Immunotherapeutics (Types of immunotherapeutics, humanisation antibody therapy, Immunotherapeutics in clinical practice).ppt

IMMUNOTHERAPEUTICS
Presented By : Nikita Bankoti
M .Pharm1st Sem
Pharmacology
1

Introduction
Immunity - The overall defence mechanism of an individual Organism against any
invading agent or pathogen is called immunity.
➢The study of immunity is called immunology.
➢When a foreign substance (antigen) is introduced into our body our body produces
antibodies against those antigen.
➢If our antibodies are able to eliminate the antigen then we are free from diseases. But,
when antibodies are not enough/able to eradicate the antigens, we suffer from a
disease.
➢Types of immunity – There are two types of immunity.
1. Natural immunity or innate immunity
2. Acquired immunity
2

Immunotherapy
➢ Treatment to stimulate or restore the ability of the immune (defence) system to fight
against infection or disease.
➢ It is also sometimes called Biologic therapyor 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 e.g. – cancer.
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. (named after William coley)
➢ Coley toxin treatment- we injected Streptococcal organism into a cancer patients in
order to cause erysipelas(red patches on the skin) and stimulate the immune system.
➢ Although he had some success, his technique was overshadowed when other forms of
cancer treatment, such as radiation therapy came into use.
➢ Since then, doctors have learned a great deal about the immune system. This has lead
to research into how it can be used to treat cancer, using many different approaches.
➢In few last decades immunotherapy has become an important part of treating several
type of cancer.
4

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

Types of Immunotherapeutics
1. Monoclonal antibody
2. Cancer vaccines therapy
3. Immune checkpoint inhibitors
4. Non-specific Immunotherapies
5. Chimeric antigen receptor (CAR) T-cell therapy
6

1. Monoclonal Antibody.
➢ These are identical immunoglobulins, generated from a single B-cell clone. These
antibodies recognize unique epitopes, or binding sites, on a single antigen.
➢ Derivation from a single B-cell clones and subsequent targeting of a single epitope is
what differentiates monoclonal antibodies from polyclonal antibodies.
➢ Monoclonal antibodies are synthetic antibodies designed to target specific molecules
on the surface of cancer cells.
➢ Mainly two type of mAbs are used in treatment of different disease, these are-
i. Conjugated Monoclonal Antibody.
a. Radio-labeledAbs
b. Chemo-labeledAbs
c. Immunotoxins
ii. Naked MonoclonalAntibody.
7

Production of Monoclonal antibodies
a) Immunization- any animal i.e. mouse are immunized 2-3 times with antigen
(subcutaneously).
➢ After several weeks when serum concentration of antibodies are optimal, the animal is
sacrificed, spleen is aseptically removed. By mechanical and enzymatical method spleen
cells are separated desired plasma cells or activated B-lymphocytes are separated
through density gradient centrifugation from other spleen cells.
b) Myeloma cells- these are mutated myeloma cells.
➢It means through mutation, HGPRT gene (hypoxanthine guanosine phosphoribosyl
transferase gene) is done non functional and these cells are unable to produce
antibodies.
c) Cell fusion- these lymphocytes are mixed with HGPRT defective myeloma cell by
polyethylene glycol (PEG) by fusion.
d) Selection of Hybridoma- when the cells are cultured in HAT medium( Hypoxanthine,
Aminopterin, Thymidine). In this medium, only hybridoma cells will be grown and other
cells will disappear with in 7-10 days.
e) Screening and purification of product OR Antibody are harvested from the culture.
8

Monoclonal Antibody Production
9

i. Conjugated monoclonal antibody-
➢ They 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 particle attached are referred to as radiolabeled and treatment with this type of
antibody is k/a Radioimmunotherapy(RIT).
➢ mAbs with chemotherapy drug attached are referred to as Chemolabeled.
➢ mAbs attached to cell toxins are called immunotoxins.
ii. Naked monoclonal antibody-
➢ They are antibodies that have no drug or radioactive material attached to them.
➢ They work by themselves.
➢They are the most common type of mAbs used to treat cancer.
➢ Naked mAbs can work in different ways, some may boost a person’s immune response against cancer cells.
While other work by blocking specific proteins that help cancer cell grow(some may do both).
➢ They are most widely used immunotherapeutics agents.
➢ Example- Herceptin (trastuzumab) is an antibody against the HER 2/ neu protein. It is used to treat breast
and stomach cancer that have large amount of this protein.
10

