "comprising" and "comprises" (singular) mean "including but not limited to", and do not exclude other elements, integers or steps. Furthermore, unless the context requires otherwise, the singular encompasses the plural; in particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Preferred features of each aspect of the invention may be combined with any of the other aspects as described above. Within the scope of the present application, it is expressly intended that the various aspects, embodiments, examples and alternatives set forth in the preceding paragraphs, claims and/or in the following description and drawings, particularly the various features thereof, may be considered separately or in any combination. That is, all aspects and/or features of any embodiment may be combined in any manner and/or combination unless such features are incompatible.