by- Pronay Mandal, M.Pharm- Pharmacology, Amrita School of Pharmacy

1. Presented By-
Pronay Mandal
1st year, M.Pharm-Pharmacology
School of Pharmacy, Amrita Vishwa Vidyapeetham

2. CONTENT
INTRODUCTION
BASICS OF IMMUNESYSTEM
STRUCTURE OF ANTIBODY
DEFINITION & CLASSIFICATION OF IMMUNETHERAPY
o Monoclonal Antibody
o Non-Specific Immunotherapies .
o Immune Checkpoint Inhibitors.
o Cancer Vaccines Therapy.
o Adoptive cell transfer/chimeric antigen receptor (CAR)/T-cell therapy.
HUMANIZATION OF ANTIBODY

3. INTRODUCTION
o Immunology is a branch of science that studies all aspects of the immune
system. Advances in this field have led to a greater understanding of the ways
that the body’s immune defences can be harnessed to treat many types of disease
like cancer.
o Immunotherapy is a type of treatment that improves the ability of the body’s
own immune system to detect and attack foreign particle like cancer cells, virus,
bacteria etc.
o Immunotherapy includes treatments that work in different ways. Some boost the
body’s immune system. Others help train the immune system to attack specific
foreign cells.
o Immunotherapeutic aims to activate the body's own defence mechanisms by
triggering and/or 'mimicking' immune responses.
o They would either bind to and neutralise protein toxins, block the attachment of
viruses to the cells, activate complement or activate natural killer (NK) cells.

4. Immune System
o The immune system is a network of cells, tissues, and organs that work
together to protect the body from foreign invaders, such as bacteria or viruses.
o The key players in defending the body are a specific type of white blood
cell (WBC) called lymphocytes. There are three major types of lymphocytes,
1. Natural killer (NK) cells
2. B cells
3. T cells
o Lymphocytes grow and develop in the bone marrow, thymus, and spleen.
They can also be found in clumps throughout the body, primarily as lymph nodes.
o A key feature of the immune system is its ability to tell the difference between
the body’s own normal cells and other substances that are foreign to the body. Every cell in the body
carries a set of distinctive proteins on its surface. These identifying surface proteins let the immune system
know that they are cells that belong to the body.
o If the surface proteins are abnormal, such as when a virus infects cells, or when cells become cancerous,
they can be recognized by the immune system. Proteins recognized by the immune system are called
antigens.
o A blood protein produced in response to and counteracting a specific antigen. Antibodies combine
chemically with substances which the body recognizes as Antigen.

6. Basics About Antibody

7. IMMUNE THERAPY
Immunotherapy is a type of biological therapy. Besides of targeting the person’s foreign
particle directly, immunotherapy trains a person’s natural immune system to recognize
foreign particle e.g., virus, bacteria, cancer cells and selectively target and kill them.
Immunotherapies do this in one of two ways:
o By enabling the immune system to mount or maintain a response.
o By suppressing factors that prevent the immune response.
 These are the types of Immune Therapy that mainly used in treatment of varies disease like
cancer,
 Monoclonal Antibody
 Non-Specific Immunotherapies .
 Immune Checkpoint Inhibitors.
 Cancer Vaccines Therapy.
 Adoptive cell transfer/chimeric antigen receptor (CAR)/T-cell therapy.

8. Monoclonal Antibody
 Monoclonal antibodies 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.
 Polyclonal antibodies are antibodies that are derived from different cell lines. They differ
in amino acid sequences.
 Mainly two types of mAbs are used in treatment of different disease, these are-
Monoclonal Antibody
Conjugated mAbs
Radiolabeled Abs Chemo labeled Abs Immunotoxins
Naked mAbs

9. Production of Monoclonal antibodies  Immunize animal.
 Isolate spleen cells
(containing antibody-
producing B cell).
 Fuse spleen cells with
myeloma cell (using PEG).
 Allow unfused B cell to die in
HAT medium.
 Add aminopterin to culture
and kill unfused myeloma
cells.
 Clone remaining cells (place 1
cell/well and allow each cell
to grow into a clones of cell).
 Screen supernatant of each
clone for presence of desired
antibody.
 Grow chosen clone of cells in
tissue culture indefinitely.
 Harvest antibody from the
culture.

10. Naked monoclonal Anti-body
 Naked mAbs are antibodies that have no drug or radioactive material attached to them. They work by
themselves. These 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.
Other work by blocking specific proteins that help cancer cells grow (some may do both).
 Naked monoclonal antibodies are the most widely used immunotherapeutic agent.
 Trastuzumab (Herceptin- against breast cancer) (against HER 2/neu protein).
 Bevacizumab (Avastin- against various cancer, e.g., colorectal, lungs, brain) (Against VEGFa & VEGFc).
 Adalimumab (Humira-against several autoimmune diseases) (Tumor necrosis factor inhibitor).
 Monoclonal antibodies achieve their therapeutic effects through various mechanisms.
1. Direct effects
2. Indirect effects
Direct effects Indirect effects
o They can have direct effects in producing apoptosis
or (programmed cell death).
o On the other hand, they can block growth factor
receptor and arrest proliferation of tumour cells.
o Their indirect effects are mainly brought about by
recruiting cytotoxic cells, such as monocytes and
macrophages.
Antibody-dependent cell mediated cytotoxicity(ADCC)
Complement-dependent cytotoxicity(CDC)

