Cellular and molecular pharmacology

1. Immunotherapeutics
Presented by : Rashmi
M.Pharm 1st sem
Pharmacology

2. Immunotherapy
• Treatment to stimulate or restore the
ability of the immune (defense) system to
fight infection and disease.
• It is also sometimes called biological
therapy or biotherapy.
• Biological therapy is thus any form of
treatment that uses the body's natural
abilities that constitute the immune system
to fight infection and disease or to protect
the body from some of the side effects of
treatment.

3. HISTORY
• William Coley, MD, a New York surgeon, first noted that getting an
infection after surgery seemed to help some cancer patients. In the
late 1800s, he began treating cancer patients by infecting them with
certain kinds of bacteria, which came to be known as coley toxins.
• Although he had some success, his technique was overshadowed
when other forms of cancer treatment, such as radiation therapy,
came into use.

5. How it works ?
• Immunotherapy includes a wide variety of treatments that work in different
ways:
• By boosting the body’s immune system in a very general way.
• Helps to train the immune system to attack cancer cells specifically.
• Giving immune system components, such as man-made immune system
proteins.

6. Types of immunotherapy
Immunotherapy
Active passive
immunotherapy immunotherapy

7. ACTIVE IMMUNOTHERAPY
• It is the type of immunotherapy that attempts to stimulate the host’s intrinsic immune
response to a disease. Further divided into:
• Specific active immunotherapy
• Non specific active immunotherapy
Specific active immunotherapy The generation of cell mediated and antibody
immune responses focused on specific antigen.
E.g. cancer vaccines • Cellular therapies • adjuvants

8. Cont.
o Cancer vaccines: cancer vaccines may contain cancer cells ,part of the cell ,
or purified tumor specific antigen.
Two categories of cancer vaccine are :
•Cell based in which the patient cancer cell is cultured with patients own
immune system cells and derived back to the same patient.
• Vector based in which the engineered virus or other vector is used to
introduce cancer specific proteins and other molecule in order to stimulate the
patient immune system to recognize the tumor cells to fight the cancer.

9. Cont.
o Cellular therapies: These are single type agent derived from the cancer patients
which are modified in the lab. to become more adapt at recognizing and killing the
tumor cells.
o This type of immunotherapy is designed to boost specific part of immune system
to cause tumor cell death E.g. lymphocyte activated killer cell therapy.
o Adjuvant immunotherapy : an adjuvant is an any material which when injected
together with an antigenic protein or other substance like monoclonal antibodies ,
cancer vaccines increases or boost the immune response to the particular system.
E.g. BCG vaccine

10. Cont.
Non specific immunotherapy: don’t target cancer cells specifically, generation of
general immune system response.
Cytokines are chemicals made by some immune system cells. They are crucial in
controlling the growth and activity of other immune system cells and blood cells in
the body.
Cytokines destruct tumour cell by two mechanism:
• direct antitumor e.g. TNF alpha,IL-6
• Indirect enhancement of antitumor response Eg;IL2 promote T-cell and NK cell
growth.

13. PASSIVE IMMUNOTHERAPY:
• Passive immunotherapy This comprised of antibodies and other immune system
component that are made outside the body and administered to the patient to
provide immunity against the disease.
• It do not stimulate a patient immune system to actively respond to a disease in the
way vaccines does.
• Adoptive Cell Therapy
• Immunomodulators
• Targeted Antibodies
• Oncolytic Virus Therapy

14. Cont.
1. Adoptive Cell Therapy: Adoptive cell therapies use immune cells to fight cancer.
There are two main approaches:
o Immune cells are isolated, expanded, and reintroduced into the cancer
patient
o Immune cells are genetically modified to “boost” their cancer-fighting ability,
and then reintroduced into the cancer patient.
o Eg.Chimeric Antigen Receptor (CAR) T-cell Therapy (Modified Cells).

15. Cont.
2. Immunomodulators: Immunomodulators regulate or “modulate” the activity
of the immune system.
• Immunomodulators can be loosely separated into four groups;
checkpoint inhibitors,( checkpoint inhibitors can reduce immune suppressive
mechanisms – increasing the immune system’s response to cancer and promoting
the elimination of cancerous cells.
The FDA approved the first checkpoint inhibitor, for the treatment of melanoma,
in 2011).

