Protein therapeutics

1. PROTEIN THERAPEUTICS
Dr. Rupendra K. Bharti

2. These are the proteins that have an effect of
healing or use inside our body, e.g
 nutrition: e.g. albumin .
 Globulins: the example is gamma globulin that
boosts our defenses against infectious diseases
(gamma globulin is a mixture of antibodies)
Synthetic proteins: antibodies against
inflammatory components (infliximab), or against
tumor components (trastuzumab)
What are therapeutic proteins?

3. • The best example of trends in the production and use
of protein therapeutics is provided by the history of
insulin in the treatment of diabetes mellitus type I
(DM-I) and type II (DM-II).
• Untreated, DM-I is a disease that leads to severe
wasting and death due to lack of the protein hormone
insulin, which signals cells to perform numerous
functions related to glucose homeostasis and
intermediary metabolism.
• In 1922, insulin was first purified from bovine and
porcine pancreas and used as a life-saving daily
injection for patients with DM-I.

4. • At least three problems hindered the
widespread use of this protein therapy:
• first, the availability of animal pancreas for
purification of insulin;
• second, the cost of insulin purification from
animal pancreas; and
• third, the immunological reaction of some
patients to animal insulin.

5. • These problems were addressed by isolating the
human insulin gene and engineering Escherichia
coli to express human insulin by using
recombinant DNA technology.
• By growing vast quantities of these bacteria,
large-scale production of human insulin was
achieved.
• The resulting insulin was abundant, inexpensive,
of low immunogenicity and free from other
animal pancreatic substances.

6. • Recombinant insulin, approved by the US FDA
in 1982, was the first commercially available
recombinant protein therapeutic, and has
been the major therapy:
• for DM-I (and a major therapy for DM-II).

8. WHY PROTEINS AGAINST SMALL DRUG
MOLECULES
• The diversity of functional groups in protein :
free thiols on cysteine residue & amine on the
N-terminus or on lysine residue
• Highly specific function- less chance of being
mimicked by simple chemical compounds.
• High specificity in action -less potential for
proteins to interfere with normal biological
processes –hence least adverse effects.

9. • The body naturally produces many of the
proteins that are used as therapeutics, &
hence are often well tolerated and are less
likely to elicit immune responses.
• Comparatively faster clinical development
and FDA approval time than that for small
molecule drugs.
• Easier to obtain far-reaching patent
protection for protein therapeutics.

10. The evolution of Protein therapeutics :
1953 First accurate model of DNA suggested
1982 Human insulin, created using recombinant DNA technology
1986 Interferon alfa and muromonab-CD3 approved
1997 First whole chimeric antibody, rituximab, and first humanized
antibody, daclizumab, approved
1993 CBER's Office of Therapeutics Research and Review (OTRR) formed
2002 Market for biotechnology products represents approximately $30
billion of $400 billion in yearly worldwide pharmaceutical sales
2006 An inhaled form of insulin (Exubera) approved, expanding protein
products into a new dosage form.

11. Classification.
• Group I: protein therapeutics with enzymatic or regulatory activity
• Ia: Replacing a protein that is deficient or abnormal
• Ib: Augmenting an existing pathway
• Ic: Providing a novel function or activity
• Group II : protein therapeutics with special targeting activity
• IIa: Interfering with a molecule or organism .
• IIb: Delivering other compounds or proteins
• Group III : protein vaccines
• IIIa: Protecting against a deleterious foreign agent.
• IIIb: Treating an autoimmune disease.
• IIIc: Treating cancer.
• Group IV : protein diagnostics

12. • Protein therapeutics replacing a protein that is
deficient or abnormal (Group Ia)

14. • Protein therapeutics augmenting an existing
pathway (Group Ib)

16. • Protein therapeutics providing a novel function
or activity(Group Ic)

18. • Protein therapeutics that interfere with a
molecule or organism (Group II a)

20. • Protein therapeutics that deliver other
compounds or proteins (Group II b)

22. • Protein vaccines (Group III )

24. • Protein diagnostics (Group IV).

26. Intracellular protein delivery
system :
• Non-covalent modification of proteins with a peptide
sequence that shows the capability to translocate
membrane rapidly, usually termed as ‘‘cell penetrating
peptide(CPP) or protein transduction domain(PTD) .
• Modification involves –
1.direct expression of recombinant fusion protein from a
vector containing DNA sequence of the CPP sequence.
2. protein or chemical conjugation of CPP to the protein
through linker such as disulfide bond linkage that is
cleavable under reductive environment.

27. To protect protein from protease degradation &
Strategy to improve delivery efficiency-
 noncovalent encapsulation of different
protein therapeutics with synthetic peptide.

28. Development of peptide based
biomaterial for delivery :
Why peptide?
1.Easy to synthesize
2.Easy characterization
3.Less toxic & has higher immunogenicity than high
molecular weight polymers.
4.Due to its amphipathic character, peptides can
associate rapidly with protein cargos in solution in
self-assembly manner, possibly through non-
covalent hydrophobic interaction.

