INTERFERONS:

“Interferon” was discovered as an antiviral activity, without reference to antigenic type or tissue
of origin. Later it was discovered that interferons produced from lymphoid, as against to
fibroblostoied. They are distinct regarding hetero specific activity using bovine or porcine cell
cultures and in certain gross chemical properties. Therefore, the distinct interferons were named
according to the tissue or organ.

Fibroblast or leukocyte subsequently it was found that lymphoid cells are capable of synthesizing
“leukocyte interferons” in response to the viral infection. That the terms fibroblast and
leucositesare applied to the human interferons, denote the antigenic properties of the interferons
but not the cell type of origin. For this reason the human interferons are further divided in to
alpha and beta (leukocyte) The most effective inducers for the interferons is the virus and the
double-helical RNA’s of natural or synthetic origin. Generally depending up on the cytogenic
studies of screening of interferon loci and biochemical studies and their messenger RNAs,
presently around fifteen IF-alpha, a three IF-beta one gamma locus. Then the interferon alpha
and beta are sub divided into

The IFN-α proteins are produced by leukocytes. They are mainly involved in innate immune
response against viral infection. They come in 14 subtypes that are called IFNA1, IFNA2,
IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17,
IFNA21. These genes for these IFN-α molecules are found together in a cluster on chromosome

The IFN-β proteins are produced in large quantities by fibroblasts. They have antiviral activity
which is mainly involved in innate immune response. Two types of IFN-β have been described,
IFN-β1 (IFNB1) and IFN-β3 (IFNB3)[4] (a gene designated IFN-β2 is actually IL-6).
Interferonbeta-1a is used a treatment for relapsing remitting multiple sclerosis

Interferons are potent natural substances composing of proteins with the molecular weight of 20-
24x103 Daltons. Some time these are glycol proteins and probably contain other post
translational modifications including of phosphorylation and disulfide linkage etc… due to the
following reasons chemical properties of interferons are determined late.
1. Generally the small amounts of the interferons are available.

2. Contamination of interferons with non interferon proteins and purification problems.

3. The multiplicity of distinct interferons gene products is possible to post-synthetic

modifications.

4. The chemical properties are unstable.

The partial amino acid sequenced data is favor the production of recombinant plasmids with
interferon sequence. Such plasmids are used to produce interferons by using bacteria.

Biological properties of interferons have biphasic mechanism as effect on cell surface receptors
and able to enter in to the cell. Interferons resemble the chemical properties like some group of
proteins and hormones like insulin, follicle-stimulating hormone, leutinizing hormone etc.

Production of interferons

By using of recombinant technology interferons are producing by using animal cells and
microorganisms. Naturally the animal cell culture is costly and inefficient to produce interferons.
In the production 103-104 IL/ML and 103-104 IU/mg will produced in to the medium. it means
few nano grams of interferons will be present in the medium. And it requires 10 5-106 IU/mg is
require to get effective purification.

The syntheses of interferons are as invitro and invivo, by the infection of virus or by the
treatment of non viral materials which are high molecular weight. In mammalian systems these
are produced by the synthetic double helical RNA, either which added from outside or produced
due to the action of viral replication.

Production of interferons by cell culturing technique.

In early days interferons are produced by viral induction, but now leucocytes as well as
nonhuman interferons are still produced efficiently using paramyxovirus inducers. Fibroblast
interferon is usually prepared in human fibroblast cultures using polyinosinic acid and
polycitidylic acid in combination with the inhibitors of RNA by the method called super
induction.
Preparation of Human IF-alpha has necessarily employed leukocytes prepared from the large
quantities of fresh blood. Blood cannot be viewed realistically as a suitable source either
qualitatively or quantitatively. Because problem arising in collection, storage, etc. where as the
primary diploid human fibroblast strain is preferred as the source of human IF- beta.