2. Therapeutic cancer vaccine.
➢Cancer vaccines may contain cancer cell, part of the cell, or purified tumor specific
antigen.
➢Two categories of cancer vaccine are-
a) Cell based- In which the patient cancer cell is cultured with patients own immune
system cells and derived back to the same patient.
b) Vector based- In which the engineered virus or other vector is used to introduced
cancer specific proteins and other molecules in order to stimulate the patient immune
system to recognize the tumor cells to fight the cancer.
11

3. Immune Checkpoint Inhibitors.
➢ Checkpoints are proteins found on T cell that regulate how T cell respond to foreign
cells.
➢ Checkpoint inhibitors are a class of immunotherapy drugs that block certain
proteins(checkpoints) that inhibit the immune system’s response against cancer cells. By
removing these brakes, the immune system can better recognize and attack the tumor.
➢ When a T cell comes close to another cell, it probes certain proteins on the surface of
that cell using a T cell receptor. If the protein of the inspected cell indicate that the cell is
foreign cell, the T cell stages an attack against it. Checkpoints signal to T cell to multiply
themselves to fight the invader. Once the invader is destroyed, checkpoints signal the T
cell to turn off and shut down the T cell multiplication response.
➢ If T cells are active for too long or react to things they should not, then they will start to
destroy healthy cells and tissues which could result in autoimmune disorder such as-
Crohn’s disease or rheumatoid arthritis.
12

➢ Two checkpoint proteins that work together to turn off (stop) the T cells after the
multiplication response are PD-1 and PD-L1. PD-1 is a checkpoint protein found on T-
cells. When PD-1 binds to PD-L1, a protein present on normal cells, it sends a message
to the immune system to leave the cell alone. As a result it reduce the production of T
cells and enables more T cell to die.
➢ T cells only expect normal cells to produce PD-L1, but sometimes cancer cell can
avoid an immune system attack by producing PD-L1 on their surfaces.
➢ Immune checkpoints inhibitors that treat blood cancer by binding to the PD-1 receptor
on T cell and blocking the interaction of PD-1 and PD-L1.
➢ EXAMPLE- Pembrolizumab (Keytruda) used to treat Hodgkin lymphoma.
13

4. Non-Specific Immunotherapies.
➢ They don’t target foreign particles specifically.
➢ They stimulate the immune system in a more general way, but this can still sometimes
lead to a better immune response against foreign particles.
i. Cytokines-
➢ cytokines are small protein made by some immune system cells.( innate macrophages,
dendritic cells, natural killer cells and the adaptive T and B lymphocytes)
➢ They are crucial in controlling the growth and activity of other immune system cells and
blood cells.
➢ The primary function of cytokines is to regulate inflammation.
➢ Cytokines are injected, either under the skin, into a muscle, or into a vein.
Cytokines destruct tumor cell by two mechanism-
1. Direct antitumor e.g.- TNF alpha, IL-6
2. Indirect enhancement of antitumor response e.g.- IL-2 promote T cell and NK cell
growth.
14

ii. Interferons-
➢ These cytokines, first discovered in the late 1950s, help the body to resist virus
infections and cancers.
➢ The types of interferons (IFN) are named after the first 3 letters of the Greek alphabet.
a. IFN- Alpha
b. IFN- Beta
c. IFN- Gamma
➢ Only IFN-alpha is used to treat cancer. It boosts the ability of certain immune cells to
attack cancer cells.
➢ It may also slow the growth of cancer cells directly, as well as the blood vessels that
tumours need to grow.
15

iii. Interleukins-
➢ Interleukins are a group of cytokines that act as chemical signals between white blood
cells.
➢ Interleukin-2 (IL-2) helps immune system cells grow and divide more quickly.
➢ When a man-made version of IL-2 was approved by the USFDA in 1992 to treat
advanced kidney cancer, it became the first true immunotherapy approved to be used
alone to treat cancer.
16