29. Challenges for protein therapeutics
• First, protein solubility, route of administration,
distribution and stability are all factors that can
hinder the successful application of a protein
therapy.
• Proteins are large molecules with both
hydrophilic and hydrophobic properties that can
make entry into cells and other compartments of
the body difficult, and the half-life of
therapeutic protein can be drastically affected
by proteases, protein-modifying chemicals or
other clearance mechanisms.

30. • Such challenges are being addressed through the
production of PEGylated versions of therapeutic
proteins.
• For example, PEG-interferon is a modified form of
interferon in which the polymer polyethylene
glycol (PEG) is added to prolong the absorption,
decrease the renal clearance, retard the
enzymatic degradation, increase the elimination
half-life and reduce the immunogenicity of
interferon.

31. • A second important challenge is that the body may
mount an immune response against the therapeutic
protein.
• In some cases, this immune response can neutralize
the protein and can even cause a harmful reaction in
the patient.
• For example, immune responses can be generated
against Group Ia therapeutic proteins used to replace a
factor that has been missing since birth, as illustrated
by the development of antifactor VIII antibodies
(inhibitors) in patients with severe haemophilia A who
are treated with recombinant human factor VIII.

32. • Recombinant technology and other advances have
allowed the development of various antibody products
that are less likely to provoke an immune response
than unmodified murine antibodies.
• In humanized antibodies, portions of the antibody that
are not critical for antigen-binding specificity are
replaced with human Ig sequences that confer stability
and biological activity on the protein but do not
provoke an anti-antibody response; and fully human
antibodies can be produced using transgenic animals .

33. • A third issue is that for a protein to be
physiologically active, post-translational
modifications such as glycosylation,
phosphorylation and proteolytic cleavage are
often required.
• These requirements may dictate the use of
specific cell types that are capable of expressing
and modifying the protein appropriately.
• In addition, recombinant proteins must be
synthesized in a genetically engineered cell type
for large scale production.

34. • The host system must produce not only
biologically active protein but also a sufficient
quantity of this protein to meet clinical
demand.
• Also, the system must allow purification and
storage of the protein in a therapeutically
active form for extended periods of time.

35. • Potential solutions could include the
development of systems in which entire
cascades of genes involved in protein folding
are induced together with the therapeutic
protein; the impetus for this work is the
observation that plasma cells, which are
natural protein production facilities, use such
gene cascades to produce large quantities of
monoclonal antibody.

36. • Although bacteria and yeast are generally
considered easy to culture, certain
mammalian cell types can be more difficult
and more costly to culture.
• Other methods of production — such as
genetically engineered animals and plants—
could provide a production advantage.

37. • Transgenic cows, goats and sheep have been
engineered to secrete protein in their milk, and
transgenic chickens that lay eggs filled with
recombinant protein are anticipated in the
future.
• Transgenic plants can inexpensively produce vast
quantities of protein without waste or
bioreactors, and potatoes can be engineered to
express recombinant proteins and thereby make
edible vaccines.

38. • A fourth important challenge is the costs
involved in developing protein therapies.
• For example, switching to recombinant
methodology from laborious purification of
placentally derived protein has allowed the
production of sufficient β-glucocerebrosidase
to treat Gaucher’s disease in many patients.

39. • A fifth issue associated with protein
therapeutics: ethics .
• For example, the possibility of efficacious but
expensive protein therapeutics for small but
severely ill patient populations, such as
patients with Gaucher’s disease, can present a
dilemma with respect to allocation of financial
resources of health-care systems.

40. • In addition, the definition of illness or disease
could be challenged by protein therapeutics
that can ‘improve upon’ conditions previously
viewed as variants of normal.
• For example, the definition of short stature
may begin to change with the possibility of
using growth hormone to increase the height
of a child.

41. Conclusion
• Recombinant human proteins make up the
majority of FDA approved biotechnology
medicines, which include monoclonal antibodies,
natural interferons, vaccines, hormones, modified
natural enzymes and various cell therapies.
• The future potential for such therapies is huge,
given the thousands of proteins produced by the
human body and the many thousands of proteins
produced by other organisms.

42. • Furthermore, recombinant proteins not only
provide alternative (or the only) treatments
for particular diseases, but can also be used in
combination with small molecule drugs to
provide additive or synergistic benefit.

43. • Treatment of EGFR-positive colon cancer is
illustrative of this point: combination therapy
with the small-molecule drug irinotecan,
which prevents DNA repair by inhibiting DNA
topoisomerase, and the recombinant
monoclonal antibody cetuximab, which binds
to and inhibits the extracellular domain of the
EGFR, results in increased survival in patients
with colorectal cancer.

44. • The therapeutic synergy between irinotecan
and cetuximab may be due to the fact that
both drugs inhibit the same EGFR signalling
pathway, with one drug (cetuximab) inhibiting
the initiation of the pathway and the other
drug (irinotecan) inhibiting a target
downstream in the pathway.

45. • With the large number of protein therapeutics
both in current clinical use and in clinical trials
for a range of disorders, one can confidently
predict that protein therapeutics will have an
expanding role in medicine for years to come.