Production of interferons by Recombinant microorganisams :

Human interferon alpha was first produced in E.coli in the year of 1980 by the biogen SA.
Currently mammalian interferons can be manufactured using recombinant technology with a
good yields of 106 IU/mL purity also very god. However the interferons are not secreted in to the
medium but we have to extracted from the cell. By the recombinant technology the production
and purity of interferons were increased.

 Iinterferon gene sequence cloned into pst1 Site of pBR 322 and is used as transform
E.coli
 Transferred E.coli pool was hybridized to a crude IFN mRNA preparation
 The hybridized mRNA was separated from the cloned DNA-mRNA hybrids and
translated in a cell free protein synthesis system.
 Each translation mixture was then assayed for IFN antiviral activity.
 Cloned with a cDNA that the hybridized with mRNA shows IFN activity.
 Positive cDNA for human IFN was identified by the repeated retesting.
 The IFN cDNA can be sub cloned in to an E.coli expression vector and expressed at high
levels.
Steps involved in interferon production

BIOLOGICAL ACTIVITIES of interferon alfa

All known subtypes of IFN-alpha show the same antiviral antiparasitic, antiproliferative
activities in suitable bioassays although they may differ in relative activities.

Human IFN-alpha is also a potent antiviral substance in murine, porcine, and bovine cell
systems. Human IFN-alpha is less active in rodent cells. Site-directed mutagenesis techniques
have been used to create some variants of certain subtypes (IFN-alpha-2) that display
approximately 100-fold enhanced antiviral activities in mouse cells.

IFN-alpha inhibits the expression of a number of cytokines in hematopoietic progenitor cells that
in turn induce a state of competence in these cells allowing them to pass from the G0 into the S
phase of the cell cycle.

The growth of some tumor cell types in vitro is inhibited by IFN-alpha which may stimulate also
the synthesis of tumor-associated cell surface antigens. In renal carcinomas IFN-alpha reduces
the expression of receptors for EGF. IFN-alpha also inhibits the growth of fibroblasts and
monocytes in vitro. IFN-alpha also inhibits the proliferation of B-cell in vitro and blocks the
synthesis of antibodies. IFN-alpha also selectively blocks the expression of some mitochondrial
genes.

IFN-alpha specifically induces the expression of a number of genes. These genes contain
regulatory DNA sequences within their promoter regions (ISRE; Interferon-stimulated response
element; interferon response sequence) that function as binding sites for a number of
transcription factors. Some of these genes are expressed also in response to other interferons.

The occurrence of spontaneous antibodies directed against IFN-alpha has been observed in
patients with certain types of autoimmune diseases, generalized virus infections, and a number of
tumors. Some inbred strains of mice appear to produce constitutively antibodies directed against
IFN-alpha or IFN-beta.

CLINICAL USE AND SIGNIFICANCE OF ALPHA INTERFERONS:

IFN-alpha is mainly employed as a standard therapy for hairy cell leukemia, metastasizing renal
carcinoma and AIDS-associated angiogenic tumors of mixed cellularity known as Kaposi
sarcomas. It is also active against a number of other tumors and viral infections. IFN-alpha is
approved by the Food and Drug Administration for the treatment of condyloma acuminata
(genital or venereal warts).

Hairy cell leukemia constitutes approximately 2 % of all leukemias. Treatment with IFN-alpha
markedly improves blood and bone marrow parameters. The number of necessary blood
transfusions is reduced and the frequency of life-threatening infections is also reduced.

Treatment of disseminated Kaposi sarcomas results in complete or partial remissions in

approximately 30-40 % of the patients. In patients with advanced malignant melanomas
treatment with a combination of IFN-alpha and chemotherapy (Dacarbazin, DTIC) has been
found to be particularly effective and to be superior to treatment with IFN-alpha alone. Complete
remissions and also a significant increase in survival times have been observed in responders.
Intralesional therapy with IFN-alpha has been found to cause almost complete disappearance of
tumors in 80 % of patients with basaliomas.

