drug targeting with fusion proteins

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Abstract

A fusion protein is a protein created by joining two or more genes which originally coded for separate proteins. Fusion proteins occur naturally, even in humans. They are also man-made using gene fusion technology. Man-made fusion proteins are being used in biological research and medical therapeutics. Indeed, currently fusion proteins, also called chimeric proteins, are being researched to be used in the medical treatment of auto-immune diseases: rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, vasculitis, and lupus are amongst the prime candidates. Gene fusion technology is also being applied for the treatment of HIV, asthma and cancer.

Key Words: gene fusion technology, fusion gene, recombinant DNA technology, exons, splicing.

Introduction

Definition

Gene fusion technology deals with the processes whereby two or more genes of interest are joined together to create a fusion gene. The fusion gene thus formed is then transcribed and subsequently translated into a single polypeptide with properties derived from each of the original proteins that the constituent genes previously coded. A fusion protein is also called a recombinant fusion protein since it is created by recombinant DNA technology. Sometimes, a fusion protein is also called a chimeric protein, after the Greek mythological monster called chimera, which was made up of parts of several animals (viz. the body of a lioness, a tail ending in a head of a snake, and a head of a goat) (Kerenyi, 1959).

Naturally Occurring Fusion Proteins

Fusion proteins occur in nature, even in humans. Several oncogenes and antibodies are typical naturally occurring fusion proteins.

Oncogenes

Some oncogenes are the product of fusion genes that result from translocations. A very typical example is the Bcr-abl fusion protein. About 95% of patients with chronic myeloid leukemia (CML) have a chromosomal abnormality called the Philadelphia chromosome (MedicineNet, 2011). This is the result of a translocation between chromosomes 22 and 9. The breakpoints occur within the BCR (breakpoint cluster region) gene in chromosome 22 and within the ABL gene in chromosome 9. The BCR-ABL fusion gene (that codes for the Bcr-abl fusion protein) is thus created by the swapping of the ABL gene on chromosome 9 to a part of the BCR gene on chromosome 22.

It has been found that the human genome is full of relic retroviral DNA sequences called HERVs, standing for Human Endogenous RetroViruses (The Medical News, 2011) (Purdom G., 2006). A transposon is a piece of DNA sequence that can move from one position to another position in the genome, hence its other name of 'jumping gene'. HERVs and transposons are held in check from doing havoc in the genome by epigenetic constraints, namely DNA methylation and histone modifications, that make the chromatin to become heterochromatized (i.e. condensed and compacted chromatin) and so silenced for transcription (Florl et al., 1999). It has been shown that in the progression of carcinogenesis, one stage involves genome-wide DNA hypomethylation and thus the stability that the epigenetic constraints give to the genome is lost, giving HERVs and transposons the chance to create chaos. For example if during the transposition, the transposon sequence, with intact exons (coding parts of a gene) replaces the terminal exons of one gene, a fusion gene is created. The fusion gene is then transcribed, spliced (a process where introns are removed from a gene leaving exons only) and translated into a fusion protein. If the fusion gene is an oncogene, an oncogenic fusion protein is created. Cancers in which fusion genes are found include several hematological cancers, sarcomas and prostate cancer.

It is hypothesized that HERVs and transposons do not only mean trouble. Indeed, it has been proposed that some of these fusion genes might have coded for novel proteins that might have been beneficial for the evolution of our species (Purdom G., 2006).

Antibodies

Antibodies are also fusion proteins which are produced by the process of VDJ recombination (Purves et al., 2003). The latter is a complex process that involves cleaving and rearranging DNA sequences that code for the antigen receptors of immunoglobulins. The DNA rearrangements result in various combinations that contribute to a wide diversity of possible antibodies, which can even range into thousands.

Man-Made Fusion Proteins

(i) Insulin was the First Man-Made Therapeutic Fusion Protein

Insulin produced by recombinant DNA technology can be considered as the first therapeutic human protein to be produced via fusion protein technology. In the original process carried out in bacteria, synthetic genes that encode the A and B subunits of insulin were constructed. Each synthetic gene was inserted into a separate vector, adjacent to the lacZ gene, the latter encoding the enzyme β-galactosidase. When transferred to the expression system, a bacterial host (namely E. coli), the lacZ gene and the synthetic gene were transcribed and translated as a unit. The product was thus a fusion protein-that is, a hybrid protein, which consisted of the amino acid sequence for β-galactosidase and the amino acid sequence for one of the insulin subunits. The fusion proteins were then purified from the bacterial extracts. On being treated with cyanogen bromide, the fusion protein was cut from the β-galactosidase. On mixing the fusion products, the two insulin subunits united, forming the insulin molecule.