11. Antibody-dependent Cellular Cytotoxicity(adcc) Or Antibody-dependent Cell-mediated Cytotoxicity
o It is a mechanism of cell-mediated immune defence whereby an effector cell of the immune
system actively lyses a target cell, whose membrane-surface antigens have been bound by
specific antibodies. It is one of the mechanisms through which antibodies, as part of the
humoral immune response, can act to limit and contain infection.
o ADCC requires an effector cell which classically is known to be natural killer (NK) cells that
typically interact with immunoglobulin G (IgG) antibodies. However, macrophages,
neutrophils and eosinophils can also mediate ADCC, such as eosinophils kills certain
parasitic worms known as helminths via IgE antibodies.
The effects against solid tumors
of trastuzumab and rituximab
monoclonal antibodies have
been shown in experiments with
mice to involve ADCC as an
important mechanism of
therapeutic action

12. Complement-dependent cytotoxicity(CDC)
o Complement-dependent cytotoxicity (CDC) is an
effector function of IgG and IgM antibodies. When
they are bound to surface antigen on target cell
(e.g. bacterial or viral infected cell), the classical
complement pathway is triggered by bonding
protein C1q to these antibodies, resulting in
formation of a membrane attack complex (MAC)
and target cell lysis.
o Complement system is efficiently activated by
human IgG1, IgG3 and IgM antibodies, weakly by
IgG2 antibodies and it is not activated by IgG4
antibodies.
o It is one mechanism of action by which therapeutic
antibodies or antibody fragments can achieve an
antitumor effect.

13. Conjugated Monoclonal Antibody
A substance made up of a monoclonal antibody chemically linked to a radioactive substances,
drug & toxin. The monoclonal antibody binds to specific proteins or receptors found on certain
types of cells, including cancer cells. The linked drug enters these cells and kills them without
harming other cells, then linked radioactive particle helps in identification. Some antibody-
drug conjugates are used to treat cancer.
Radiolabeled Antibody
Radiolabelled antibodies have small radioactive particles
attached to them.
o Radiolabelled antibodies were studied first for tumour
detection by single-photon imaging.
o In the meantime, radiolabelled antibodies were shown
to be effective in the treatment of lymphoma.
 Ibritumomab tiuxetan (Zevalin) is an example of a
radiolabelled mAb. This is an antibody against the
CD20 antigen, which is found on lymphocytes called B
cells.

14. Chemo labeled Antibody
o Chemo labeled antibodies are mAbs have powerful chemotherapy (or other) drugs attached to
them.
o Chemotherapeutic agents can be attached to monoclonal antibodies to deliver high
concentrations of these toxic substances directly to the tumour cells
 Examples include: Brentuximab vedotin (Adcetris), an antibody that targets the CD30 antigen
(found on lymphocytes), attached to a chemo drug called MMAE [Monomethyl auristatin E
(MMAE) - Antimitotic Agent].

16. o In this technology, ScFv domains of monoclonal
antibodies are linked to a drug activating enzyme.
o These ScFv domains are capable of selectively
binding to the target cells.
 The objective of antibody directed enzyme prodrug
therapy (ADEPT) is to selectively deliver
chemotherapy to cancer sites. The basic principle of
ADEPT is to target an enzyme to tumours by
attaching it to an antibody directed to a tumour
associated antigen. After clearance of enzyme from
blood, a non-toxic prodrug is given. The targeted
enzyme converts a non-toxic prodrug into a potent
cell killing agent within tumours to achieve effective
therapy without normal tissue toxicity.
Antibody-directed Enzyme Prodrug Therapy (ADEPT)

17. Immune Checkpoint Inhibitors
o Checkpoints are proteins found on T cells that regulate how T cells respond to foreign cells.
o 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
proteins of the inspected cell indicate that the cell is foreign, the T cell stages an attack against it. Checkpoints signal to T
cells to multiply themselves to fight the invader. Once the invader is destroyed, checkpoints signal the T cells to turn off and
shut down the T-cell multiplication response.
o If T cells are active for too long or react to things they should not, they will start to destroy healthy cells and tissues, which
could result in autoimmune disorders such as Crohn’s disease or rheumatoid arthritis.
o 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 on normal cells, it sends a
message to the immune system to leave the cell alone. As a result, it reduces the production of T cells and enables more T
cells to die.
o T cells only expect normal cells to produce PD-L1, but sometimes cancer cells can avoid an immune system attack by
producing PD-L1 on their surfaces.
o Immune checkpoint inhibitors that treat blood cancer by binding to the PD-1 receptor on T cells and blocking the
interaction of PD-1 and PD-L1.
Pembrolizumab (Keytruda) Nivolumab (Opdivo)
Approved for the treatment of adult and paediatric patients with
refractory Hodgkin lymphoma, or those who have relapsed after 3
or more prior lines of therapy. Pembrolizumab has also been
approved for the treatment of adult and paediatric patients with
refractory primary mediastinal large B-cell lymphoma, or those
who have relapsed after 2 or more prior lines of therapy
is approved for the treatment of adults with Hodgkin
lymphoma that has relapsed or progressed after
autologous hematopoietic stem cell transplantation (auto-
HSCT) and brentuximab vedotin (Adcetris), or after three
or more lines of systemic therapy including auto-HSCT

