The document discusses monoclonal antibodies, including their discovery, structure, functions, types, production, targets for cancer treatment, and mechanisms of action. It provides details on how monoclonal antibodies can directly kill tumor cells or induce immune-mediated killing through mechanisms like phagocytosis, complement activation, and antibody-dependent cell cytotoxicity. The document also examines immune checkpoint inhibitors, immunotoxins, bispecific antibodies, immunoliposomes, antibody fragments, and an animal model study on monoclonal antibody treatment for colorectal cancer.

1. DOCTORAL SEMINAR- II
Presenter:
SUMIYAH RASOOL
Doctoral Student
Dept. of Microbiology & Immunology
Major Advisor:
Dr. Md. Isfaqul Hussain
Assistant Professor
Dept. of Microbiology & Immunology

2. What Are Antibodies?
structurally related
Glycoproteins
An antibody is a protein
used by the immune
system to identify &
neutralize foreign objects
like bacteria & viruses.
Introduction

3. ANTIBODIES...
Monoclonal antibodies
are identical because
they were produced by
one type of immune
cell, all clones of a
single parent cell.
Polyclonal antibodies
are derived from
different cell lines.
They differ in amino
acid sequence.
Introduction

4. • Animals and humans have the ability to make
antibodies able to
▫ recognize virtually any antigenic determinant
(epitope)
▫ to discriminate between even similar epitopes
• The remarkable specificity of antibodies makes
them promising agents in the field of
therapeutics.
Introduction

5. DISCOVERY!!!
George Kohler & Cesar Milstein in
1975 shared the Nobel Prize in
Physiology or Medicine in 1984
for discovery of hybridoma
technology.

6. Monoclonal Antibodies
Clones of a single parent cell.
MAbs are an integral part of targeted therapy approach
for various diseases which result in decrease in adverse
effects and increase in efficacy.
They target various receptors or various growth factors
on the cell surface and modulate their vital functions
and cause cell death by various mechanisms.

7. Functions
Antibodies have two major functions:
• Recognize and bind antigen
• Induce immune responses after binding
The variable region mediates binding
• Affinity for a given antigen
• confers absolute specificity for an antigen
The constant region mediates immune response after
binding
• Different classes of constant regions generate
different isotypes
• Different isotypes of antibody have differing
properties

8. Production of Monoclonal Antibodies

9. Types of Monoclonal Antibodies
 Murine mAbs:
• Rodent mAbs with excellent
affinities and specificities.
• Lead to allergic or immune
complex hypersensitivities.
Human mAbs:
• Completely human origin

10. Chimeric mAbs:
• Combine the human constant regions
with the intact rodent variable regions.
• Affinity and specificity unchanged.
• Also cause human antichimeric antibody
response (30% murine resource).
Humanized mAbs:
These Contain :-
•CDRs of the rodent variable region
•Human variable region framework.
Types of Monoclonal Antibodies

11. Evolution Of Monoclonal Antibody
Ist
generation
mAb
2nd generation mab
daclizumab

12. Cancer and immune system
• Connection between cancer and the immune
system was first uncovered by Dr. William B.
Coley who is regarded as the “father of cancer
immunotherapy.”
• He observed dramatic disappearance of
malignant tumors in cancer patients who had
contracted acute streptococcal infections.

13. Contd.
• Dr. Coley developed mixture of killed bacteria
that became known as Coley’s mixed bacterial
toxin.
• Bacterial products of which it was composed
had acted as immune potentiators they had
stimulated certain immune cells to kill the
cancer.

