MONOCLONAL ANITBODIES_MR PAYAAM VOHRA NIPER MOHALI.ppt

MONOCLONAL ANTIBODY
HYBIRIDOMA TECHNOLOGY

1. Submitted by: Mr. Payaam Vohra
M.S(Pharm.) Pharmacology and
Toxicology, NIPER S.A.S Nagar

2. BECOMES
FUNCTIONING OF THE IMMUNE SYSTEM
HUMORAL (ANTIBODY MEDIATED) IMMUNE RESPONSE CELL MEDIATED IMMUNE RESPONSE
ANTIGEN (1
ST
EXPOSURE)
ANTIGENS
DISPLAYED
BY
INFECTED
CELLS
ACTIVATE
CYTOTOXIC
T CELL
GIVES RISE TO
ACTIVE
CYTOTOXIC T
CELL
ENGULFED BY
STIMULATES
MACROPHAGE
APC
HELPER
T CELLS
STIMULATES
MEMORY
HELPER T
CELLS
MEMORY
T CELLS
MEMORY
B CELLS
PLASMA
CELLS
STIMULATES STIMULATES
B CELLS
FREE
ANTIGENS
DIRECTLY
ACTIVATE
STIMULATES
GIVES RISE TO
SECRETE ANTIBODIES
STIMULATES
ANTIGEN (2
nd
EXPOSURE)
STIMULATES

3. Polyclonal antibodies Monoclonal Antibodies
Produced by: Many B cell clones A single B cell clone
Bind to:
Multiple epitopes of all
antigens usedin the
immunization
A single epitope of a single
antigen
Antibody class:
A mixture of different
Ab classes(isotypes)
All of a single Ab class
Ag-binding sites:
A mixture of Abs with
different antigen-binding
sites
All Abs. have the same antigen
binding site
DIFFERENCE BETWEEN MONOCLONAL AND POLYCLONAL ANTIBODY

4. Monoclonal antibodies(mAbor moAb)
Monoclonal antibodies are:
monospecificantibodies that are identical because they are
produced by one type of immune cell (a single parent cell).
Antibodies produced from a single clone of B cells.
Produced by fusing a B cell secreting the desired antibody with
a myeloma cell capable of growing indefinitely in tissue culture.
Monoclonal antibodies all have identical antigen-binding sites.
i.e. they all bind to the same epitopewith the same affinity.
They are all of the same antibody class (isotype).
Ebell MH, Barry HC, Baduni K, Grasso G. Clinically important benefits and harms of monoclonal antibodies targeting amyloid for the treatment of Alzheimer disease: a systematic
review and meta-analysis. The Annals of Family Medicine. 2024 Jan 1;22(1):50-62.

5. Hybridoma
are cells that have been engineered to produce a desired
antibodyin large amounts.
To produce monoclonal antibodies, B-cellsare removed
from the spleen of an animal that has been challenged with
the relevant antigen
These B-cells are then fused with myelomatumor cells that
can grow indefinitely in culture
This fusion is performed by making the cell membranes
more permeable.
The fused hybrid cells (hybridomas), being cancer cells,
will multiplyrapidly and indefinitelyand will produce
large amounts of the desired antibodies
Alejandra WP, Irene JP, Antonio GS, Patricia RG, Elizabeth TA, Aleman-Aguilar JP, Rebeca GV. Production of monoclonal
antibodies for therapeutic purposes: A review. International Immunopharmacology. 2023 Jul 1;120:110376.

6. Myeloma cells lack certain enzymes so that
they can not use hypoxanthine, aminopterin,
and thymidine(HATmedium) as a source for
nucleic acid biosynthesis and will die in
culture.
Only B cells that have fused with the
engineered myeloma cells will survive in
culture when grown in HAT medium.
Hybridoma Selection
The “HAT Trick”

7. Practical steps in monoclonal antibody production:
1) Immunize animal
2) Isolate spleen cells (containing antibody-producing B cells)
3) Fuse spleen cells with myeloma cells (e.g. using PEG -polyethylene glycol)
4) Allow unfused B cells to die
5) Add aminopterinto culture to kill unfused myeloma cells
6) Clone remaining cells (place 1 cell/well and allow each cell to grow into a clone of
cells)
7) Screen supernatant of each clone for presence of the desired antibody.
8) Grow the chosen clone of cells in tissue culture indefinitely.
9) Harvest antibody from the culture supernatant.

