UNIT 5

ANIMAL CELL CULTURE: PRODUCTION OF SECONDARY

METABOLITES/PRODUCTS:

INTERFERONS:

Interferons(IFNs) are proteins made and released by host cells in response to the presence of
pathogenssuch as viruses, bacteria, parasites or tumor cells. They allow for communication
between cells to trigger the protective defenses of the immune system that eradicate pathogens or
tumors.

IFNs belong to the large class of glycoproteins known as cytokines. Interferons are named after
their ability to "interfere" with viral replication within host cells. IFNs have other functions: they
activate immune cells, such as natural killer cells and macrophages; they increase recognition of
infection or tumor cells by up-regulating antigen presentation to T lymphocytes; and they
increase the ability of uninfected host cells to resist new infection by virus. Certain symptoms,
such as aching muscles and fever, are related to the production of IFNs during infection.

Functions
All interferons share several common effects; they are antiviral agents and can fight tumors. As
an infected cell dies from a cytolytic virus, viral particles are released that can infect nearby
cells. However, the infected cell can warn neighboring cells of a viral presence by releasing
interferon. The neighboring cells, in response to interferon, produce large amounts of an
enzymeknown as protein kinase R(PKR). This enzyme phosphorylates a protein known as eIF-2
in response to new viral infections; the phosphorylated eIF-2 forms an inactive complex with
another protein, called eIF2B, to reduce protein synthesis within the cell. Another cellular
enzyme, RNAse L— also induced following PKR activation—destroys RNA within the cells to
further reduce protein synthesis of both viral and host genes. Inhibited protein synthesis destroys
both the virus and infected host cells. In addition, interferons induce production of hundreds of
other proteins—known collectively as interferon-stimulated genes (ISGs)—that have roles in
combating viruses. They also limit viral spread by increasing p53 activity, which kills virus-
infected cells by promoting apoptosis. The effect of IFN on p53 is also linked to its protective
role against certain cancers.

Another function of interferons is to upregulate major histocompatibility complex molecules,

MHC Iand MHC II, and increase immunoproteasomeactivity. Higher MHC I expression
increases presentation of viral peptides to cytotoxic T cells, while the immunoproteasome
processes viral peptides for loading onto the MHC I molecule, thereby increasing the recognition
and killing of infected cells. Higher MHC II expression increases presentation of viral peptides
to helper T cells; these cells release cytokines (such as more interferons and interleukins, among
others) that signal to and co-ordinate the activity of other immune cells.

Interferons, such as interferon gamma, directly activate other immune cells, such as macrophages
and natural killer cells. Interferons can inflame the tongue and cause dysfunction in taste bud
cells, restructuring or killing taste buds entirely.

Interferon therapy

The immune effects of interferons have been exploited to treat several diseases. Agents that
activate the immune system, such as small imidazoquinoline molecules that activate TLR7, can
induce IFN-α. Imidazoquinoline is the main ingredient of Aldara (Imiquimod) cream, a treatment
approved in the United States by the Food and Drug Administration (FDA) for actinic keratosis,
superficial basal cell carcinoma, papilloma and external genital warts. Synthetic IFNs are also
made, and administered as antiviral, antiseptic and anticarcinogenic drugs, and to treat some
autoimmune diseases.

New research has shown that imiquimod's anti-proliferative effect is totally independent of
immune system activation or function. Imiquimod exerts its effect by increasing levels of the
opioid growth factor receptor (OGFr). Blocking OGFr function with siRNA technology resulted
in loss of any antiproliferative effect of imiquimod.

Interferon beta-1a and interferon beta-1b are used to treat and control multiple sclerosis, an
autoimmune disorder. This treatment is effective for slowing disease progression and activity in
relapsing-remitting multiple sclerosis and reducing attacks in secondary progressive multiple
sclerosis.

Interferon therapy is used (in combination with chemotherapy and radiation) as a treatment for
many cancers. This treatment is most effective for treating hematological malignancy; leukemia
and lymphomas including hairy cell leukemia, chronic myeloid leukemia, nodular lymphoma,
cutaneous T-cell lymphoma.[21]Patients with recurrent melanomas receive recombinant IFN-α2b.
Type I IFNs have a therapeutic potential for the treatment of a wide variety of leukemias and
solid tumors due to their antiproliferative and apoptotic effects, their anti-angiogenic effects and
their ability to modulate an immune response specifically activating dendritic cells, cytolytic T
cells and NK cells. Research in this area is receiving intensive investigation. Interferon a 2b is
also being used for treatment of ocular surface squamous neoplasia (OSSN) in the form of
perilesional injection followed by topical interferon a 2b drops at Lahore General Hospital Eye
unit II.

