Hybridoma technology

monoclonal antibodies
Hybridoma technology
nomenclature of monoclonal antibodies
applications

1. BY: UDITI HANDA (ASST. PROFESSOR)
M.PHARM (PH.D)
GGSCOP, YAMUNANAGAR

¡Subject: Pharmaceutical Biotechnology
(BP-605T) |
Topic: Hybridoma Technology
sm

2. What are antibodies?
An antibody is a protein used the immune
SE to identify and neutralize foreign
| cts like bacteria and viruses. Ea >
ant recognizes a specific antigen unique
to its target.

Monoclonal antibodies (mAb) are antibodies
that are identical because prey were pruducsa
one type of immune cell, all ciones of a
single parent cell.
poycions antibodies are antibodies that are
rived from different cell lines. They differ in
amino acid sequence.

immunoglobulin (19) are structurally related
glycoproteins that function as antibodies

5. _ Monodional Antibodies

* Technique for Monoclonal Antibody Production was
discovered by..

Georges J F Koehler and Cesar Milstein in 1975.

7. Isolation of
Plasma cell

8. Fusion of Plasma and Mons ;

0

—

Plasma Myeloma E
Fused Fused. idoma Unfused Unfused

Plasma Myeloma - dados = Plasma Myeloma

9. HAT Media

est.

cell o Cell

Salvage

pathway nn
2 . Nucleotides

aa de novo
pathway

10. -Sugars and.
amino acids

af Baras

pathway

11. Fused Fused
Plasma = Myeloma

HGPRT+ >:

8 e

Hybridoma Unfused

HGPRT+

Se

Plasma
HGPRT+

Unfused i

Myeloma 0

SE

12. Fess e

Fused Fused Hybridoma Unfused Unfused

Mets Myelom Plasma _Myeloma _
=> HGPRT+ ESE

13. HAT Selection

Denovo Pathway ml DNA synthesis ——— a Salwage Pathway

(HGPRT gene required for this Pathway)
H= Hypoxanthine (Required for Sak
UNFUSED, A N ( TS UNFUSED
Bcal A = Aminopterin (Block Denovo y
Myeloma Cell
T = Thymidine (Required for Salwag« ay
(HAT Medium) ye
Denovo Pathway Salwage Pathway Denovo Pathway Salwage Pathway
Blocked by : Blocked by No Salwage Pathway
Amenopterin | X. Died, because! x Amenopterin Because Myeloma
Present in It has short Present in Cells has no
HAT medium Life span HAT medium HGPRT gene
HGPRT =
Unfused 8-cell can not survive Unfused Myeloma cell can not survive
Blocked by Amenopterin ve, À
Present in HAT medium

FUSED ey SalwagePathway we
todo In hybridoma cell, HGPRT gene comes
From B-Bell partner, so cell survive in HAT

14. Fused
Hybridoma
Cells

Culture tubes

Monoclonal
antibody
Selected against

The desired antigen

Hybridoma célls
Secretes monoclonal
Antibodies in media | } YY

invo Placed in mice peritoneal
production | Cavity to produce
| Monoclonal Abs

irom cell
in vitro | Placedin fermenters } selected for further production
production | to produce Monoclonal Abs of monoclonal antibodies

15. “Production of Monoclonal Antibodies

Myeloma 3. Selection of Hybridomas 4. Isolation of

hybridomas of single
Celts (HAT Selection)
(HGPRY, Ig PS Le) ed

2. Cell Fusion » y”,
ia 5. = of
B cells © Lt =

so suit EEE E

1. immunization m

| 50 - a
of mouse
ep. 7. Characterization and ik Hicoinn anil fropenation

Storage

16. By: a ee Heb
L >

(ira Teck a

"Nomenclature ef nammoclomal Ard leodres
d e

Monocle: Prods
a A Gone wal
m Kb. ax not
a q £talte
Saget eo eel T
if a abet
| vant te ED a a en
GER 5 huwou = u
gr ori tust Sf boten a sold,
Z 30 st
[2-9] TL +

