monoclonal antibodies in therapeutics in pharmacology ppt , especially for MD Pharmacology students.

1. Monoclonal Antibodies in
Therapeautics
DR. SARJU R. ZILATE

2. Monoclonal Antibodies
Monoclonal antibodies (MAb) are antibodies that are
identical because they are produced by one type of immune
cell; all are clones of a single parent cell.
Monoclonal antibodies (MAbs) are an integral part of
targeted therapy approach for various diseases which
result in decrease in adverse effects and increase in
efficacy.
They target various receptors or various growth factors on
the cell surface and modulate their vital functions and
cause cell death by various mechanisms.

4. The Structure of an Antibody

5. Antibody Function
Antibodies have two major functions:
•Recognize and bind antigen
•Induce immune responses after binding
The variable region mediates binding
•Affinity for a given antigen
•confers absolute specificity for an antigen
The constant region mediates immune response after
binding
•Different classes of constant regions generate
different isotypes
•Different isotypes of antibody have differing
properties

6. Production of monoclonal antibodies
• Since 1975, when Keller and Milstein developed
hybridoma technology,
• technological strides towards the production of antibodies
have been made.
• The first success in generating human mAbs (hmAbs)
with predefined specificity was conducted in 1980
through
• the fusion of human spleen cells from patients with
human myelomas.

7. • Since then, several major methods have been
established to generate hmAbs, including
• 1) immortalization of antigen-specific human B cells;
• 2) acquisition of antigen-specific human B cells via
phage display technology;
• 3) the production of hmAbs from transgenic mice;
• 4) single human B cell cloning techniques to directly
clone and express immunoglobulin (Ig) genes in vitro
from antigen-specific B cells.

9. Conventional hybridoma technology
• Step 1: Immunization of mice
• Mice are immunized with an antigen (attached to
adjuvant).
• The antigen can be whole cells, membrane fragment, or
complex molecules.
• Mice serum’s are screened using various techniques such
as ELISA.
• When sufficient titre is reached the mice are euthanized
and spleen is removed as a source of cells for cell fusion.

10. • Step 2: Preparation of Myeloma Cells
• Myeloma cells are immortalized cells that are capable of
dividing indefinitely.
• These cells are treated with 8-azaguanine to ensure
sensitivity to HAT

11. • Step 3: Fusion of myeloma cells with Spleen cells:
• spleen cells harvested from mice are fused with myeloma
cells. ,through co-centrifuging in polyethylene glycol.
• Cells are plated in selection medium
• Hypoxanthine-aminipterin-thymidine (HAT) selection
medium –blocks nucleotide synthesis.
• Only fused cells with grow on HAT. Cells are distributed on
feeder cells (murine bone-marrow) to promote growth of
the hybridomal cells.

12. • Step 4: Cloning of Hybridoma cells .
• A mouse is inoculated with the cell and thereby becomes a
factory for producing the MAbs.
• Ascites fluid collected from the mouse
• Step 5: Antibody is screened and purified:
• Antibodies are screened using specific Ag binding.
Advantage of in vivo
• Step 6: Desired antibodies are cloned:
• This is done in vitro on culture bottle

13. Immortalization of antigen-specific human
B cell and hybridoma technology
• Hybridoma technology has contributed to virtually all
areas of biology and medicine
• and has been greatly refined since its introduction in 1975.
• human antibody-secreting cells can be immortalized by
fusion of antibody-producing human B lymphocytes with
either mouse or human myeloma or lymphoblastoid cells
by infection with the EBV.

14. Phage display(1985)
• successfully used
• to select antigen-specific variable region genes and
• to express functional antibody fragments with unique
specificity.
• the library of diverse human immunoglobulin-heavy
chain variable (VH) gene and light chain variable (VL)
gene segments are prepared by reverse transcription of
mRNA from B cells and PCR amplification.

