PRODUCTION OF GROWTH FACTORS, CYTOKINES, AND CHEMOKINES

Types of growth factors, cytokines and chemokines

- Growth factors include cytokines and hormones that bind to specific receptors on the surface of their
target cells.
- Cytokines are primarily secreted from leukocytes. (production of which leukocytes… depends on
stimulation)
- Cytokines that are secreted from lymphocytes are called lymphokines and those secreted by
monocytes or macrophages are called monokines.
- Growth factors are often used interchangeably with cytokines.
- Traditionally, cytokines are associated with hematopoietic cells and the immune system, including
lymphocytes, spleen, thymus, and lymph nodes.
- However, some of the same signaling proteins are also being used by other cells and tissues.
- Growth factors imply positive effects on cell division.
- Cytokines are neutral in terms of their functions.
- Some cytokines promote cell growth or proliferation, such as granulocyte colony stimulating factor
(GCSF) and granulocyte macrophage colony stimulating factor (GM-CSF).
- Other cytokines exhibit inhibitory effects on these processes, such as Fas ligand as “death” signal
causing target cells to undergo programmed cell death or apoptosis.
- Chemokines are a family of pro-inflammatory activation-inducible cytokines or small protein signals
secreted by cells.
- Chemokines induce directed chemotaxis in nearby responsive cells and therefore the name
chemotactic cytokines.

Type Representative growth factors, cytokines and chemokines

Adipose-derived cytokine Leptin

Immunoregulatory lymphokine IL-2, IL-9, IL-10, IL-13, IL-15

Interferon INF-α, INF-β, INF-γ, INF-λ

Osteoinductive cytokine BMP-2

TGF superfamily TGF-α, TGF-β1 to TGF-β4, GDF-1 to GDF-15, BMP-1 to BMP-

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Functions of growth factors, cytokines and chemokines
- Growth factors bind to receptors on the cell surface and subsequently activate cellular proliferation or
differentiation.
- Many growth factors stimulate cellular division in numerous cell type, whereas others are specific to
particular cell types.
- Promote cell differentiation and maturation, whereas varies between growth factors. For example,
bone morphogenic proteins stimulate bone cell differentiation, whereas vascular endothelial growth
factors stimulate blood vessel differentiation.
- Cytokines stimulate the humoral and cellular immune responses and activate phagocytic cells.
- Each cytokine binds to a specific cell-surface receptor
- Upregulation or downregulation of gene expression, resulting in the production of other cytokines
- The major functions of cytokines could be classified into three categories:
(1) autocrine: cytokine acts on the cells that secrete it;
(2) paracrine: cytokine action is restricted to the immediate vicinities of cytokine’s secretion;
(3) endocrine: cytokine diffuses to distant regions of the body to affect different tissues.
- Chemokines induce directed chemotaxis in nearby responsive cells. They are released from various
cells in response to bacteria and viruses infection and in response to agents that cause physical damage
such as silica or urate crystals.
- The main functions of chemokines are chemoattractants for leukocytes.
- They help to recruit monocytes, neutrophils, and other effector cells from the blood to the sites of
infection or damage.
- They serve to guide cells involved in innate immunity and in the adaptive immune system. Some
chemokines have other roles in the development of lymphocytes, migration, and the growth of new
blood vessels.
- cytokines: mostly from animals, cytokines’ function is mostly control the proliferation, especially B
cells and T cells
- endocrine: go into blood stream → act on distant regions (e.g., some IL, stimulators produced in bone
marrow → go to lymph node → go to blood stream → act)

Some drugs involving growth factors, cytokines and chemokines

- IFN-1β and TNF-α, are being replaced by next-generation drugs developed from studies using more
refined approaches, causing the knowledge of growth factors/cytokines/chemokine networks to become
more clarified.