16. Cont.
Cytokines (These molecular “messengers” enable immune cells to
communicate and mount a coordinated response to a target antigen , the first
to be approved was a cytokine called interferon-alpha 2 (IFN-α2) back in
1986).
Drugs: Immunostimulants(Levamisole, thalidomide, BCG, Interferons,)
Immunosuppressant (Cyclosporine, Tacrolimus, glucocorticoid ,
Cytotoxic drugs etc.)
Adjuvants

17. Cont.
3. Monoclonal antibodies :Many copies of a specific Antibody can be made in the lab.
These are known as Monoclonal Antibodies (mAbs or moAbs).
• These Antibodies can be useful in fighting diseases because they can designed specifically to
only target a certain antigen, such as one that is found on cancer cells.
• Over the past 15years, the US FDA has approved about a mAbs to treat certain cancers.
• Two types of monoclonal antibodies are used in cancer treatment:
• Naked mAbs are antibodies that work by themselves.
• Conjugated mAbs are those joined to a chemotherapy drug, radioactive particles, or a
toxin.

18. Cont.
• Naked Monoclonal Antibodies : Naked mAbs can work in different ways.
• Some may boost a person’s immune response against cancer cells.
• Other work by blocking specific proteins that help cancer cells grow ( some
may do both).
• For example – Herceptin (trastuzumab) is an antibody against the HER
2/neu protein. It is used to treat breast and stomach cancers that have large
amounts of this protein.

19. Cont.
• Conjugated mAbs: are also sometimes referred to as tagged, labeled or loaded
Antibodies.
• They can be divided into groups depending on what they are linked to.
• mAbs with radioactive particles attached are referred to as radiolabeled, and
treatment with this type of Antibody is known as radioimmunotherapy (RIT).
• mAbs with chemotherapy drugs attached are referred to as Chemolabeled.
• mAbs attached to cell toxins are called immunotoxins.

21. Cont.
4. Oncolytic Virus Therapy: Viruses are infectious agents that are capable of
infecting living cells, hijacking their genetic machinery, which allows the viruses to
replicate inside of them.
• Modified versions of viruses can be created to target and attack cancer cells. These
are termed “oncolytic viruses” as they are designed to target cancer specifically.
• The viruses can be engineered to decrease their ability to infect “normal” cells and
they can also be used as delivery vehicles, transporting therapeutic payloads to
cancer cells.
• The first oncolytic virus therapy was approved by the FDA in 2015 – T-VEC for
treatment of melanoma.

22. HUMANISATION ANTIBODY THERAPY

23. What are antibodies?
• An antibody is a protein used by the
immune system to identify and neutralize
foreign objects like bacteria and viruses.
Each antibody recognizes a specific
antigen unique to its target.
• Monoclonal antibodies (mAbs) are
antibodies that are identical because they
were produced by one type of immune
cell.
• Polyclonal antibodies are antibodies that
are derived from different cell lines..

24. Humanized antibody
• Humanized antibodies are antibodies from non-human species whose
protein sequences have been modified to increase their similarity to antibody
variants produced naturally in humans.
• The process of "humanization" is usually applied to monoclonal antibodies
developed for administration to humans (for example, antibodies developed
as anti-cancer drugs).

25. Why humanization is necessary ?
• Humanization can be necessary when the process of developing a specific
antibody involves generation in a non-human immune system (such as that in
mice).
• The protein sequences of antibodies produced in this way are partially
distinct from homologous antibodies occurring naturally in humans, and are
therefore potentially immunogenic when administered to human patients.

26. Cont.
• Murine antibodies had several shortfalls which include –
i. A short half-life in vivo (due to host immune rejection).
ii. Limited penetration into target cells (such as tumor site).
iii. Being 100% murine proteins, and are recognized as ‘foreign’ by the human body
and therefore they are rejected by an antibody-mediated immune response
(human anti-murine antibodies –HAMA; produced by the host) .
• To overcome these problems novel chimeric and humanized antibodies have been
developed.