18. Therapeutic Cancer Vaccine
In addition to traditional vaccines, there are two types of cancer vaccines,
A preventive cancer vaccine is given to prevent cancer from developing in healthy people.
For example, the hepatitis B vaccine is given to children to protect against a hepatitis B viral
infection, which can lead to liver cancer.
A therapeutic cancer vaccine is given to treat an existing cancer by aiming a stronger and
faster response from the immune system.

19. Non-specific Immunotherapies
 Non-specific immunotherapies don’t target foreign particle 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.
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.
Interferons
 Interferons are chemicals that help the body resist virus infections and cancers. The types of interferon (IFN)
are named after the first 3 letters of the Greek alphabet.
 Only IFN-alfa 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.
IFNA10 IFNa-4-Fc FNA alpha-4 IFNA alpha-5
IFN-alpha-7 IFN beta IFN gamma IFN omega
Various Types of Interferons

20. Interleukins
o Interleukins are a group of cytokines that act as chemical signals between white blood cells.
o Interleukin-2 (IL-2) helps immune system cells grow and divide more quickly. A man-made
version of IL-2 is approved to treat advanced kidney cancer and metastatic melanoma.
o IL-2 can be used as a single drug treatment for these cancers, or it can be combined with
chemotherapy or with other cytokines such as interferon-alfa.
o Side effects of IL-2 can include flu-like symptoms such as chills, fever, fatigue, and
confusion. Most people gain weight. Some have nausea, vomiting, or diarrhoea. Many people
develop low blood pressure.
o Other interleukins, such as IL-7, IL-12, and IL-21, are now being studied for use against
cancer too, both as adjuvants and as stand-alone agents.

21. Adoptive Cell Transfer
Adoptive cell transfer is a type of
immunotherapy that uses a patient’s
own T cells 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
able to target and kill cancer cells in
their bodies. Several types of
adoptive cell transfer therapies have
been developed, but to date, the one
that has advanced the furthest in
clinical development is called
chimeric antigen receptor (CAR) T-
cell therapy.

22. Tisagenlecleucel (Kymriah) Axicabtagene ciloleucel (Yescarta)
It is approved for the treatment of patients age 25
years and younger with B-cell precursor acute
lymphoblastic leukaemia that is either refractory
or in a second or later relapse. It is also approved
for adult patients with relapsed or refractory large
B-cell lymphoma after 2 or more lines of systemic
therapy.
It is approved for the treatment of adult patients
with either relapsed or refractory large B-cell
lymphoma after two or more lines of systemic
therapy, including diffuse large B-cell lymphoma
not otherwise specified, primary mediastinal large
B-cell lymphoma, high-grade B-cell lymphoma,
and diffuse large B cell lymphoma (DLBCL)
arising from follicular lymphoma.

23. Humanization of Antibody
o 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).
o 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 therefore potentially immunogenic to human patients but may cause toxicity.
o The International Non-proprietary Names of humanized antibodies end in -zumab, as- omalizumab
(see Nomenclature of monoclonal antibodies).
Chimeric mAb are usually produced from
transgenic mice and/or hybridoma
technology.
Humanised (zuMab) is produced by
genetic engineering, CDR grafting. V
gene cloning, and eukaryotic expression.

24. Methods of Humanization
CDR Grafting (Antibody Reshaping)
A flow chart with the three
main decision-making points
of a typical CDR grafting
protocol.
(1) definition of regions
determining the specificity
of the donor antibody and
hence target for grafting,
(2) source of human
sequences to be utilized as FR
donors,
(3) selection of residues
outside of the region defining
the specificity and thus target
for back mutation to restore or
improve the affinity of the
humanized antibody.

25. 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 immortalisation is the antibody produced are only specific to the antigen from the
infected organism (as humans cannot be immunised to produce the desired antibodies).

26. Hybridoma Technology Using Transgenic Mice
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 immunisation.
The steps thereafter are similar to producing traditional
monoclonal antibodies.
The B cells from immunised transgenic mice are fused with
myeloma cells derived from in vitro cell culture to produce
immortalised hybridoma.
These hybridomas are then screened for desired specificity.
Once these specific hybridomas are produced, it is possible
to generate hybrid-hybridomas

27. Recombinant Antibodies by Cloning V Region Gene
 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 cDNA responsible for
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 humanised/chimeric antibodies.
 Cloning of mouse variable genes into human constant-
region genes generates chimeric as well as humanised
antibodies depending on the size of the clone.

28. Immunotherapeutic in clinical practice
The WHO appointed body for International Non-proprietary Names (INN) has given different suffixes
to these antibodies to identify their origins,
 Chimeric antibodies (Names-xiMabs)
 Humanized antibodies (Names -zuMabs)
 Fully human antibodies (Names -muMabs)