2. content
• What are monoclonal antibodies
• Types of mAb
• Preparation of mAb
• Hybridoma
• Phage display
June 2021
m.Pharm 1st year 2
3. What are monoclonal
antibodies
Mono clonal
Identical immunoglobulin .
Generated from single B-cell clone
Target single epitome /binding site on single antigen
Poly clonal
Collection of many immunoglobulin
generated from multiple B-cell clone
target multiple binding site /epitome on
single antigen
june2021
m.Pharm 1st year
3
7. Types of mAb
• Murine (-o mab): entirely derived from a murine
source. They can lead to an allergic reaction in humans.
• Chimeric (-xi mab): the variable regions are of murine
origins whereas the constant regions are human. These
can also cause an allergy.
• Humanized (-zu mab): mostly derived from a human
source except for the part of the antibody which binds to
its target.(CDR-murine)
• Human (-u mab) : entirely derived from a human
source
7/11/2021
Footer Text 7
16. Steps for production of
mAb
1. Immunize animal
2. Isolate spleen cells (containing antibody-producing B cell)
3. Fuse spleen cells with myeloma cell (using PEG)
4. Allow un-fused B cell to die
5. Add hypoxanthine-aminopterin-thymidine (HAT) to culture and kill un-fused
myeloma cells
• The cells that survive are the ones that express hypoxanthine guanine
phosphoribosyl transferase (HGPRT) and are called hybridoma cells.
• Hybridoma cells become visible after 4 days and
• are grown for about 3 weeks.
6. Clone remaining cells (place 1 cell/well and allow each cell to grow into a clones
of cell)
7. Screen supernatant of each clone for presence of desired antibody
8. Grow chosen clone of cells in tissue culture indefinitely
9. Harvest antibody from the culture.
june2021
m.Pharm 1st year 16
18. Phage
Display
• George P. Smith in 1985
• First phage display derived mAb
Adalimumab (Humira),
an anti-tumor necrosis factor α (TNFα) human antibody
• Display of protein of interest on surface of bacteriophage
• a gene encoding that protein is inserted into phage coat
protein gene .
ne2021ju
m.pharm1st year 18
19. Steps
1) Construct phage
display library
2) Binding
3) Washing
4) Elusion
5) Amplification
(infect E.coli)
6) Repeated cycle 2-3
times for selection of best
binding sequence.
June2021
m.pharm1st year 19
20. Steps
• STEP1: Construct phage display library Recombinant DNA technology is used to
incorporate foreign cDNA into viral DNA. Different sets of genes are inserted into
the genomes of multiple phages. Spliced into gene for a coat protein, so that the
protein will be displayed on the outside of phage particles, and these separate
phages will only display one protein, peptide, or antibody. Collections of these
phages can comprise libraries, such as antibody phage library, protein phage
library, or random phage library.
• STEP2: Binding; These libraries are exposed to selected targets and only some
phages will interact with targets. The target is for which specific ligands planned
to be identified such as immobilized protein, cell surface protein or vascular
endothelium.
• STEP3: Washing; Unbound phages can be washed away, and only those which
showing affinity for the receptors was left.
• STEP4: Elution; Recovery of the target bound phage by elution.
• STEP5: Amplification : Eluted phages showing specificity are used to infect new
host cells for amplification, or direct bacterial infection and amplification of the
recovered phage.
• Back to step 1, repeated cycle 2-3 times for stepwise selection of best binding
sequence.
7/11/2021
Footer Text 20
22. transgenic mouse
• Panitumumab ,anti-epidermal growth
factor receptor (EGFR),
• Human Immunoglobulin gene is inserted
into genome ,replacing the endogenous Ig
genes and making these animals capable
of synthesizing fully human antibodies
upon immunization .
7/11/2021
Footer Text 22
23. Examples of mAb
mAb Type technology target indication
Ibritumomab tiuxetan Murine hybridoma CD20 Non-Hodgkin
lymphoma
Cetuximab Chimeric
IgG1
Hybridoma EGFR Colorectal
cancer
Pertuzumab Humanized
IgG1
hybridoma HER2 Breast cancer
Ramucirumab Human IgG1 Phase display VEGFR2 Gastric cancer
Daratumumab Human IgG1 Transgenic
mice
CD38 Multiple
myeloma
7/11/2021
Footer Text 23
24. Drug targeting by mAb
Antibody drug conjugate
• Antibody drug conjugates consist of an
antibody that targets a cancer-specific marker
conjugated
to the small molecule drug.
• The antibody enhances delivery to the tumor
site, increasing the efficacy of the small
molecule and leave s normal cells unaffected.
• It enhances tumor specificity
June 2021
m.Pharm 1st year 24
B cell –B lymphocyte – mature in bone marrow – produce immunoglobulin
150 kDa =150000g/mol
complementarity-determining region (CDR0 binds to a specific epitope on antigen
Fc = fragment crystallizable region
Fab = fragment antigen-binding ( CL ,VL, CH ,VH region )
Murine - o-
Chimeric -xi-
Humanized -zu-
Human -u-
Myloma cells are cancerous immune cell
1) animals (usually mice or rats) are immunized by injection with a soluble antigen that
is mixed with an adjuvant.
2) The antigen-specific producing plasma cells from the spleen are then
Isolated
3) and are fused with a cancerous immune cell (called a myeloma cell) using fusing reagents
such as HVJ or PEG .
4) The fused cells are then cultured in a growth medium supplemented
with hypoxanthine-aminopterin-thymidine (HAT) medium.
5) The cells that survive are the ones that express hypoxanthine guanine phosphoribosyl transferase (HGPRT) and are called hybridoma
cells.
6) Hybridoma cells become visible after 4 days and are grown for about 3 weeks.
7) The culture supernatant is eventually screened for secretion of the desired antibody (assays include Western blot
and enzyme-linked immunosorbent assay (ELISA)) .
8) Given the rapid proliferation of myeloma cells
and the production of specific antibodies, these two properties lead to the feasibility to produce mAbs
in vitro.
9) Plasma and myeloma cells can also be fused using an electrical field (pearl-chain formation)
or laser radiation in order to enhance fusion efficiency
The fused cells are then cultured in a growth medium supplemented
with hypoxanthine-aminopterin-thymidine (HAT) medium.
5) The cells that survive are the ones that express hypoxanthine guanine phosphoribosyl transferase (HGPRT) and are called hybridoma
cells.
6) Hybridoma cells become visible after 4 days and are grown for about 3 weeks.
There is connection between Genotype and phenotype .
scFv = single chain variable fragment (fusion protein of VH and VL of Ig )
VL = variable region of light chain
Linker peptide in red
Vh = variable region of heavy chain
Phagemid vector = DNA based cloning vector
has bactoriophage and plasmid properties
has genetic sequence that signals for packaging into capsid of a bacteriophage
cDNA = complimentary DNA
Panning = step to screen the antibody candidates based on binding property to target molecules.
The
affinity screening process for antibody libraries is called
Biopanning
Target antigen is immobilized on solid surface such as Polystyrene surfaces
with high protein binding capacity, magnetic beads
with protein G/A, streptavidin, maleimide or N -hydroxysuccinimide