14. How do cancer cells differ from normal?
 Clonal in origin
 Deregulated growth and lifespan
 Altered tissue affinity
 Resistance to control via apoptotic signals
 Change in surface phenotype and markers
 Structural and biochemical changes
 Presence of tumour-specific antigens

15.  Cytokines – T cell growth factors that stimulate
immune response (ie: interferons, interleukins).
 Allogeneic HSCT – tumor cells eliminated through
high-dose chemotherapy followed by graft-versus-
tumor effect.
 Oral molecular target drugs (e.g tyrosine kinase
inhibitors, signal transduction inhibitors, etc.).
Cancer Immunotherapy: A New Era

16.  Immune checkpoint inhibitors – “release the brakes”
on the immune system (ie: ipilimumab, nivolumab,
pembrolizumab).
 CAR-T therapy – chimeric antigen receptor (CAR) T
cells.
 Immunovirus – therapeutic cancer vaccine.
 Autologous cellular immunotherapy – activated APCs
are reinfused into patient to direct immune cells vs
tumor cell (ie: sipuleucel-T)
Cancer Immunotherapy: A New Era

17.  Monoclonal Antibodies – direct or indirect immune
response
▫ Rituximab – immune-mediated cytotoxic response
▫ cetuximab – block signal pathways needed for cell growth
▫ Bevacizumab – block angiogenesis needed for oxygen &
nutrients
▫ Brentuximab vedotin – antibody drug conjugate (ADC)
▫ Tositumomab 131
I – antibody combined with radioactive
particle
Cancer Immunotherapy: A New Era

18. Antibodies against cancer
Cancer cells share many similarities with the
normal host cells and this presents a challenge
for achieving selective cytotoxicity.
Chemotherapeutic monoclonal antibodies were
engineered with the predicted advantage of
specificity, thus acting as ‘targeting missiles’
toward cancer cells.
~Yoon et al ,2010

19. Contd.
Antibody therapy for cancer has become established
over the past 15 years & is now one of the most
successful & important strategies for treating
patients with haematological malignancies &
tumours.
Ab therapy targets those cancer antigens that are :-
• abundant
• accessible
• expressed homogeneously, consistently and
exclusively on the surface of cancer cells.

20. Antibody-based therapies offer many advantages
due to
• Long half-life,
• Good tolerance and
• Broad extracellular fluid bio-distribution of these
biomolecules.
13 antibodies already approved by the FDA for
various oncological indications, a pipeline of 165 new
anticancer mAbs are in clinical trials: 89 (54%) in
phase I, 64 (39%) in phase II and 12 (7%) in phase III
studies. ~Reichert and Dhimolea, 2012
Contd.

21. oEGFR, HER2 and CD20
are among the top five
most frequently
targeted.
oThese three antigens
are targets for a total
of 18 mAbs in clinical
study, as well as eight
marketed mAbs.
~Nelson, 2010
Antibody targets

22. MAbs targeting EGFR
oEGFR is the target for three marketed anticancer
mAbs: cetuximab, panitumumab and nimotuzumab
and seven mAbs are currently undergoing clinical
study.
oFunction through ;
1. Blocking ligand–receptor interactions.
2. Antibody-dependent cell mediated cytotoxicity

24. • The humanized IgG1
trastuzumab (Herceptin) is
FDA-approved as a
treatment for HER2
overexpressed in breast
cancer and metastatic
gastric or gastroesophageal
junction adenocarcinoma.
MAbs targeting HER2

25. oCD20 is the target of four
marketed mAbs
(rituximab, ofatumumab,
ibritumomab tiuxetan,
tositumomab) and five
mAbs currently in study.
MAbs targeting CD20

26. Mechanism of
Action

27. Direct Tumor cell Killing
By receptor agonist activity:- Ab binding to a tumour
cell surface receptor and activating it, leading to
apoptosis.
By receptor antagonist activity:- Ab binding to a cell
surface receptor and blocking dimerization, kinase
activation and downstream signalling, leading to
reduced proliferation and apoptosis.
Antibody binding to an enzyme can lead to
neutralization, signalling abrogation & cell death.
Conjugated antibodies can be used to deliver a
payload (such as a drug, toxin, small interfering
RNA or radioisotope) to a tumour cell.

28. Direct Tumor cell Killing

29. Immune–mediated killing
Carried out by the induction of
o Phagocytosis
o Complement activation
o Antibody-dependent cell cytotoxicity
Genetically modified T cells
being targeted to the tumour by
single chain variable fragment.
T cells being activated by Ab-
mediated crosspresentation of
antigen to dendritic cells.
 Inhibition of T cell inhibitory
receptors, such as CTLA4.

