monoclonal antibodies , molecular mechanism of action .pptx

MONOCLONAL ANTIBODIES
SCIENTIFIC OVERVIEW WITH THERAPEUTIC APPLICATIONS
AND MECHANISTIC INSIGHTS
PART 1
Supervised by:
Prof. Dr. Kawa Dizaye
Head of the Department of
Pharmacology, Medical Physics,
and Biochemistry
Hawler Medical University (HMU)
Prepared by:
Omer Muhammed Asaad
MSc candidate in Pharmacology

OUTLINE
Introduction to Monoclonal Antibodies
History
Structure of Monoclonal Antibodies
Production of monoclonal antibodies
classification of monoclonal antibodies
Mechanisms of Action of mAbs
Comparison with Small Molecule Drugs
Advantages & Disadvantages of Monoclonal Antibodies

INTRODUCTION TO MONOCLONAL ANTIBODIES
Monoclonal antibodies (mAbs) are laboratory-produced molecules engineered to
serve as substitute antibodies that can restore, enhance, or mimic the immune system’s
attack on cells.
They are designed to bind to antigens.
Other terms for this therapy include “biologics,” “biological therapy,” “biologicals,”
and “biopharmaceuticals.”
mAbs have revolutionized the treatment of various diseases, including cancers,
autoimmune disorders, and infectious diseases. Hansel et al., 2010.

HISTORY
1900s: Paul Ehrlich proposed the “magic bullet” concept — targeting
disease selectively.
1975: Köhler & Milstein developed hybridomas to produce specific,
immortal antibodies (Nobel 1984).
1988: Gregory Winter humanized antibodies to reduce immune reactions.
Lee Nadler: First to treat a human with monoclonal antibodies; discovered
B-cell antigens.

STRUCTURE OF MONOCLONAL ANTIBODIES
Y-shaped glycoproteins composed of two identical heavy chains
and two identical light chains.
Each arm of the Y contains a variable region that binds to a specific
antigen.
The stem of the Y, known as the Fc region, determines the
antibody’s class and can bind to cell receptors or complement
proteins.
Fab region  antigen-binding site
Fc region  binds to effector (immune cells such as helper T cell)
Hansel et al., 2010.

PRODUCTION OF MONOCLONAL ANTIBODIES BY
HYBRIDOMA TECHNIQUE
1. Antigen injected into mouse to stimulate B-cell
antibody production.
2. Spleen B-cells fused with immortal myeloma
cells using PEG.
3. Fused cells (hybridomas) selected in HAT
medium; only hybrids survive.
4. Hybridomas screened for specific antibody
production.
5. Selected clones cultured; antibodies harvested
and purified.
Note: HAT medium = Hypoxanthine – Aminopterin – Thymidine
Normal B-cells: Cannot grow long-term in HAT medium, only Hybridoma cells can.
Hybridoma = B-cell (antibody-producing) + Myeloma cell (immortal) used to make monoclonal antibodies.
→

CLASSIFICATION OF MONOCLONAL ANTIBODIES
Murine (mouse)  100% murine
Chimeric  70% human, 30% murine
Humanized  90% human, 10% murine
Fully human  100% human
First-generation mAbs:
1. Fully murine (mouse-derived) antibodies:
Caused immune reactions in 50–75% of patients.
Had short plasma half-life.
Could not activate human complement.
2. Chimeric mAbs:
Combine murine Fab (antigen-binding) + human Fc
(constant) regions.
Extended half-life (5x longer) due to human Fc
domain.
Improved compatibility and function in human body.

CLASSIFICATION OF MONOCLONAL ANTIBODIES
Second-generation mAbs:
3. Humanized mAbs:
Mostly human IgG structure.
Only the hypervariable (antigen-binding) regions are
murine.
Example: Trastuzumab (Herceptin)
4. Fully Human mAbs:
Derived from transgenic mice that carry human
immunoglobulin genes.
Antibody engineering technologies.
Still may be immunogenic, despite being “fully human”.
(Harding et al., 2010).