Figure 1. Flow diagram showing the main processes involved in the synthesis of recombinant

human insulin (e.g. Humulin) using E.coli as expression host cells. (Purves et al., 2003)

(ii) Manufacture of other Man-Made Fusion Proteins

Since recombinant insulin came into the arena of biotechnology, well over 200 recombinant products have entered the market worldwide (Ratledge & Kristiansen, 2007). One road of research was the synthesis of chimeric proteins to be used for drug targeting.

Generally, the creation of a fusion protein involves the removal of the stop codon from a cDNA (complementary DNA) sequence coding for the first protein, and then adding the cDNA sequence of the second protein through ligation using the enzyme ligase (Ratledge & Kristiansen, 2007). The engineered protein can include the full sequence of both original proteins, or only a portion of either as long as the portion chosen gives the desired bioactive effect. The new recombinant DNA sequence is subsequently introduced into an expression system where it will be expressed as a single protein. Generally, linker peptides (also called "spacer" peptides) that link the two proteins to be fused, are added. These linker peptides have a twofold function. First it makes it more likely that the two proteins fold into their 3-dimensional configuration and so exhibit their normal biological behaviour. Moreover, linker peptides also may be used to enable the fused protein to be purified in the down-streaming processes (the steps by which biomolecules, including fusion proteins, are clarified, concentrated, purified and formulated into a marketable product) (Ratledge & Kristiansen, 2007).

(iii) Choice of Expression Systems

Fusion proteins may be expressed in cell-based or cell-free systems (McCornack et al., 2008). Cell-based expression systems used include mammalian (including human), insect, yeast or bacterial cells (refer to Table 1). The expression system used depends on (i) the type of fusion protein that needs to be synthesized and (ii) its amount. Bacterial cells are often used when a large-scale fusion protein expression is needed. This is because bacterial cells could be cultured in a cost-effective manner in large numbers in large bioreactors, yielding a high production of the desired fusion protein. The drawback of using bacterial cells is that such cells, being prokaryotic, do not have the ability to perform the post-translational modifications of eukaryotic cells. Thus if the fusion protein needs to undergo glycosylation (the addition of a carbohydrate moiety) or disulphide bond formation, mammalian or insect cells are opted for. For some fusion proteins, such post-translational processing is important for they influence the proteins stability, folding, solubility (Ratledge & Kristiansen, 2007) and therefore their correct bioactive and therapeutic effects. Indeed, if not present they can make the protein immunogenic and cause serious side-effects (including anaphylaxis) when used therapeutically. Yeast cells could also be used to express human proteins that are correctly folded and disulphide-bridged. However, glycosylation still differs from that seen in mammalian cells.

Type of Expression System

Name

Mammalian cells

Chinese hamster ovary (CHO) cells

Baby hamster kidney (BHK) cells

Mouse myeloma (NSO, SP/O) cells

Human cells

Insect cell/baculovirus

Sf-9, Sf-21 (Ovarian tissue of Spodoptera frugiperda)

SL-2, SL-3 (Drosophila melanogaster)

Yeast cells

Saccharomyces cerevisiae

Pichia pastoris

Bacterial cells

Escherichia coli

Bacillusspecies

Table 1. Choice of Expression System. (Ratledge & Kristiansen, 2007)

Some Typical Therapeutic Uses Of Man-Made Fusion Proteins

(i) Immunotoxins in Cancer Treatment

The aim of an immunotoxic type of fusion protein is drug targeting, i.e. to kill target cells such as cancer cells, while leaving normal tissues unharmed. Generally, immunotoxins combine several important features to have this 'magic bullet' effect (Figure 2). The antibody moiety makes the immunotoxin to bind specifically to a cell surface antigen (like for example a tumour-associated antigen) which is expressed on the target cell only. An internalization process then helps with the delivery of the toxic moiety to the cytoplasm. Once inside the cytoplasm, the toxic moiety inhibits a vital cell function (like protein synthesis) causing the cell to die by apoptosis (programmed cell death).

Figure 2. General Structure and Mode Of Action of an Immunotoxic Chimeric Protein. (???)

Some definitions are appropriate at this stage, since it is very useful to know the difference between apoptosis, necrosis and cytotoxicity when it comes to choose promising immunotoxic fusion-proteins. Necrosis or apoptosis are the two main mechanisms by which cell death can occur. Moreover, some chemical compounds are described to be cytotoxic to the cell, implying that they cause the cell to die. Necrosis ("accidental" cell death") is a pathological process that leads to cellular death due to a physcial or chemcial insult (Rode et al., 2005). Conversely, apoptosis is one form of "programmed" cell death. It is a chain of physiological events through which unwanted or useless cells are removed. Such events commonly occur during the development of an organism (Rode et al., 2005) or when the cell receives chemical cues that its normal biological processes are not going to work well, e.g. apoptosis occurs (when mistakes that cannot be corrected) occur during the phase of DNA replication of the cell cycle. In apoptosis, the cell participates actively in its own demise, hence its nickname "cellular suicide".