Both hepatitis B and hepatitis C are treated with IFN-α, often in combination with other antiviral
drugs. Some of those treated with interferon have a sustained virological response and can
eliminate hepatitis virus. The most harmful strain—hepatitis C genotype I virus—can be treated
with a 60-80% success rate with the current standard-of-care treatment of interferon-α, ribavirin
and recently approved protease inhibitors such as Telaprevir (Incivek) or Boceprevir (Victrelis).
Biopsies of patients given the treatment show reductions in liver damage and cirrhosis. Some
evidence shows giving interferon immediately following infection can prevent chronic hepatitis
C, although diagnosis early in infection is difficult since physical symptoms are sparse in early
hepatitis C infection. Control of chronic hepatitis C by IFN is associated with reduced
hepatocellular carcinoma.

Administered intranasally in very low doses, interferon is extensively used in Eastern Europe and
Russia as a method to prevent and treat viral respiratory diseases such as cold and flu. However,
mechanisms of such action of interferon are not well understood; it is thought that doses must be
larger by several orders of magnitude to have any effect on the virus. Although most scientists
are skeptical of any claims of good efficacy recent findings suggest that interferon applied to
mucosa may act as an adjuvant against influenza virus, boosting the specific immune system
response against the virus. A flu vaccine that uses interferon as adjuvant is currently under
clinical trials in the US.

When used in the systemic therapy, IFNs are mostly administered by an intramuscular injection.
The injection of IFNs in the muscle, in the vein, or under skin is generally well tolerated. The
most frequent adverse effects are flu-like symptoms: increased body temperature, feeling ill,
fatigue, headache, muscle pain, convulsion, dizziness, hair thinning, and depression. Erythema,
pain and hardness on the spot of injection are also frequently observed. IFN therapy causes
immunosuppression, in particular through neutropenia and can result in some infections
manifesting in unusual ways.
 Insulin:

The hormone insulin is produced by the (J-cells of islets of Langerhans of pancreas. Human
insulin contains 51 amino acids, arranged in two polypeptide chains. The chain A has 21 amino
acids while B has 30 amino acids. Both are held together by disulfide bonds.

In the absence of insulin, glucose accumulates in the blood stream at higher concentration,
usually when the blood glucose concentration exceeds about 180 mg/dl, glucose is excreted into
urine. The patients of diabetes are weak and tired since the production of energy (i.e. ATP) is
very much depressed.

The more serious complications of uncontrolled diabetes include kidney damage (nephropathy),
eye damage (retinopathy), nerve diseases (neuropathy) and circulatory diseases (atherosclerosis,
stroke). In fact, diabetes is the third leading cause of death (after heart disease and cancer) in
many developed countries.

Production of recombinant insulin:

Attempts to produce insulin by recombinant DNA technology started in late 1970s. The basic
technique consisted of inserting human insulin gene and the promoter gene of lac operon on to
the plasmids of E. coli. By this method human insulin was produced. It was in July 1980,
seventeen human volunteers were, for the first time, administered recombinant insulin for
treatment of diabetes at Guy’s Hospital, London.

And in fact, insulin was the first ever pharmaceutical product of recombinant DNA technology
administered to humans. Recombinant insulin worked well, and this gave hope to scientists that
DNA technology could be successfully employed to produce substances of medical and
commercial importance. An approval, by the concerned authorities, for using recombinant
insulin for the treatment of diabetes mellitus was given in 1982. And in 1986, Eli Lilly Company
received approval to market human insulin under the trade name Humulin.

Technique for recombinant insulin production:

The original technique of insulin synthesis in E. coli has undergone several changes, for
improving the yield, e.g. addition of signal peptide, synthesis of A and B chains separately etc.
The genes for insulin A chain and B chain are separately inserted to the plasmids of two different
E. coli cultures.

The lac operon system (consisting of inducer gene, promoter gene, operator gene and structural
gene Z for β-galactosidase) is used for expression of both the genes. The presence of lactose in
the culture medium induces the synthesis of insulin A and B chains in separate cultures. The so
formed insulin chains can be isolated, purified and joined together to give a full-pledged human
insulin.

Second generation recombinant insulin’s:

After injecting the insulin, the plasma concentration of insulin rises slowly. And for this reason,
insulin injection has to be done at last 15 minutes before a meal. Further, decrease in the insulin
level is also slow, exposing the patients to a danger of hyper-insulinemia. All this is due to the
existence of therapeutic insulin as a hexamer (six molecules associated), which dissociates
slowly to the biologically active dimer or monomer.

Attempts have been made in recent years to produce second generation insulin by site- directed
mutagenesis and protein engineering. The second generation recombinant proteins are termed as
muteins. A large number of insulin muteins have been constructed with an objective of faster
dissociation of hexamers to biologically active forms. Among these is insulin lispro, with
modified amino acid residues of the B-chain of insulin. Insulin lispro can be injected
immediately before a meal as it attains the pharmacologically efficient levels very fast.

Human Growth Hormone:

Growth hormone is produced by the pituitary gland. It regulates the growth and development.
Growth hormone stimulates overall body growth by increasing the cellular uptake of amino
acids, and protein synthesis, and promoting the use of fat as body fuel.

Insufficient human growth hormone (hGH) in young children results in retarded growth,
clinically referred to as pituitary dwarfism. The child usually is less than four feet in height, and
has chubby face and abundant fat around the waist.

