MONOCLONAL ANTIBODIES-AN OVERVIEW

1. Dr. K. Raja Rajeswari,
Associate Professor, Dept. of Pharmaceutics,
Pulla Reddy Institute of Pharmacy

2. Introduction
In 1890, Behring and Kitasato found that rabbits that are immune to tetanus had
‘substances’ ( identified as immuno-globulin or antibody) in their blood capable of
destroying tetanus toxin. This fraction of blood is called anti-toxin.
Paul Ehrlich laid down the first theoritical principles of antibody-antigen
interactions and termed the antibodies as magic bullets in 1928 since they accurately
target and neutralize their antigens.
Antibodies or immunoglobulins are serum glycoproteins synthesized and secreted by
plasma cells derived from β-lymphocytes stimulated in response to foreign substances
called antigens.

3. CLONAL SELECTION THEORY:
 Developed by Macfarlane Burnet, Niels Jerne, David Talmage and Joshna
Lederberg in 1957
It explains the production of a specific antibody against an antigen. i.e., each B-
cell makes antibody of a single kind
Antibody is a protein used by the immune system to identify and neutralize
foreign objects like bacteria and viruses. ...
Monoclonal antibodies (mAb) are antibodies that are identical because they
were produced by one type of immune cell, all clones of a single parent cell.
mAb were produced by Kohler and Milstein for which they were awarded Nobel
prize in 1984.
Polyclonal antibodies: Antibodies that are derived from different cell lines which
differ in amino acid sequence.

4. Property Polyclonal Antibody Monoclonal Antibody
Useful antibody
content
Low High
Composition Heterogenous Homogenous
Specificity Multiple; variable form
animal to animal
Defined and consistent
Cross –reaction with
other antibodies
Partial with antigens
bearing common
antigenic determinants
Usually absent but
complete, if antibody
binds to common
determinant
Class and Subclass of
antibody
Typical mixture of
classes and subclasses
Only one, may be any
Supply limited abundant

5. Characters of mAb:
 Single type of antibody that are identical and directed against a specific
epitope (antigen, antigenic determinant).
Produced by B cell clones of a single parent or a single hybridoma cell line
(a
hybridoma cell line is formed by the fusion of one B cell lymphocyte with a
mylenoma cell. Few mylenoma cells synthesize single mAb naturally.
Production of Monoclonal Antibodies:
The first mAb produced using hybridom a technology using mouse
spleenocytes (B cells) was Orthoclone OKT3 in 1986 ( ORTHOBIOTECH) .

6. Methods:
 Hybridoma technology:-steps involved are---
 Immunization
 Spleenectomy
 Fusion
 Selection of Hybrid
Cloning by dilution
Screening
Production
Chimeric Monoclonal Antibodies
Humanized Monoclonal Antibodies
Human Monoclonal Antibodies –produced by two approaches:--
 Phage display
 Human Antibody mouse

7. Hybridoma technology:
Involves fusion of different types of cells to give a hybrid cell.
It is a part of modern biotechnology.
Steps:
1.Immunization: A mouse is immunized by injecting intradermally or
by SC/IV route. Immune response is stimulated with an adjuvant
like aluminium salt.
2. Spleenectomy: Removal of spleen which is a concentrated source of B cells.
The spleen is dissociated mechanicall or enzymatically into single
spleenocytes.
3. Fusion: Spleenocytes are mixed with an immortal cell line ( myeloma cells ) in a
ratio of 5:1 to 2:1 and allowed to fuse in the presence of an equal amount
of (50%W/W) of fusionogenic agent like PEG.
4. Selection of hybrid: Fusion products (hybrids) are formed during fusion.
Formed products may be
spleenocyte-spleenocyte hybrid,
myeloma cell-myeloma cell hybrid
spleenocyte- cell-myeloma cell hybrid
or no hybrids are formed.

8. Selective medium like HAT (Hypoxanthine-Aminopterine-Thymidine ), by
Littlefield is used to select the desired hybrid cells (spleenocyte- cell-myeloma cell
hybrid).
Hypoxanthine is used for the synthesis of purine nucleotides.
Thymidine is used for the synthesis of pyrimidine nucelotides.
The selective nature of medium depends upon biosynthesis of nucleotides by
mammalian cells.

9. Pathways of synthesis of nucleotides:
1.De novo pathway:
The nitrogenous bases purines and pyrimidines are built up from simple
precursors like amino acids, formyl tetrahydrofolate and carbon dioxide.
In spite of many enzymes that involve in the assembly of nitrogenous bases
and their synthesis to form nucleosides and nucleotides, an enzyme
dihydro folate reductase is required for the generation of tetra hydro folate.,
one of the precursors.
The enzyme dihydro folate reductase is competitively inhibited by
aminopterine and methotrexate (formerly called amethopterine ) of HAT
medium (either of which present in the medium.
Hence this pathway is not used in the synthesis of nucleotides.