Potential applications of tristetraprolin family proteins in inflammation-related diseases by

causing cytokine mRNA instability
- Tristetraprolin (TTP) and related proteins are well-established factors that destabilize cytokine
mRNAs.
- TTP is the best-understood member of a small family of tandem CCCH zinc finger proteins.
- Similar zinc finger sequences are found in numerous species in the GenBank database ranging from
human to yeast and plants (including human, cow, mouse, rat, sheep, chimpanzee, dog, horse, pig,
Xenopus, carp, zebrafish, Drosophila, C. elegans, baker’s yeast, fi ssion yeast, oyster, rice, and
Arabidopsis.) In humans, three members of this family have been characterized: TTP (ZFP36, TIS11,
G0S24, and NUP475), ZFP36L1 (TIS11B, cMG1, ERF1, BRF1, and Berg36), and ZFP36L2
(TIS11D, ERF2, and BRF2).
They are encoded by different genes and their patterns of cell- and tissue-specific expression and
agonist-stimulated expression are quite different.
Formulations of growth factors, cytokines and chemokines
- The formulations for growth factors, cytokines, and chemokines include low concentrations of
nontoxic detergents, such as Tween 80, and osmotic agents, such as sorbitol.
- Additional agents are also included for providing optimum pH for their activity. Some salts are
usually included in the formulations. A number of formulations for growth factors, cytokines, and
chemokines exist.
- These formulations are based on saline, liposome, conjugation, and coadministration.
- The goals of developing optimum formulations are designed to protect the activities of these proteins
and to effectively deliver the biologics to the targets.
- Liposome help in deliver the drug/gene/any material have interaction with liposome
- Use saline, don’t use distilled water because:
+ normal fluid in body (osmotic pressure)
+ problems in intravenous
- Look for some conjugation: some cytokines are easy to be degraded → conjugate with some
component (polyethylene glycol-PEG)
Coadministration: use cytokine with other at the same time
e.g., MTX: methotrexate → anticancer
IL2/IFN-alpha/histamine: help in inflammation
- Use nontoxic detergent (tween 80) and osmotic agents (avoid osmotic pressure)
- IL-8: help in pro-inflammation, let the cancer tumor become progress and go to blood stream (4th
stage of cancer?)
Delivery of growth factors, cytokines and chemokines
A number of routes for the delivery of growth factors, cytokines, and chemokines have been studied in
detail. Those routes include:
(1) oral administration;
(2) intravenous injection;
(3) intraperitoneal injection;
(4) intramuscular injection;
(5) subcutaneous injection. Most of the delivery methods for these compounds are accomplished
through injection because of their protein nature.

Pharmacokinetics of growth factors, cytokines and chemokines

- Numerous published papers exist dealing with the pharmacokinetics of growth factors, cytokines, and
chemokines.
- Those studies were performed using cells tissues, animals, and patients.
- The experiments tested the various formulations and delivery methods mentioned above.

Summary of the Formulation, Delivery and Pharmacokinetics for Growth factors, Cytokines
and Chemokines 

Drug Experimental Formulation Delivery Pharmacokinetics

system

Apo2L/TRAIL Mice model of Apo2L/TRAIL Intravenous Intact [125I]-Apo2L/TRAIL was

 human colon injection detectable in the solid tumor at
carcinoma all time points and was the only
tissue in which radioactivity
transiently increased over time.
Apo2L/TRAIL was stable in the
circulation, localized to human
solid xenograft tumors, and was
primarily eliminated through the
kidney

Epo  Mice myeloma Fc-Epo (NDS) Fc-Epo (NDS) was secreted

cells  fusion protein almost exclusively as a dimer,
was relatively stable to the
removal of N-linked
oligosaccharides, had much
improved pharmacokinetic
properties, and had a
significantly improved effect on
RBC production

bFGF Rat bFGF, bio- Intravenous The brain uptake of the [125I]-
bFGF and bio- injection bio-bFGF/OX26-SA was
bFGF/OX26- increased five-fold, although the
SA conjugation uptake of the conjugate by
to a blood-brain peripheral tissues was decreased
barrier peptide relative to the unconjugated bio-
drug delivery bFGF.
vector