30. Vascular and stromal cell ablation

31.  Vascular and stromal cell ablation can be
induced by:-
ovasculature receptor antagonism
ostromal cell inhibition
odelivery of a toxin to stromal cells
odelivery of a toxin to the vasculature
Vascular and stromal cell ablation

32. Immune checkpoint inhibitors
Immune system relies on multiple checkpoints to
avoid over activation on healthy cells.
Tumor cells hijack these checkpoints to escape
detection.
CTLA-4 & PD-1 are upregulated on T cell surface in
some cancers.

33. Immune checkpoint inhibitors
PD-1 : PD-L1 interaction results in T cell
suppression (anergy, exhaustion, death).
Inhibiting CTLA-4 & PD-1 can “release the
brakes” .
Checkpoint inhibitors don’t attack the tumor,
they set the T cells straight.

34. Immune checkpoint inhibitors

35. Immunotherapy: Checkpoint inhibitors

36. Immunotoxins :
Monoclonal antibodies combined to some antitumor
toxic agents.
oRadiolabeled : e.g. Ibritumomab tiuxetan
• Against the CD20 antigen which is found on
lymphocytes called B cells.

37. oChemolabeled
• Also known as antibody-drug conjugates (ADCs).
• The drug is often too powerful to be used on its own
as it would cause too many side effects if not
attached to an antibody. Brentuximab vedotin

38. Bispecific (BiTE) Antibodies
Made up of parts of 2 different mAbs .
Attach to 2 different proteins at the same time.
Brings the cancer cells and immune cells together,
which is thought to cause the immune system to
attack the cancer cells.

40. Immunoliposomes
Generated by coupling of antibodies to the
liposomal surface .
Designed to assure delivery to the target.
 Avoid cellular drug resistance mechanisms.
Facilitate intracellular penetration by receptor-
mediated endocytosis thus increasing intracellular
drug levels.

41. Immunoliposomes

42. Antibody Fragments

43. Better penetration of tumors as compared with full-
length IgG molecules.
Because of the lack of an Fc region, antibody
fragments and/or domains can have reduced biological
activity unless they are modified.
The antibody fragment currently undergoing Phase
II/III studies.
~Wong et al, 2017
Antibody Fragments

44. Animal Model study

45. • Sears and colleagues treated 20 patients with anti-
colorectal cancer mouse monoclonal antibody of the
IgG2a class.
• The antibody had been shown to mediate lysis of
colorectal carcinoma cells in tissue culture by human
or mouse effector cells and specifically inhibited the
growth of human colon carcinomas xenografted in
athymic nude mice.
• The investigators concluded that three patients had a
definite response to the monoclonal antibody
therapy. Yet two of these patients received
simultaneous radiation or chemotherapy.
Human study

46. Challenges of Monoclonal Antibody
Low immunogenicity of xenogeneic antibodies.
 Unscrupulous antigen shedding into circulation
causing bystander effects.
Inordinate vasculature in the tumor making targeting
through intravenous injection to site of tumor
challenging.
 Elevated hydrostatic pressure at the tumor site
limits the penetrating power of antibodies.

47. Side effects of mAbs which are usually mild and are
often more like an allergic reaction.
Some mAbs can also have other side effects that are
related to the antigens they target.
e.g. a mAb that targets tumor blood vessel growth
can cause side effects such as high blood pressure,
bleeding, blood clots, etc.
Production cost is estimated at twice that required
for conventional drugs.
Challenges of Monoclonal Antibody

48. Future Prospects
With the success rate of bringing these drugs to the
market being better than that of small molecular
drugs, it is expected that pharmaceutical companies
will continue to progress toward more specific, less
toxic and more cost-effective mAbs.
Most mAbs currently in the market as cancer drugs
are canonical. So wide variety of noncanonical mAbs,
like bispecific antibodies, engineered antibodies and
antibody fragments and/or domains should be
developed.

49. THANKSTHANKS