Moderate and high doses of IFN-alpha are one of the most effective forms of treatment of
metastasizing renal carcinomas. Response rates of combinations of vinblastin and IFN-alpha are
approximately 25 % higher than those with interferon alone. Response rates have been reported
to be improved by combining IFN-alpha with antineoplastic agents or other cytokines.
Combination therapy with systemically administered IL2 and IFN-alpha has resulted in long-
term remissions in 30 % of patients with metastatic renal cell carcinoma.
Treatment of CML with IFN-alpha causes hematological remissions in most patients and has
been shown to cause a complete elimination of the PH1-(Philadelphia chromosome)-positive
cells in the bone marrow of some patients.

Prospective studies are now under way to evaluate the effectiveness of IFN-alpha in the
treatment of non-Hodgkin lymphomas, cutaneous T-cell lymphomas (Mycosis fungoides, Sézary
syndrome), multiple myelomas, condylomata acuminata and chronic active hepatitis B.

Some patients treated with genetically engineered recombinant IFN-alpha-2 have been shown to
develop neutralizing antibodies against interferon. Increasing levels of antibodies correlate with
increasing reoccurrence of the disease. Therefore, patients should be monitored for the presence
and clinical relevance of IFN-alpha antibodies to determine those who could respond to
alternative treatment. It has been assumed that the recombinant protein may possess an altered
tertiary structure leading to the exposure of a novel immunoreactive epitope not normally
recognized in natural IFN-alpha. Continuation of the treatment with natural purified IFN-alpha
leads to the disappearance of antibodies and also causes remissions.

In many instances a combination of the various interferons has been found to cause synergistic
effects. The antiviral/antiproliferative/antitumor properties of IFN-alpha is potentiated by febrile
temperatures.

BIOLOGICAL ACTIVITIES OF INTERFERON BETA:

In contrast to IFN-alpha IFN-beta is strictly species-specific. IFN-beta of other species is inactive

in human cells.

IFN-beta is involved in the regulation of unspecific humoral immune responses and immune
responses against viral infections. IFN-beta increases the expression of HLA class 1 antigens and
blocks the expression of HLA class 2 antigens stimulated by IFN-gamma. IFN-beta stimulates
the activity of NK-cells and hence also antibody-dependent cytotoxicity. The activity of T
suppressor cells elicited by several stimuli is stimulated also by IFN-beta. IFN-beta enhances the
synthesis of the low affinity IgE receptor CD23. In activated monocytes. IFN-beta induces the
synthesis of neopterin. It also enhances serum concentrations of Beta-2-Microglobulin. IFN-beta
selectively inhibits the expression of some mitochondrial genes. IFN-beta shows antiproliferative
activity against a number of cell lines established from solid tumors.

CLINICAL USE AND SIGNIFICANCE OF INTERFERON BETA

IFN-beta can be used for topic treatment of condylomata acuminata. It is also suitable for the
prophylactic use following surgical removal of large condylomas. Some studies suggest that
IFN-beta tends to prevent disease activity in patients with multiple sclerosis.

IFN-beta in combination with IFN-alpha has been used in the treatment of chronic active
hepatitis B and appears to be most promising if the disease has not lasted longer than 5 years.
The antiviral activity of IFN-beta is demonstrated also in the treatment of severe childhood viral
encephalitis.

A combination treatment in combination with acyclovir is more effective than treatment with
acyclovir alone.

IFN-beta is a lipophilic molecule that should be particularly useful for local tumor therapy due to
its specific pharmacokinetics. It is hardly removed from the tumor tissues after intralesional
administration and hence also shows little systemic side effects. Head and neck squamous
carcinomas, mammary and cervical carcinomas, and also malignant melanomas respond well to
treatment with IFN-beta. IFN-beta also appears to be very promising for the adjuvant therapy of
malignant melanomas with a high potential for metastasis. Response rates have been reported to
be improved by combining IFN-beta with antineoplastic agents or other cytokines.

In many instances a combination of the various interferons has been found to cause synergistic
effects. The antiviral/antiproliferative/antitumor properties of IFN-beta is potentiated by febrile
temperatures.