MECHANISMS OF ACTION OF MABS
Direct Mechanisms (Signaling pathway blockage) :
Blocking ligand-receptor interactions to inhibit cell
signaling.
Inducing apoptosis directly by binding to specific antigens.
Indirect Mechanisms:
Antibody-Dependent Cellular Cytotoxicity (ADCC):
Recruitment of immune cells to destroy target cells.
Complement-Dependent Cytotoxicity (CDC): Activation of
the complement system leading to cell lysis.
Antibody-dependent cellular phagocytosis (ADCP):

ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY
(ADCC):
In antibody-dependent cellular cytotoxicity (ADCC),
the Fc receptor
γ (Fc R or FCGR) on
γ immune
effector cells binds to the Fc region of an antibody,
which is attached to a specific target cell.
Immune cells like natural killer cells, macrophages,
monocytes, and eosinophils can mediate ADCC.
Once Fc receptor
γ engages with the antibody, its
ITAM domain is phosphorylated, leading to
effector cell activation.
This activation triggers the release of substances
such as perforin, granzyme, lyase, and
TNF, which contribute to the targeted cell’s
destruction.

COMPLEMENT-DEPENDENT CYTOTOXICITY (CDC)
In complement-dependent cytotoxicity (CDC), C1q
binds to antibodies, which triggers the complement
cascade, resulting in the formation of a membrane
attack complex (MAC)
(C5b to C9) on the surface of target cells, and
further resulting in a classical pathway of
complement activation.

ANTIBODY-DEPENDENT CELLULAR PHAGOCYTOSIS
(ADCP)
Antibody-dependent cellular phagocytosis
(ADCP) is a key MOA for many
antibody-based therapies.
It is defined as a highly regulated process in
which an antibody eliminates binding target and
initiates phagocytosis by linking its Fc domain to
a specific receptor on the phagocytic cell.
Unlike ADCC, ADCP can mediate monocytes,
macrophages, neutrophils and dendritic cells via
FcγRIIa, FcγRI and FcγRIIIa, where FcγRIIa
(CD32a) on macrophages represents the major
pathway.

KEY DIFFERENCES OF ADCC/CDC/ADCP

BISPECIFIC MONOCLONAL ANTIBODY
• Engineered antibodies that can simultaneously bind two different
antigens or epitopes, enhancing therapeutic specificity and efficacy
(Kontermann, 2012).
• Mechanism of Action:
• Redirect immune cells (e.g., T cells) to cancer cells.
• Block two signaling pathways simultaneously.
• Crosslink receptors for synergistic inhibition (Spiess et al., 2015).
Advantages:
• Enhanced specificity
• Reduced resistance
• Lower off-target toxicity (Suresh et al., 2021)
Approved bsAbs:
Blinatumomab (Blincyto): Targets CD19/CD3 for B-cell precursor
acute lymphoblastic leukemia (ALL).
Teclistamab (Tecvayli): Targets BCMA/CD3 for multiple myeloma.

mAbs VS. SMALL MOLECULE DRUGS
Which one is better?!
Small Molecule Drugs limitations

COMPARISON WITH SMALL MOLECULE DRUGS
(TRADITIONAL PHARMACEUTICAL DRUGS)
mAbs are structurally more complex than
small-molecule agents & lower molecular
weight biologics.
mAbs are typically higher molecular
weight proteins (~150kDa) with complex
secondary and tertiary structures subject
to post-translational modiﬁcations.

COMPARISON WITH SMALL MOLECULE DRUGS
(TRADITIONAL PHARMACEUTICAL DRUGS)
Molecular Size:
mAbs: Large (~150 kDa).
Small Molecules: Small (<1 kDa).
Specificity:
mAbs: High specificity to target antigens.
Small Molecules: Often less specific, may affect multiple
targets.
Pharmacokinetics
mAbs: Poor oral bioavailability; mostly parenteral
administration.
Slow absorption (unless IV), long half-lives.
Volume of distribution often small due to high target affinity.
Pharmacodynamics
mAbs: High-affinity binding, slow on/off kinetics.
May be internalized, altering target cells.
Dose-response may be bell-shaped or U-shaped (not linear).
Optimal dosing may be narrow and not proportional.
Elimination
Not metabolized/excreted like small molecules.
Cleared via lysosomal degradation after lymphatic uptake.
Some smaller mAbs (<69 kDa) cleared by kidneys.
Immunogenicity (if present) can increase clearance.
Drug Interactions & Safety
Fewer drug interactions and general toxicity issues.
May still cause serious biological side effects.