10. 2. Salvage pathway: (Salvage means to save)
Pre –formed bases of hydrolytic degradation of nucleic acids and nucleotides are
used as substrates to synthesize nucleotides using hypoxanthine-guanine-
phosphoribosyl transferase (HGPRT) and thymidine kinase (TK) .
Hence it is considered as simple and economic pathway .
HGPRT
Hypoxanthine---------------------->Ionosinate ((precursor for purines(adenylate and
guanylate))
TK (phosphorylation)
Thymidine ( a nucleoside) ---------------------->Thymidilate ( a nucleotide)
Mammalian cells have functional genes to code for HGPRT and TK for the
survival of cells in HAT medium using this pathway.

11. Key points:
 Normal animal adult cells cannot survive in vitro for longer periods
due
to aging or Senescence (programmed natural cell death). Thus B cells
are
cultured in vitro in HAT medium though they possess functional genes
for HGPRT and TK that die out. Myeloma cells (cancer cells) are
cultured indefinitely.
 In Hybridoma technology myeloma cells that are non-functional or
are
deficient in HGPRT and /or TK gens are used. Thus they die out in the
medium.

12. A fusion product (hybrid) –deficient of HGPRT/TK genes and B cells-with
functional HGPRT +
and TK +
genes are able to survive and grow
indefinitely. Thus producing an antibody, a property of B cells.
Medium allows selective growth of hybridoma cells while original cells
cannot survive.

13. Cloning by dilution:
Cells proliferating in HAT medium consists of hybridoma cells and all
secrete antibodies.
Cells are screened to determine the specific antibody by diluting the
cultured cells into 96 –well plastic plates.
Dilution is done such that only one positive cell is found per well and it is
allowed to grow repeatedly to form a clone.

14. Screening:
Methods like ELISA(Enzyme Linked-Immuno Sorbent Assay), RIA(Radio
Immuno Assay), FIA (Fluro Immuno Assay) are used to identify the desired or
specific antibody producing cell.
The Cells identified are grown for the production of antibodies or frozen and
stored .
Production:
Single positive cell s are grown using tissue culture ( produces 10-100 µg/ml)
or
by injecting into mice.(form an ascetic tumor and secrete antibody ( about 1-
25 mg/ml) in ascetic fluid surrounding lungs and also in plasma. But
Also by Immobilized cell reactors (hollow fiber reactor) and traditional
suspended cell fermentors cultures are done .

15. Other methods:
Genetic engineering of CHO cells
 Pharming (production in animal milk)
Production in eggs, plants, bacteria, virus etc
Novel approaches ( to overcome the severe immune reactions in humans
caused by murine mAB) chimeric mAb, Humanized mAb are used.

16. Structure of Antibodies:
Chemically antibodies are glycoproteins with attached carbohydrates
and termed as immunoglobulins(Ig).
Antibody molecules have two basic functions:
-- -Binding with antigen (antigen binding sites)---Fab
--- participation in the effector functions ( fragment that mediates effector
functions. (crystallizable fragment-Fc
•The two fragments are separated by cleavage of the antibody molecule by
treatment a prteolytic enzyme-papain

17.  Two large and small pairs of polypeptide chains.
Large pair is composed of two heavy chains (H), each with a molecular
weight of 50 kdaltons.
Small pair is composed of two light chains (L) each with a Mol.Wt of 25
kdaltons.
 L & H bonded by a disulphide linkage.
H chains are linked with atleast one disulphide bond.
Fab consists of entire L chains and the amino terminal halves of H chains.
Fc consists of carboxyl terminal halves of both H chains.

18. L chains are made up of 214 amino acid residues and H chains with 446.
 L chain consists of variable region (from 1 to 108 amino acid residues) and a
constant region (from 109-214 amino acid residues)
 H chain consists of variable region (from 1 to 108 amino acid residues) and
a constant region (from 109-446 amino acid residues)
 Three hyper variable segments (CDR- complementarity determining
regions) in L&H.
 These 6 CDRs determine the antibody specificity and affinity that contacts
with the epitope (specific region of antigen contacted by the antibody)
 Other parts of the variable regions that remain relatively unchanged serve
as
Frame work for proper orientation of CDRs to maintain a close contact with
epitope.

20. Chimeric mAb:
Part of the genes encoding for the antibody are derived from mouse and partly
from human,--- resulting in the formation of antibodies to possess properties of
both.
They are created by chimeric genes by selecting genes encoding for variable
regions from selected hybridoma murine and genes encoding for constant
regions from human B cells.
Created Chimeric gene—introduced into Chinese hamster ovary (CHO) cells
for the gene to be expressed.
.