28. Methods of humanization of antibodies
• Humanized antibodies are produced by:
i. Grafting complementarity determining regions (CDRs)
ii. Memory B cell immortalization
iii. Hybridoma technology using transgenic mouse
iv. Hybrid-hybridoma
v. Human antibody display
vi. Recombinant antibodies by cloning v-region genes

29. CDR grafting (antibody reshaping)
Complementarity–determining regions (CDRs) of murine Antibody ( variable regions)
Grafted into variable regions of human antibody
which were then joined to constant regions of human antibody
Reshaped ( humanized ) antibody .

31. Memory B cell immortalisation
• This technique involves isolation of human memory B cells from peripheral
blood mononuclear cells (PBMCs) of infected patients.
• These B cells are then immortalised using Epstein Barr Virus (EBV) in the
presence of a polyclonal B cell activator (mostly CpG oligodeoxynucleotide).
• These transformed cells are capable of producing a human monoclonal
antibody with desired antigen specificity.
• Finally the culture supernatants are screened directly for specific antibodies.
Positive cultures are further cloned and fully humanised.

32. Hybridoma technology using transgenic mouse
• This technology is a slightly modified version of traditional hybridoma technology.
Here, the hybridoma is produced from the spleen cells of transgenic mice in which
the immunoglobulin genes are knocked out and replaced with human counterparts.
This is followed by the antigen immunization.
• The steps thereafter are similar to producing traditional monoclonal antibodies.
• The B cells from immunized transgenic mice are fused with myeloma cells derived
from in vitro cell culture to produce immortalized hybridoma.
• These hybridomas are then screened for desired specificity. Once these specific
hybridomas are produced, it is possible to generate hybrid-hybridomas.

34. Hybrid-hybridoma
• These hybrid-hybridomas are obtained by fusion of two cells which contain the genetic
information necessary for production of two different antibodies. In fact the formation of
hybrid-hybridoma paved the first steps towards humanization of mouse antibodies.
• They can also secrete chimeric antibodies made up of two non-identical halves. This new
class of immunotherapeutic agents is also called bi-specific antibodies.
• This is beneficial in therapeutics as one arm of the bi-specific antibody binds to one antigen,
the second arm binds to another.
• For example one arm of the antibody may bind to a marker molecule and the second to a
target cell, creating an entirely new way of detecting and/or destroying tumor cells.
• This also has potentials in cancer immunotherapy as one arm of this chimeric antibody
locks onto the tumor cells while the other may bind to a killer T cell to activate the
destruction of tumor cells.

38. Recombinant antibodies by cloning v-region genes
• The functional structure of the antigen-binding site is determined by genes of both
heavy (H) and light (L) variable (V) domains.
• Therefore in this technology, the V domain is extracted from mouse monoclonal
cell lines and cloned into a mammalian expression vector. Then the vector is
transfected into mammalian cells [usually Chinese hamster ovary (CHO) cells] which
can generate the humanized/chimeric antibodies.
• Cloning of mouse variable genes into human constant-region genes generates
chimeric as well as humanized antibodies depending on the size of the clone.

39. Monoclonal antibodies approved for therapeutic use
• Generic name Antibody format Antigen Approved indication
• Muromomab Murine, IgG2a Allograft rejection in allogeneic renal transplantation
• Abciximab1 Chimeric, IgG1 Maintenance of coronary patency
• Rituximab2 Chimeric, IgG1 positive B-cell non-Hodgkin's lymphoma
• Daclizumab Humanized, IgG1 Allograft rejection
• Basiliximab Chimeric, IgG1 Allograft rejection
• Palivizumab Humanized, IgG1 Respiratory syncytial virus (RSV inhibitor) in children
• Infliximab Chimeric, IgG1 TNFα Crohn's disease and rheumatoid arthritis
• Trastuzumab Humanized, IgG1 Metastatic breast cancer
• Etanercept3 Autoimmune diseases such as ankylosing spondylitis
• Gemtuzumab4 Humanized, IgG4 CD33-positive acute myeloid leukemia

40. Thank you