8. Purification of monoclonal Antibodies
Contaminants:
Media components: hormones, growth factors, transferrins……etc
Viral, bacterial,endotoxins….etc
Methods of purification:
1.Filtration for larger particles
2.Ultrafiltration esp. for low concentration samples
3.Chromatography:
•Ionexchange chromatography (either cationor anion)can be
used (Most impurities are usually anions)
•Size exclusion chromatography

9. PossibleFusion
Products
+
PlasmaCells From
ImmunizedAnimal
Myelomacells
Unfused Fused Hybridcells Fused Unfused
plasmacellsplasmacells myelomacellsmyeloma

10. PossibleFusionProducts
+
MyelomaHGPRT Deficient
AndIgDeficient
Ig
-
HGPRT
-
PlasmaCells From
ImmunizedAnimal

Senescence

CanUseSalvage
Pathway
NoSenescence

HATMedium

Senescence

HATMedium
Ig
+
HGPRT
+

11. Suffix Nature of Monoclonal antibody
-mab monoclonal antibody
-mo-mab mouse mab
-xi-mab chimeric mab
-zu-mab humanized mab
-mu-mab human mab
-tu-xx-mab tumor-directed XX mab
-li-xx-mab immune-directed XX mab
-cl-xx-mab cardiovascular-directed XX mab
-vi-xx-mab virus-directed XX mab
Types of Monoclonal Antibodies
Kinch MS, Kraft Z, Schwartz T. Monoclonal antibodies: Trends in therapeutic success and commercial focus. Drug Discovery
Today. 2023 Jan 1;28(1):103415.

12. BY SOURCE
› Murine
› Chimeric
› Humanized
› Human
BY APPLICATION
> In Vivo
> In Vitro
BY END-USE
> Speciality Centres
>Hospitals
> Others
BY REGION
North America
› Europe
› Asia Pacific
› Latin America
› Middle East &
Africa
BY PRODUCTION
> Oncology
> Autoimmune
Diseases
> Infectious Diseases
> Neurological
Diseases
> Others
MARKET PLAYERS
> Abbott Laboratories Amgen Inc
> AstraZeneca plc
> Bayer AG
> Biogen Inc.
> Bristol Myers
> Squibb
> Daiichi Sankyo Company Ltd
> Eli Lilly and Company
> Hoffman-La Roche Ltd.
> GlaxoSmithKline plc
> Johnson & Johnson Services, Inc
> Merck & Co., Inc.
> Merck KGaA
> Novo Nordisk A/S
MARKET INSIGHTS OF MONOCLONAL ANTIBODIES

13. GLOBAL MARKET VALUE OF MONOCLONAL ANTIBODIES
Kinch MS, Kraft Z, Schwartz T. Monoclonal antibodies: Trends in therapeutic success and commercial focus. Drug Discovery Today.
2023 Jan 1;28(1):103415.

14. GLOBAL TRENDS IN MONOCLONAL ANTIBODIES MARKETING

15. Antibody Brand name Approval dateType Target Approved treatment(s)
Abciximab ReoPro 1994 chimeric inhibition of glycoprotein llb/illa Cardiovascular disease
Adalimumab Humira 2002 human inhibition of TNF-a signaling Several auto-immune disorders
Alemtuzumab Campath 2001 humanized CD52 Chronic lymphocytic leukemia
Basiliximab Simulect 1998 chimeric IL-2Ra receptor (CD25) Transplant rejection
Bevacizumab Avastin 2004 humanized Vascular endothelial growth factor (VEGF)Colorectal cancer
Cetuximab Erbitux 2004 chimeric epidermal growth factor receptor Colorectal cancer, Head and neck cancer
Certolizumab pegolCimzia 2008 humanized inhibition of TNF-a signaling Crohn's disease
Daclizumab Zenapax 1997 humanized IL-2Rg receptor (CD25) Transplant rejection
Eculizumab Soliris 2007 humanized Complement system protein C5 Paroxysmal nocturnal hemoglobinuria
Efalizumab Raptiva 2002 humanized CD11a Psoriasis
Gemtuzumab Mylotarg 2000 humanized CD33 Acute myelogenous leukemia (with calicheamicin)
Ibritumomab
tiuxetan
Zevalin 2002 murine CD20 Non-Hodgkin lymphoma (with yttrium-90 or indium-11
Infliximab Remicade 1998 chimeric inhibition of TNF-a signaling Several autoimmune disorders
Muromonab-CD3Orthoclone OKT31986 murine T cell CD3 Receptor Transplant rejection
Natalizumab Tysabri 2006 humanized alpha-4 (a4) integrin, Multiple sclerosis and Crohn's disease
Omalizumab Xolair 2004 humanized immunoglobulin E (lgE) mainly allergy-related asthma
Palivizumab Synagis 1998 humanized an epitope of the RSV F protein Respiratory Syncytial Virus
Panitumumab Vectibix 2006 human epidermal growth factor receptor Colorectal cancer
Ranibizumab Lucentis 2006 humanized Vascular endothelial growth factor A (VEGF-A)Macular degeneration
Rituximab Rituxan, Mabthera1997 chimeric CD20 Non-Hodgkin lymphoma
Tositumomab Bexxar 2003 murine CD20 Non-Hodgkin lymphoma
Trastuzumab Herceptin 1998 humanized ErbB2 Breast cancer
RETROSPECTIVE STUDIES OF MONOCLONAL ANTIBODIES
Kelley B. The history and potential future of monoclonal antibody therapeutics development and manufacturing in four eras. InmAbs 2024 Dec 31 (Vol. 16, No. 1, p. 2373330). Taylor & Francis.