Cytokines as biopharmaceuticals
- Cytokines, in many ways, constitute the single most important group of biopharmaceutical
substances.
- As coordinators of the immune and inflammatory response, manipulation of cytokine activity can
have a major influence on the body’s response to a variety of medical conditions.
- Administration of certain cytokines can enhance the immune response against a wide range of
infectious agents and cancer cells.
- EPO has proven effective in stimulating red blood cell production in anaemic persons. Growth factors
have obvious potential in promoting wound healing. And neurotrophic factors display some clinical
promise in the abatement of certain neurodegenerative diseases.
- A better understanding of the molecular principles underlining cytokine biology may also provide
new knowledge-based strategies aimed at defeating certain viral pathogens.
- These pathogens appear to establish an infection successfully, at least in part, by producing specific
proteins that thwart the normal cytokine-based immunological response.
- The cowpox virus, for example, produces an IL-1-binding protein, and the shope fibroma virus
produces a TNF-binding protein.
- The Epstein–Barr virus, on the other hand, produces a protein homologous to IL-10.
- Inhibiting the biological activity of such cytokines may provide effective therapies for such
conditions.
- Some cytokines have already gained approval for medical use. Many more are currently undergoing
clinical or preclinical trials.

Interferon biotechnology
IFN: cytokine produced when body is infected → APCs stimulated and produce IFN (but can produce
undesired products)
→ Produce recombinant IFN:
- Materials: plasmid, bacteria (E. coli host), target gene, enzyme, inducer, probe (short oligonucleotide:
help to screen DNA for interferon in DNA hybridization)
- Method: packaging
+ Target gene: RT-PCR to get cDNA → get correct modification (overcome intron extron problems)
→ DNA hybridization and southern blotting (DNA probe), screen mRNA: northern blot
+ Introduce into vector (pBR322) → get 6000 clones → 12 pools with 512 clones each (for easier
detection)
+ Pool is hybridized with mRNA for IFN (DNA-mRNA hybridization): double strand DNA is
converted into single strand (denature 60-65oC) and complementary with mRNA → incubate and
give signals
+ Detect: kill virus or not (e.g., use to treat HBV)
+ Divide positive to pools → finally get one clone only
+ Subcloned into E. coli expression vector
1. Isolation of Interferon cDNAs
- Isolate either the genes or cDNA for human proteins. In some cases, the target protein is isolated and
a portion of amino acid sequence is determined.
- From this information, a DNA coding sequence is deduced. The appropriate oligonucleotide is
synthesized and used as a DNA hybridization probe to isolate the gene or cDNA from either a genomic
or cDNA library.
- Alternatively, antibodies are raised against the purified protein and used to screen a gene expression
library.
- For human proteins that are synthesized primarily in a single tissue, a cDNA library from mRNA of
this tissue is enriched for the target DNA sequence.
- When the IFN cDNAs were initially isolated in the early 1980s, very little were known about the
encoded proteins (interferon was originally thought to be a single protein), so a novel scheme had to be
devised to overcome the scarcity of both the mRNAs and the proteins.
The isolation of IFN cDNAs included the following steps
1. Size-fractionated mRNA was isolated from human leukocytes, reverse transcribed, and inserted
into the PstI-site of plasmid pBR322.
2. The 6,000 clones that were produced following transformation of E. coli were divided into 12
pools of 512 clones each. Pools of clones rather than individual clones were tested to speed up
the identification process.
 3. Each pool was hybridized to a crude IFN mRNA preparation. (hybridization: chuyển từ double
strand thành single strand → dùng probe complement với ssDNA để tìm)
4. The hybridized input mRNA was separated from the cloned DNA-mRNA hybrids and
translated in a cell-free protein synthesis system.
5. Each translation mixture was then assayed for IFN antiviral activity. The pools that showed IFN
activity contained a clone with a cDNA that hybridized to IFN mRNA.
6. Positive pools were divided into eight subgroups of 64 clones each and retested. This
subgrouping process was repeated until a clone with the complete cDNA for a human IFN was
identified.
7. Subsequently, whenever large quantities of the IFN are required, the IFN cDNA can be
subcloned into an E. coli expression vector and expressed at high levels.

2. Human Interferons
- After the isolation of the first IFN gene, researchers found that there are a number of different IFNs.
- On the basis of chemical and biological properties, the IFNs can be classified, as noted earlier, into
three different groups: IFN-alpha, IFN-beta, and IFN-gamma.