Cytotoxicity implies the killing-property of a chemical. Unlike apoptosis or necrosis, the term cytotoxicity does not specifiy how the cell's death is brought about. For example, a cytotoxic compound can kill a cell by necrosis or apoptosis.

Necrosis is not good. Generally, if a compound causes necrotic death it would have little potential as a therapeutic agent. This is because necrotic cells burst and release their cytoplasmic content (including lysosomal enzymes) into the surrounding extracellular fluid, causing an intense inflammatory response that is potentially harmful to neighbouring, non-target cells. On the contrary, apoptosis produces a natural discrete form of 'cell suicide' which does not cause inflammation. Thus usually, immunotoxic fusion proteins that cause apoptotic death are opted for.

Cell-surface receptors are being used as targets for man-made immunotoxic fusion proteins. A number of cell-surface receptors (refer to Table 2) have already been identified on tumour cells. Some are only expressed on cancer cells while others are expressed relatively more on cancer cells than on normal cells. For example, cells of most human carcinomas of the colon, breast, ovary, and lung (non-small cell) have been found to express abundantly the Lewisy (Ley) cell surface receptor (Hellstrom et al., 2000). Normal tissues however have a low expression of this receptor. This is conducive to use this receptor to target cancer cells expressing it without harming normal tissues and so minimizing collateral side-effects.

Tumour-Associated Antigens

GnRH (gonadotropin releasing hormone) binding receptor

IL-2 receptor (interleukin 2 receptor)

IL-4 receptor (interleukin 4 receptor)

IL-6 receptor (interleukin 6 receptor)

IL-13 receptor (interleukin 13 receptor)

TR receptor (transferrin receptor)

EGFR (epidermal growth factor receptors)

e.g. HER2/neu (also known as ErbB-2)

= Human Epidermal growth factor Receptor 2

Lewisy (Ley) receptor

CD7 (Cluster of differentiation-7)

CD19 (Cluster of differentiation-19)

CD22 (Cluster of differentiation-22)

CD25 (Cluster of differentiation-25)

CD30 (Cluster of differentiation-30)

CD33 (Cluster of differentiation-33)

CD56 (Cluster of differentiation-56) (also called NCAM)

Mesothelin antigen

Mucin carbohydrate

Table 2. Some Cell Surface Antigens Expressed

On Tumour Cells That Are Targeted By Immunotoxins.

A breakthrough in the development of man-made immunotoxic fusion proteins was the introduction of hybridoma technology, pioneered in the 1970s by Kohler and Milstein (Ratledge & Kristiansen, 2007). This made monoclonal antibodies (mAbs) to become available in limitless supply. This was the era of the "first generation" of immunotoxins. These immunotoxins were fusion proteins linking mAbs to potent protein toxins. The toxins were derived from plants or bacteria and some examples included ricin, abrin, saporin, Pseudomonas aeruginosa exotoxin (PE), cholera toxin (CT) and Diphtheria toxin (DT) (Table 3). Their immunotoxic effect was remarkable in vitro but not in vivo. Indeed, in animals or humans, many exhibited poor anti-tumor effects and excessive toxicity.

Name of Toxin

Action of Toxin

Plant Toxin

Ricin

Abrin

Modeccin

Gelonin

Saporin

protein synthesis inhibition

Bacterial Toxin

Diphteria toxin (DT)

Pseudomonas aeruginousa exotoxin A (PE)

Cholera toxin

Fungal Toxin

Alpha-sacrin

Table 3. Protein Toxins Used In 'First Generation' Immunotoxins.

Thus this lead to the synthesis of "second generation" immunotoxins. These were fully recombinant antibody-toxin chimeric molecules. Generally, they consisted of a single-chain antibody that was genetically fused to a truncated version of either Diphtheria toxin (DT) or Pseudomonas aeruginosa exotoxin (PE). The truncated version generally consists of the toxin molecule lacking certain domains (condensed globular regions of proteins that form part of the protein's tertiary structure).

These domains can be eliminated without affecting the toxicity of the toxin once inside the targeted cell. The eliminated domains usually show intrinsic cell binding capacity enabling the toxin molecule to bind to non-targeted cells. Hence their absence in the truncated version of the toxin causes less side-effects during treatment.