16. Company Name Monoclonal productIndications FDA Approval
Ortho Biotech Orthoclone-OKT® Organ Transplant Rejection1986
J&J/Eli Lilly ReoPro Acute Cardiac Conditions1994
BiogenIdec/Genentech/Ro
che
Rituxan Non-Hodgkin's Lymphoma 1997
BiogenIdec Zevalin" Non-Hodgkin's Lymphoma 2002
PDLI Zenapax® Acute Transplant Rejection1997
MedImmune/Abbott Synagis Viral Respiratory Disease1998
Genentech/Roche HerceptinG Breast Cancer 1998
J & . J Remicade Crohn's, Rheumatoid Arthritis1998
Novartis Simulect Acute Transplant Rejection1998
Wyeth Mylotarg™ Acute Myleoid Leukemia 2000
Schering/ILEXOncologyCampath Chronic Lymphocytic 2001
Abbott/CAT Humira" Leukemia Rheumatoid Arthritis2002
Novartis/Genentech/Tano
x
Xolair Asthma 2003
Genentech/Xoma Raptiva Psoriasis 2003
Corixa/GlaxoSmithKlineBexxar® Non-Hodgkin's Lymphoma 2003
BMS/ImClone SystemsErbitux TM Colorectal Cancer 2004
Kelley B. The history and
potential future of monoclonal
antibody therapeutics
development and manufacturing
in four eras. InmAbs 2024 Dec
31 (Vol. 16, No. 1, p. 2373330).
Taylor & Francis.

17. Target Generic Name Indication
Alkaline phosphatase asfotasealfa Hypophosphatasia
Amyloid beta gantenerumab Alzheimer's Disease
Anti-amyloid solanezumab Alzheimer's Disease
Amyloid protein A NEOD001 Amyloidosis, Primary
C. difficile enterotoxin A and Bactoxumab C. difficile associated disease
C. difficile enterotoxin Bbezlotoxumab C. difficile associated disease
Calcitonin gene-related peptideLY2951742 Hypercholesterolemia, familial
CCR4" mogamulizumab Lymphoma, Cutaneous T-cell
Cell adhesion molecules elotuzumab Cancer, Multiple Myleloma
Cyclic ADP ribose hydrolasedaratumumab, HuMax-38 Cancer, Multiple Myleloma
Dabigatran idarucizumab Dabigatran Overdose
Folate receptor alpha farletuzumab Cancer, Ovarian
Glycoprotein, Immune cellibalizumab HIV Infection
HER3b patritumab Cancer, Lung, Non-small Cell
IL-2 receptor daclizumab (high-yield process)Multiple Sclerosis
IL-4 receptor dupilumab Asthma, moderate/severe
IL-5 mepolizumab Hypereosinophilic Syndrome
IL-5 receptor, alpha benralizumab Asthma, moderate/severe
IL-13 lebrikizumab Asthma, moderate/severe
IL-13 tralokinumab Asthma, moderate/severe
Mucin 5AC clivatuzumabtetraxetan Cancer, Pancreatic
Nerve growth factor fulranumab Pain, Osteoarthritis
PCSK-9 alirocumab Hypercholesterolemia, familial
PD-1L atezolizumab Cancer, Lung, Non-small Cell
Phosphatidyl-serine bavituximab Cancer, Lung, Non-small Cell
SALI* epratuzumab Systemic Lupus Erythematosus
Targets of
Monoclonal
antibodies
Alejandra WP, Irene JP, Antonio GS,
Patricia RG, Elizabeth TA, Aleman-
Aguilar JP, Rebeca GV. Production
of monoclonal antibodies for
therapeutic purposes: A review.
International Immunopharmacology.
2023 Jul 1;120:110376.