Epithelial growth factor

- EGF, a 6 kDa, 53 amino acid unglycosylated peptide, was one of the first growth factors discovered,
also found in cancer cells (breast cancer), used to treat burn.
- Promote premature tooth eruption and eyelid opening in neonatal mice. EGF has subsequently proven
to exert a powerful mitogenic effect on many cell types, and its receptor is expressed by most cells.
- Its influence on endothelial cells, epithelial cells and fibroblasts is particularly noteworthy, and the
skin appears to be its major physiological target. It stimulates growth of the epidermal layer.
- Along with several other growth factors, EGF plays a role in the wound healing process, rendering a
potential medical application obvious.
- EGF may also find a novel agricultural application in sheep.
- Administration of EGF to sheep has a transient effect on the wool follicle bulb cell, which results in a
weakening of the root that holds the wool in place.

The EGF receptor. The N-terminal, extracellular region of the receptor contains 622 amino acids.
It displays two cysteine-rich regions, between which the ligand-binding domain is located. A 23 amino
acid hydrophobic domain spans the plasma membrane. The receptor cytoplasmic region contains some
542 amino acids. It displays a tyrosine kinase domain, which includes several tyrosine
autophosphorylation sites, and an actin-binding domain that may facilitate interaction with the cell
cytoskeleton.

Fibroblast growth factors

- FGFs constitute a family of about 20 proteins (numbered consecutively FGF-1 to FGF-20), a
molecular mass in the region of 18–28 kDa and induce a range of mitogenic, chemotactic and
angiogenic responses.
- Classification as an FGF is based upon structural similarity. All display a 140 amino acid central core
that is highly homologous between all family members.
- All FGFs also tightly bind heparin and heparin-like glycosaminoglycans found in the extracellular
matrix. → Purify several such FGFs via heparin affinity chromatography (heparin can be used to purify
FGF easily).
- Several newer members have little/no effect upon fibroblasts.
- Keratinocyte growth factor (FGF-7) is thus far the only member of the FGF family to have gained
approval for general medical use

Recombinant human basic fibroblast growth factor

- The DNA sequences encoding human and bovine acidic and basic fibroblast growth factors (FGF)
can be recombinantly expressed to obtain practical amounts of proteins useful in effecting wound
healing and related tissue repair.
- A number of candidate factors have been identified on the basis of the ability of extracts from various
tissues, such as brain, pituitary, and hypothalamus, to stimulate the mitosis of cultured cell lines.
+ Platelet-derived growth factor (PDGF) + Fibroblast growth factor (FGF)
+ Macrophage-derived growth factor + Hypothalamus-derived growth factor
(MDGF) (HDGF)
+ Epidermal growth factor (EGF) + Retina-derived growth factor (RDGF)
+ Tumor anglogenesis factor (TAF) + Heparin-binding growth factor (HGF)
+ Endothelial cell growth factor (ECGF)

Therapeutic hormones
- Hormones are amongst the most important group of regulatory molecules produced by the body.
- This describes what is now termed a true endocrine hormone.
- Under such a broad definition, all cytokines, for example, could be considered hormones. The
delineation between a cytokine and a hormone is already quite fuzzy using any definition.
- True endocrine hormones, however, remain a fairly well defined group.
- Virtually all of the hormones used therapeutically (discussed below) fit into this grouping. Examples
include insulin, glucagon, GH and the gonadotrophins.

Production of human insulin by recombinant DNA technology

- The initial approach to recombinant insulin production taken entailed inserting the nucleotide
sequence coding for the insulin A- and B-chains into two different E. coli cells (both strain K12).
- These cells were then cultured separately in large-scale fermentation vessels, with subsequent
chromatographic purification of the insulin chains produced.
- The A- and B-chains are then incubated together under appropriate oxidizing conditions in order to
promote interchain disulfide bond formation, forming ‘human insulin crb’
- An alternative method (developed in the Eli Lilly research laboratories), entails inserting a nucleotide
sequence coding for human proinsulin into recombinant E. coli. This is followed by purification of the
expressed proinsulin and subsequent proteolytic excision of the C peptide in vitro.
- ‘human insulin prb’.
- Although recombinant product produced by either method is identical in sequence to native insulin,
any impurities present will be host microbial-cell-derived and, hence, potentially highly immunogenic.
- Stringent purification of the recombinant product must thus be undertaken.
- This entails several chromatographic steps (often gel filtration and ion exchange, along with
additional steps that exploit differences in molecular hydrophobicity, e.g. hydrophobic interaction
chromatography or reverse-phase chromatography).
The growth hormone receptor
- GH induces its characteristic biological effects by binding to a specific cell surface receptor.
- The human receptor is a single-chain 620 amino acid transmembrane polypeptide. Sequence analysis
indicates it is a member of the haemopoietic receptor superfamily (which includes receptors for several
interleukins, GM-CSF and EPO).
- Soluble GH-binding proteins (GHBPs) are also found in the circulation. In humans, these GHBPs are
generated by enzymatic cleavage of the integral membrane receptor, releasing the GH-binding
extracellular domain. The exact physiological role of these binding proteins remains to be elucidated.
- In serum, GH binds to two such GHBPs, an action that prolongs the hormone’s plasma half-life.