5. Adoptive cell therapy.
➢ Adoptive cell transfer is a type of immunotherapy that uses
a patient’s own T cell to help fight cancer.
➢ The T cells are taken from the patient’s blood or from the
tumour itself and treated in the laboratory with substances
to make them better to target and kill cancer cells in their
bodies.
➢ There are two main approaches :-
a. Immune cells are isolated, expanded and reintroduced
into the cancer patient.
b. Immune cells are genetically modified to “boost” their
cancer-fighting ability, and
then reintroduced into the cancer patient.
EXAMPLE- Chimeric antigen receptor (CAR) T-cell therapy.
17

Humanisation antibody therapy
Antibody – 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.
Humanized Antibody- They 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. (e.g- antibodies developed as anti-cancer drugs)
18

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 (able to produce an immune response) when administered to human
patients.
19

Murine antibodies had several shortfalls which includes-
➢ A short half-life within the living body (i.e. in-vivo) due to host immune rejection.
➢ limited penetration into target cells (such as tumor site).
➢ Being 100% murine proteins, they are recognized as foreign by the human body and
therefore they are rejected by an antibody-mediated immune response (i.e. HAMA-
human anti-murine antibodies, produced by the host).
20

Methods of Humanization antibodies
1. CDRs grafting (complementarity determining regions)
2. Hybridoma technology using transgenic mice
3. Hybrid-Hybridoma
4. Memory B-cell immortalization
5. Recombinant antibodies by cloning V region gene
21

1. CDRs Grafting.
➢ The complementarity determining regions are part of the ‘variable ends’ of an antibody
which are responsible for that very antibody to bind to a specific antigen.
➢ Also known as Antibody reshaping.
Steps involved are-
complementarity determining regions(CDRs) of murine antibody variable regions.
Grafted into variable regions of human antibody.
Whichwere then joined to constant regions of human antibody.
Humanized reshaped antibody produced.
22

2. Hybridoma technology using transgenic mouse.
a) Immunization- any animal i.e. mouse are immunized 2-3 times with antigen
(subcutaneously).
• After several weeks when serum concentration of antibodies are optimal, the animal is
sacrificed, spleen is aseptically removed. By mechanical and enzymatical method spleen
cells are separated desired plasma cells or activated B-lymphocytes are separated through
density gradient centrifugation from other spleen cells.
b) Myeloma cells- these are mutated myeloma cells.
• It means through mutation, HGPRT gene (hypoxanthine guanosine phosphoribosyl
transferase gene) is done non functional and these cells are unable to produce antibodies.
c) Cell fusion- these lymphocytes are mixed with HGPRT defective myeloma cell by
polyethylene glycol (PEG) by fusion.
d) Selection of Hybridoma- when the cells are cultured in HAT medium( Hypoxanthine,
Aminopterin, Thymidine). In this medium, only hybridoma cells will be grown and other cells
will disappear with in 7-10 days.
e) Screening and purification of product OR Antibody are harvested from the culture.
23

3. Immortalizing memory B cell.
➢ 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 oligo-deoxy- nucleotide).
➢ 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.
➢ The main limitation of B cell immortalization is the antibody produced are only specific
to the antigen from the infected organism.
24

Immunotherapeutics in clinical practice
➢Emil von Behring and Wernicke found that animals could be cured of Diphtheria and
an injection of sera produced by animals immunized with an attenuated form of
diphtheria successfully treated a child with Diphtheria.
➢ Emil von Behring discovered Diphtheria and tetanus vaccine.(passive immunology)
➢ In 1891, William B. Coley (father of Immunotherapy), Injected bacteria into a patient
with cancer and found that due to bacteria infection the patient’s immune system got
stimulated and helped in shrink patient tumour.
➢ Drugs like Thalidomide, Lenalidomide can boost immune system (immunomodulatory
agents). They are used to treat myeloma (cancer of plasma cells).
➢ Checkpoints inhibitors have shown a remarkable antitumour efficacy in a broad
spectrum of malignancy (presence of cancerous cells that have ability to spread).
25

Immunotherapeutics (Types of immunotherapeutics, humanisation antibody therapy, Immunotherapeutics in clinical practice).ppt

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Immunotherapeutics (Types of immunotherapeutics, humanisation antibody therapy, Immunotherapeutics in clinical practice).ppt