18. Monoclonal Antibody Brand Name Approval DateIndication Manufacturer
Teplizumab-mzwv Tzield November 2022Type 1 diabetes (delaying onset in at-risk individuals) Provention Bio
Sotrovimab Xevudy December 2022COVID-19 (mild to moderate)
GlaxoSmithKline &
Vir Biotechnology
Mosunetuzumab-axgb Lunsumio December 2022Relapsed/refractory follicular lymphoma Genentech
Bimekizumab Bimzelx August 2022 Plaque psoriasis UCB Pharma
Faricimab-svoa Vabysmo January 2022 age-related macular degeneration (AMD), Diabetic macular edema (DME)Genentech
Marstacimab Marstaca April 2023 Hemophilia A or B Pfizer
Tebentafusp-tebn Kimmtrak January 2022 Uveal melanoma Immunocore
Odevixibat Bylvay July 2021 Pruritus in patients with progressive familial intrahepatic cholestasisAlbireo Pharma
Tralokinumab-ldrm Adtralza January 2022 Moderate-to-severe atopic dermatitis Leo Pharma
Tirzepatide Mounjaro May 2022 Type 2 diabetes Eli Lilly
Bebtelovimab No brand nameFebruary 2022COVID-19 Eli Lilly
Lanadelumab-flyo Takhzyro August 2022 Hereditary angioedema (HAE) Takeda
Lecanemab-irmb Leqembi January 2023 Alzheimer's disease Eisai & Biogen
Dostarlimab-gxly Jemperli April 2021 Endometrial cancer GlaxoSmithKline
Nivolumab + RelatlimabOpdualag March 2022 Unresectable or metastatic melanoma Bristol Myers Squibb
LIST OF RECENTLY APPROVED MONOCLONAL ANTIBODIES
Kaplon H, Crescioli S, Chenoweth A, Visweswaraiah J, Reichert JM. Antibodies to watch in 2023. InMAbs 2023 Dec 31 (Vol. 15, No. 1, p. 2153410). Taylor & Francis.

19. Antibody Name Target(s) Indication(s)
First EU
Approval
Year
First US Approval
Year
Datopotamab
deruxtecan
TROP-2
Non-small cell lung
cancer
Review Review
ZenocutuzumabHER2, HER3
NRG1+ non-small cell
and pancreatic cancer
NA Review
Nemolizumab IL-31R-alpha Dermatitis Review Review
Zanidatamab Pending Pending Pending Pending
FUTURE INSIGHTS OF MONOCLONAL ANTIBODIES
Crescioli S, Kaplon H, Chenoweth A, Wang L, Visweswaraiah J, Reichert JM. Antibodies to watch in 2024. InMAbs 2024 Dec 31 (Vol. 16, No. 1, p.
2297450). Taylor & Francis.

20. FUTURE SCOPE

21. Measuring protein and drug levels in serum
Typing tissue and blood
Identifying infectious agents
Identifying clusters of differentiation (CDs) for
classification and follow-up therapy of leukemias and
lymphomas
Identifying tumor metastasis
Identifying and quantifying hormones
Immunoaffinity Purification
Uses of monoclonal Abs

22. Applications of Monoclonal Antibodies
•Diagnostic Applications
•Therapeutic Applications
Transplant rejection
Cardiovascular disease
Cancer
Infectious Diseases
Inflammatory disease
•Clinical Applications
Purification of drugs, Imaging the target
•Future Applications
Fight against Bioterrorism

23. Diagnostic Applications (Immuno-ctochemistry)
1.Tissues and tumors can be classified based on their
expression of certain defined markers: e.g. Prostate
specific antigen(PSA)
2.Classification of tissues and tumors
3.Distinguishing morphologically similar lesions, like
mesotheliomaand adenocarcinoma
4.Detecting small quantities of metastatic cancer and
MDR

24. THANK YOU !