Research is continuing and is aimed mainly at finding new selective targets (i.e. receptors) on tumour cells and finding new ways of killing these cells by effective cytotoxic reagents.

Fusion Protein

Tumour

GnRH-based

Gonadotropin Releasing

Hormone-DNA Fragmentation

Factor 40 Chimeric Protein(GnRH-DFF40)

Breast, prostatic, pancreatic, endometrial and ovarian carcinoma.

(specifically target and kill adenocarcinomas)

IL-4 receptor based

IL-4(38-37)-PE38KDEL

Circular permuted IL-4-Pseudomonas exotoxin fusion protein.

MPM (Malignant pleural mesothelioma)

Human medulloblastoma tumours

CD-25 receptor based

RFT5-SMpt-dgA

Hodgkin's disease (22)

CD-22 receptor based

RFB4(dsFv)-PE38 or (BL22)

B cell Leukemia (22)

Mesothelin receptor based

SSIP (SS1(dsFv)-PE38)

Mesothelioma

Ovarian carcinoma

Pancreatic carcinoma

NCAM receptor based

N901-bR

Small cell lung cancer (SCLC)

erbB2 recpetor based

ScFv(FRP5)-ETA

Cutaneous metastases of colon and breast cancers

IL-13 receptor based

IL13-PE38QQR

Metastatic renal cell carcinoma

Table 4.

Compared to solid tumors, hematologic malignancies are the most sensitive diseases when treated with immunotoxins (Kreitman, 2006). This is due to two reasons. First the malignant cells are inside the vascular system where they can be easily reached by the immunotoxin that is given intravenously. Secondly, patients do not make neutralizing antibodies against the toxin because they often have associated immunosuppression.

(ii) Chimeric Proteins in the Treatment of Auto-Immune Diseases

Currently chimeric proteins are also being researched to be used medically in the treatment of auto-immune disease such as rheumatoid arthritis (including juvenile rheumatoid arthritis), psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, vasculitis, and lupus amongst others. Etanercept (trade name Enbrel) is an example of one such chimeric protein that is being used to this end.

Tumour necrosis factor (TNF) is a cytokine involved in systemic inflammation and is a member of a group of cytokines that stimulate the acute phase reaction of inflammation (Wikipedia). Its main role is to regulate immune cells. Aberration in its regulation (in particular, its overproduction) have been implicated in a variety of autoimmune disease. Etanercept treats autoimmune diseases by interfering with the tumour necrosis factor (TNF).

Figure 3. Domain Structure of Etanercept. (Ratledge & Kristiansen, 2007)

Etanercept is an "immunoadhesin" fusion protein consisting of the extracellular ligand binding portion of the human tumor necrosis factor receptor (TNFR) linked to the Fc portion of a human IgG1. (figure 3). The first part of the chimeric construct blocks the action of the receptor whereas the second part helps to stabilize this bioactive protein in circulation Ratledge & Kristiansen, 2007).

(iii) Gene Fusion Technology being applied for the Treatment of HIV

When the immune system is weakened by infection with HIV-1 (human immunodeficiency virus), the symptoms of AIDS (acquired immunodeficiency syndrome) may result. Specifically, HIV infects and kills cells of the immune system that carry a cell-surface receptor known as CD4. An HIV surface protein known as gp120 binds to the CD4 receptor and allows the virus to enter the cell. The gene that encodes the CD4 protein has now been cloned and it is envisaged to be used along with recombinant DNA techniques to combat HIV infection. In one strategy, the CD4 gene has been fused with a gene encoding a bacterial toxin. The resulting fusion protein contains a CD4 region that binds to gp120 on the surface of HIV-infected cells and a toxin region that then kills the infected cell. In tissue culture experiments, cells infected with HIV are killed by this fusion protein, whereas uninfected cells survive.

Figure 4. HIV Adsorption Process. (De Clercq, 2003)

gp120 is a glycoprotein in the HIV envelope.

gp120 locks with CD4 receptor in the cell membrane of the host cell.

This facilitates the locking of gp120 with co-receptor CXCR4, thus anchoring the virus to the cell.

(iv) Chimeric Proteins in the Treatment of Asthma

Here chimeric proteins are being used to target Interleukin (IL)-4 and Interleukin (IL)-13 receptors. These receptors are expressed on T-helper type 2 (Th2) cells which are associated with the pathogenesis of atopy in asthma. Specifically, the cytokines IL-4 and IL-13 bind to their respective receptor, which then bring about a transduction of events leading to the inflammation. Research is ongoing to use chimeric proteins that immuno-neutralise these receptors and so modulate the asthmatic phenotype, especially the uncontrolled and severe one (Oh et al., 2010), (Blease et al., 2001).

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