• Monoclonal antibody (mAb) preparations now dominate the biopharmaceutical market.
• They represent the majority of the top-selling drugs in recent years (Evans et al., 2021).
• As of 2025, over 86 monoclonal antibody therapeutics have been approved by the FDA, with more than
200 globally approved or in review, highlighting their dominant role in modern biopharmaceuticals.
(Jiang et al., 2024; Kaplon & Reichert, 2023).

ADVANTAGES & DISADVANTAGES
OF MONOCLONAL ANTIBODIES
Advantages
1. High target specificity reduces off-target
effects.
2. Long half-life allows for less frequent dosing.
3. Ability to engage the immune system for
enhanced therapeutic effects.
(Hansel et al., 2010)
Disadvantages
1. High production costs and
complex manufacturing
processes.
2. Potential for immunogenicity
leading to adverse reactions.
3. Limited tissue penetration due
to large molecular size.

TRASTUZUMAB
Targets the HER2 receptor overexpressed in certain breast cancers.
Mechanism: Inhibits HER2-mediated signaling pathways and induces ADCC.
Clinical Impact: Improves survival rates in HER2-positive breast cancer patients.

RITUXIMAB
Targets CD20 antigen on B cells.
Mechanism: Induces B cell lysis through CDC and ADCC.
Clinical Use: Treatment of non-Hodgkin’s lymphoma and chronic lymphocytic leukemia.

REFERENCES
1. Hansel, T.T., Kropshofer, H., Singer, T., Mitchell, J.A., & George, A.J.T. (2010). The safety and side effects of monoclonal antibodies. Nature Reviews
Drug Discovery, 9(4), 325–338.
2. Kciuk, M., Kołat, D., Kałuzińska-Kołat, Ż., & Kontek, R. (2023). Comparison of small-molecule inhibitors and monoclonal antibodies. ResearchGate.
3. Jiang, S., Gao, X., Liu, Y., & Chen, Y. (2024). Recent advances in therapeutic monoclonal antibodies: FDA approvals and clinical trends. Frontiers in
Pharmacology, 15, Article 11729449.
4. Kaplon, H. & Reichert, J.M. (2023). Antibodies to watch in 2023. mAbs, 15(1), e2193136.
5. Mellstedt, H., 2013. Clinical considerations for biosimilar antibodies. Eur. J. Cancer Suppl. 11, 1–11. https://doi.org/10.1016/S1359-
6349(13)70001-6
6. Mokobi, F., 2022. Monoclonal Antibodies- Definition, Types, Production, Applications [WWW Document]. URL
https://microbenotes.com/monoclonal-antibodies-types-uses-and-limitations/ (accessed 5.18.25).
7. Schematic representation of the mechanism of action of trastuzumab and... [WWW Document], n.d. . ResearchGate. URL
https://www.researchgate.net/figure/Schematic-representation-of-the-mechanism-of-action-of-trastuzumab-and-pathogenesis-
of_fig4_323612013 (accessed 5.21.25).
8. Kontermann, R.E. (2012). Dual targeting strategies with bispecific antibodies. mAbs, 4(2), pp.182–197.
9. Spiess, C., Zhai, Q. and Carter, P.J. (2015). Alternative molecular formats and therapeutic applications for bispecific antibodies. Molecular
Immunology, 67(2), pp.95–106.
10. Suresh, S., Lee, J. and Ghosh, P. (2021). Bispecific antibodies in cancer immunotherapy: Advances and challenges. Cancer Treatment Reviews, 96,
102175.

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