Therapeutic uses of growth hormone

- GH has a potentially wide range of therapeutic uses. To date, however, its major application has been
for the treatment of short stature.
- hGH extracted from human pituitary glands was first used to treat pituitary dwarfi sm (i.e. caused by
suboptimal pituitary GH secretion) in 1958. It has subsequently proven effective in the treatment of
short stature caused by a variety of other conditions, including:
+ Turner’s syndrome
+ Idiopathic short stature
+ Chronic renal failure.
- The use of hGH extracted from the pituitaries of deceased human donors came to an abrupt end in
1985, when a link between treatment and Creutzfeld–Jacob disease (CJD, a rare, but fatal, neurological
disorder) was discovered.
- Fortunately, several recombinant hGH (rhGH) preparations were coming on-stream at that time and
now all hGH preparations used clinically are derived from recombinant sources.
- Currently, in excess of 20 000 people are in receipt of rhGH therapy
- rhGH was first produced in E. coli in the early 1980s.
- In vitro analysis, including tryptic peptide mapping, amino acid analysis and comparative
immunoassays, shows the native and recombinant forms of the molecule to be identical.
- Clinical trials in humans have also confirmed that the recombinant version promotes identical
biological responses to the native hormone.
- rhGH was first purified (on a laboratory scale) by Genentech scientists.
- A somewhat similar strategy is likely used in its process-scale purification.
(bệnh bò điên (spongiform, encephalopathy: gây tổn thương não), gây bởi prion bền với nhiệt
Production of rhGH in E. coli (as an intracellular protein). Subsequent to fermentation, the cells are
collected by centrifugation or fi ltration. After homogenization, nucleic acids and some membrane
constituents are precipitated by the addition of polyethyleneimine. Ammonium sulfate precipitation of
the supernatant concentrates the crude rhGH preparation.
How can we have E. coli express hGH?

Medical and veterinary applications of gonadotrophins

- Because of their central role in maintaining reproductive function, the therapeutic potential of
gonadotrophins in treating subfertility/some forms of infertility was obvious.
- Gonadotrophins are also used to induce a superovulatory response in various animal species.
- The market for these hormones, though modest by pharmaceutical standards, is, none the less,
substantial.
- Normally, they have in pregnant urine (low price: contamination of virus), recombinant (high price)
Overview of the procedure by which hCG may be purified from the urine of pregnant females at
laboratory scale. Production-scale systems would be at least partially based upon such a purification
strategy. Although initial concentration steps could involve precipitation, the use of ultrafiltration
would now be more common
Collect urine → concentrate → remove undesired products by anion exchange chromatography and gel
filtration
Recombinant gonadotrophins
- Gonadotrophins are now also produced by recombinant DNA technology (look for gene → cDNA →
introduce into host (CHO) → produce).
- The genes, or cDNAs coding for gonadotrophins from several species, have been identified and
expressed in various recombinant host systems, particularly mammalian cell lines. rhFSH produced in
CHO cells has proven clinically effective.
- Although exhibiting an amino acid sequence identical to the human molecule, its carbohydrate
composition differs slightly.
- When administered to humans, the preparation is well tolerated and yields no unexpected side effects.
It does not elicit an immunological response, and its plasma half-life is similar to the native hormone.
- rhFSH has proven efficacious in stimulating follicular growth in females suffering from
hypogonadotrophic hypogonadism and is effective in the treatment of males suffering from similar
conditions.
- Medicines Agency via the centralized application procedure. Recombinant gonadotrophins approved
for general medical use.