Traditional treatment for dwarfism:

The children of pituitary dwarfism were treated with regular injections of growth hormone
extracted from the brains of deceased humans. It may be noted that only human growth hormone
is effective for treatment of dwarfism. (This is in contrast to diabetes where animal insulin’s are
employed).

At least eight pituitary glands from cadavers must be extracted to get hGH adequate for treating a
dwarf child just for one year! And such treatment has to be continued for 8-10 years!! Further,
administration hGH isolated from human brains exposes the children to a great risk of
transmitting the cadaver brain diseases (through virus or viral-like agents) e.g. Creuzfeldt- Jacob
(CJ) syndrome characterized by convulsions, wasting of muscle etc.

Production of recombinant hGH:

Biotechnologists can now produce hGH by genetic engineering. The technique adopted is quite
comparable with that of insulin production. The procedure essentially consists of inserting hGH
gene into E. coli plasmid, culturing the cells and isolation of the hGH from the extracellular
medium.

Limitation in hGH production:

The hGH is a protein comprised of 191 amino acids. During the course of its natural synthesis in
the body, hGH is tagged with a single peptide (with 26 amino acids). The signal peptide is
removed during secretion to release the active hGH for biological functions. The entire process
of hGH synthesis goes on in an orderly fashion in the body.

However, signal peptide interrupts hGH production by recombinant technology. The

complementary DNA (cDNA) synthesized from the mRNA encoding hGH is inserted into the
plasmid. The plasmid containing E. coli when cultured, produces full length hGH along with
signal peptide. But E. coli cannot remove the signal peptide.

Further, it is also quite difficult to get rid of signal peptide by various other means. Theoretically,
cDNA encoding signal peptide can be cut to solve these problems. Unfortunately, there is no
restriction endonuclease to do this job, hence this is not possible.

A novel approach for hGH production:

Biotechnologists have resolved the problem of signal peptide interruption by a novel approach.
The base sequence in cDNA encoding signal peptide (26 amino acids) plus the neighbouring 24
amino acids (i.e a. total 50 amino acids) is cut by restriction endonuclease ECoRI.

Production of Recombinant Human Growth Hormone

Now a gene (cDNA) for 24 amino acid sequence of hGH (that has been deleted) is freshly
synthesized and ligated to the remaining hGH cDNA. The so constituted cDNA, attached to a
vector, is inserted into a bacterium such as E. coli for culture and production of hGH. In this
manner, the biologically functional hGH can be produced by DNA technology. Recombinant
hGH was approved for human use in 1985. It is marketed as Protropin by Gene-tech Company
and Humatrope by Eri Lilly Company.

Tissue Plasminogen Activator:

Tissue plasminogen activator (tPA) is a naturally occurring protease enzyme that helps to
dissolve blood clots. tPA is a boon for patients suffering from thrombosis. The majority of
natural deaths worldwide are due to a blockade of cerebral or coronary artery by a blood clot,
technically called as thrombus. The phenomenon of thrombus blockage of blood vessels is
referred to as thrombosis.

Chemically, thrombus consists of a network of fibrin, formed from the fibrinogen. In the normal
circumstances, plasmin degrades fibrin and dissolves blood clots. This plasmin is actually
produced by activation of plasminogen by tissue plasminogen activator.

Role of Tissue Plasminogen Activator

The natural biological systems is however, not that efficient to remove the blood clots through
this machinery. Tissue plasminogen activator is very useful as a therapeutic agent in dissolving
blood clots (thrombi) by activating plasminogen. By removing the arterial, thrombi, the possible
damage caused by them on heart and brain could be reduced.

Production of recombinant tPA:

DNA technologists synthesized the complementary DNA (cDNA) molecule for tissue
plasminogen activator. This cDNA was then attached to a synthetic plasmid and introduced into
mammalian cells. They were cultured and tPA-producing cells were selected by using
methotrexate to the medium.

tPA-producing cells were transferred to an industrial tank (fermenter). tPA, secreted into the
culture medium, is isolated for therapeutic purpose. It may be noted here that tPA was the first
pharmaceutical product to be produced by mammalian cell culture.

Recombinant tPA has been in use since 1987 for treatment of patients with acute myocardial
infarction or stroke. Gene-tech was the first to market tPA with a trade name Activase.

Erythropoietin:

Erythropoietin is a hormone synthesized by the kidneys. It stimulates the stem cells of bone
marrow to produce mature erythrocytes. Biotechnologists were successful in producing
recombinant erythropoietin. An approval for its therapeutic use in humans was obtained in the
year 1989. Amgen Inc. first marked erythropoietin with a trade name Epogen. It is useful in
treating the patients with severe anemia that accompanies kidney disease.

Another firm Ortho-Biotech company produced Procrit, a genetically engineered erythropoietin

in 1997. Procrit acts like the natural hormone and stimulates the production of erythrocytes. It is
used in anemic patients undergoing non-cardiac, nonvascular surgery. Procrit administration
before surgery serves as an alternative to blood transfusion. However, therapeutic use of procrit
is quite expensive, hence not widely used.