Ss = fe

zu

17. Y Name of a monoclonal antibody is divided into
4 parts :

Vv Prefix

Y Target subsystem

Y Origin subsystem
Y Suffix

18. Types of mAbs

Human Murine Chimeric Humanized
-umab -momab -ximab -zumab

Purple denotes human component orange murine component

19. Differences in antibodies
ized antibody

20. u vine > Mo + Pr anınal
= Geel I => Se-50 | —
N H@maniGed > ZU > 80-30 ko 2 sohich related Lo one TE |
EE ea gere) GES
FA +
Vers ET TEE
1 a a taa en
Li + Rate - ian
599 Bases
zk a} LU ha -. ed _ A este > Rs Fer platas
= 3
{ Trastubr malo = La Lone pa Fr =
Panitufauymabl > nimes >42
Ly re I AR i Etap D PES
: = twas, re
| Ld ME 4
ok y sb

LL > ue mm )
+

>
CAL home vt )

> ogainet view)
zs

e
E BERN
‘ig

© 31 pa ¿lu malo
a Ada Emu mab R ti . +
certo) zumal = y ad
Er ftkivale a

CETTE SE

Ea pala)
Se
Gr Reet vans
Benbhuwab,
E girls.

21. 2. Target subsystem - Identified by specific letters 2. Target subsystem - Identified by specific letters

co (1)- colonic tumor
ba(c) - Bacterial me (1)- Melanoma
Koons ma(r)- mammary tumor
fu(ng) fungal go (t)- Testicular tumor
CAUSA go(v)- ovarian tumor
COMENTES pr(o)- Prostate tumor

my(D-musculoskeletal JS tu(m) miscellaneous tumor

22. Denosumab

23. Denogumab

1-Bone> RANKL inhibitor; osteoporosis
2-Human

24. Bevacizumab

25. Bevacizumab

1-Cardiovascular> VEGF inhibitor
2-Humanized

26. Adalimumab

27. Adalimumab

1-Immunomodulator> TNF-a inhibitor
2-Human

28. Few Commercially available mAb approved by FDA
| Name | Trade name ge A Use

Abciximab ReoPro CDga (integrin alpha-llb) Platelet aggregation Inhibitor

Adalimubab Humira TNF-alpha Rheumatoid arthritis, Crohn's
Disease, Plaque psoriaris,
psoriatic Arthritis

Alirocumab Praluent PCSKg Hypercholesterolemia

Avilumab Bavencio PD-La Cancer

Benralizumab Facenra CD125 Asthma

Daclizumab Zenapax CDa5 Organ transplant rejection

Daratumubab Darzalex CD-38 Multiple Myeloma

__ https://en.wikipedia.org/wiki/List of therapeutic monoclonal antibodies
=

29. Applications of mABs

r Diagnostic applications
1 MAb is used to detect pregnancy as early as a
week or two after conception by reacting with
human chorionic gonadotrophin
2 HIV diagnostic kits
| Rapid dia sis of hepatitis, influenza, herpes,
Fan & een yala Econ E
| identification & characterization of tumor specific
antigen: classification of cancer
| Imaging: Localization of cancer
o Arcitumomab : colorectal carcinoma
o Nofetumomab : small cell lung cancer

30. i Antiplatelet therapy
r infectious diseases
Cc Asthma

| Osteoporosis

31. References:

Shivanand P. (2010). “Hybridoma technology for production of monoclonal antibodies"
International Joumat of Pharmaceutical Sciences Review and Research vol 2, issue 2 (017)

U. Marx et al. (1997)"Monoclonal Antibody Production" The Report and Recommendations of
ECVAM Workshop ATLA 25, 121.137,

Edward A. Greenfield, (2014) "Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory
Press, 2™ ed. Chapter?

dustin K.H. Liu, (2014) "The history of monoclonal antibody development Progress, remaining
challengesand future innovations” Annals of Medicine and Surgery (2014) 113-116

Andrew 5., Otavia C., (2014) “Monoclonal antibodies for the therapy of cancer” Simpson and
Caballero BMC Proceedings 2014, 8 (Suppl 4)

WHO Guidelines on evaluation of monoclonal antibodies as similar biotherapeutic products,
2016

Bishwjit Ghosal, Research project on “Study of Monoclonal Market and it’s potential in India”,
NIPER