21. Properties of Chimeric mAb:
1. Antigen specificity (due to Fab) is identical to murine antibodies.
2. Effector function( due to Fc) and in vivo half life are identical to human antibodies
3. Less immunogenic than murine mAb.
4. Potential to stimulate the production of human anti-chimeric antibodies(HACA)

22. Humanized mAb:
This is constructed by utilizing only the sequences ( of Fab that make contact with
the epitope on an antigen on CDR and the adjoining providing the frame work)
from the murine antibody gene that encodes for the hyper variable region and
utilizes the rest of the gene sequences for frame work and Fc from human
immunoglobulin IgGI sequence.
The resulting antibody is 90% human and data concerning its immunogenecity
are lacking.
It can be further developed to produce fully non-immunogenic human antibodies.

23. Human mAb:
Produced by two approaches.
1. Phage display
2. Human antibody mouse
Phage display
Used to construct 1-2 million genes encoding for variable regions of L & H
chains with a predominant linker between them to express them as a part of
single polypeptide chain. They are called mini antibodies or single chain
variable fragments (ScFv). These genes are spliced upstream of gP3 gene( one
of the constituent proteins of the outer coat of the phage) in DNA of phage
such as M13.

24. Such phages containing the recombinant gene ( Scfv-gP3) are allowed to replicate
in bacterial cell culture where they ( M13 bacteriophages) express the antibody
fragments as part of the outer protein coat resulting in the generation of
Phage display antibody library. The gene with strong affinity is selected from this
library. Such phages also generate human antibodies.
Human antibody mouse:
The genes of human antibodies encoding for H & L chains are inactivated
( Knocked out) by homologous DNA recombination are inserted in mice ( due to
lack of B Cells in mice).
These human monoclonal antibody mice are immunized with an antigen to
stimulate the production of antibodies.

25. Spleenocytes of immunized mice are collected and allowed to fuse with myeloma
cells to form hybridomas.
The hybridomas are screened and the one producing best desired mAb is selected
for the production of fully human mAb.
Applications:
Therapeutic
Diagnostic
Investigational and analytical
Drug targeting
Abzymes (catalytic antibodies)

26. Generic name Brand
name
Manufacturer Use
Abciximab Reopro Centocor anticoagulant
Basiliximab Simulect Novartis Prophylaxis of acute organ (renal)
transplantation rejection
Daclizumab Zenepax Hoffman La-Roche Prevention of acute kidney
transplantation rejection
Endreolomab Panorex Centocor Treatment of colorectal cancer
Gemtuzumab Mylotarg Glazo Celltech Treatment of acute myeloid leukemia
Infliximab Remicade Centocor Treatment of Crohn’s disease.
Rheumatoid arthritis
Muromomab Orthoclone
(OKT3)
Ortho Biotech Reversal of acute kidney
transplantation rejection
Palivizumab Synagis MedimmuneAbbott Prophylaxis of lower tract disease
caused by synctial virus
Rituximab Rituxan
Mabthera
Genentech
Hoffman La-Roche
Treatment of Non-Hodgkins
lymphoma
Transtuzumab Herceptin Genentech Treatment of metastatic breast cancer
Therapeutic

27. Generic name Brand
name
Manufacturer Use in the detection of
Coprumab ProstaScint - Prostate adenocarcinoma
Imiciromab
Pentetate
MyoScint - Myocardial infarction
Nofetumomab Verluma - Small-cell lung cancer
Satumomab
Pendetide
OncoScint - Colorectal and ovarian cancer
Technitium-99-
arcitumomab
CEA-Scan - Metastatic colorectal cancer
Diagnostic

28. Available (Mouse/Human) monoclonal antibodies of clinical interest
Normal Blood group antigens, lymphocyte subpopulation, major,
histocompaibility complex, sperm oocyte, neurons & trophoblast
antigens.
Pathogens
a. bacteria
Strptococcus, E. Coli, Pneumococcus, S. typhi. S. paratyphi –A & B,
Salmonella, N. meningitis, N. gonorrhoea, M. leprae, M. tuberculosis etc
b. Virus Herpes simplex virus, HBV, Influenza virus-A, Foot and mouth diseases
virus, Polio virus, rota virus, measles, rinderpest, rabies, murine
leukemia, bovine leukemia, HIV, EBV
c. Parasites P.falciparum
d. Mycoplasma Toxoplasma gondii
Hormones Human chronic gonadotrpin, LH, Gonadotropin releasing hormone,
progesterone, oestradiol α 17β, vasopressin, growth hormone, gastrin,
insulin, adrenocorticotrophin etc
Tumour associated
antigens
Leukemia, ovarian carcinoma, colon carcinoma, germ cell tumor, terato
carcinoma, glioblastoma, hodgkins disease, lung and mammary cancer,
melanoma, osteocarcinoma, prostate cancer, neuroblastoma, etc
others Trasferin receptors, tetanus toxoid, interferons, interleukins,
immunoglobulins, oestrogen and progesterone receptors etc