15. • The gene encoding single chain variable fragment (scFv)
can be created by randomly combining VH and VL gene
segments using PCR.
• After bio-panning the phages bound to a range of antigens
or haptens, antigen-specific antibody fragments with good
affinities can be identified.
• One phage display-derived mAb has been approved by the
FDA and at least 35 human mAbs generated by phage
display technology have entered into clinical development.
Phage display(1985)

16. Transgenic mice
• Genetically engineered mice expressing human
antibody repertoires were first reported for
generating hmAbs using the hybridoma technology in
1994.
• disruption of endogenous mouse
Ig-heavy chain and
Igκ-light chain loci together
• with the introduction of
human Ig-heavy chain and
Igκ-light chain genes.

17. Generation of mAbs from single human B cells
by Ig gene cloning and expression in vitro
This approach requires the
•isolation of individual B cells from mouse
spleen or human PBMCs using magnetic
microbeads conjugated with B-cell-selective
markers

18. •The light-chain and heavy-chain genes
•coding for variable fragments of antibody
•in each cell
•are separately amplified by RT-PCR
•and then combined with the sequences by an
overlapping PCR technique.
•For each cell,
•Ig heavy- and corresponding Ig light-chain gene
transcripts are amplified by nested RT-PCR and
•cloned into mammalian expression vectors to
produce hmAb with defined specificity in vitro.

19. Types of mAbs
19
Purple denotes human component orange murine component

20. EVOLUTION OF MONOCLONAL ANTIBODY
1. TRANSGENIC
2. LIBRARIES
a.BACTERIOPHAGE
b. mRNA
c. Cell Surface
Ist
generation
mab
2nd generation mab
daclizumab

21. Types of monoclonal antibodies
1. Murine source MAbs: rodent MAbs with excellent
affinities and specificities generated using
conventional hybridoma technology.
2. Chimeric MAbs: chimers combine the human
constant regions with the intact rodent variable
regions. Affinity and specificity unchanged.
3. Humanized MAbs: contained only the CDRs of the
rodent variable region grafted onto human variable
region framework
4. Recombinant DNA engineered MAbs

22. Murine
Derived from mice
Patients treated with murine mAbs develop a human
antimouse antibody (HAMA) response
Rapid clearance of the mAb
Poor tumour penetration
Hypersensitivity reactions
90Y-ibritumomab
131I -Tositumomab
22

23. Chimeric Abs
Antigen binding parts (variable region) of mouse
Ab with effector parts (constant region) of
human
–Infliximab
–Abciximab
–Rituximab
23

24. Humanized
Human Ab with complimentary determining
region
(CDR) or hypervariable region from non human
source
–Daclizumab
–Trastuzumab
24

25. Human Abs
Recombinant DNA technology:
Genes for variable Fab portion of human Abs is
inserted in genome of bacteriophages & replicated
Mixed with Ag & complementary Ab producing
phages selected
e.g. Adalimumab
25

26. Nomenclature of Monoclonal Antibodies
Prefix Target Source Suffix
variable
-o(s)- bone -u- human
-mab
-vi(r)- viral -o- mouse
-ba(c)- bacterial -a- rat
-li(m)- immune -e- hamster
-le(s)- infectious lesions -i- primate
-ci(r)- cardiovascular -xi- chimeric
-mu(l)- musculoskeletal -zu- humanized
-ki(n)- interleukin -axo- rat/murine hybrid
-co(l)- colonic tumor
-me(l)- melanoma
-ma(r)- mammary tumor
-go(t)- testicular tumor
-go(v)- ovarian tumor
-pr(o)- prostate tumor
-tu(m)- miscellaneous tumor
-neu(r)- nervous system
-tox(a)- toxin as target

27. Nomenclature
Suffix
–Human: -umab
–Humanized: -zumab
–Murine: -momab
–Chimeric: -ximab
27

28. Examples
•ab- + -ci- + -xi- + -mab: chimeric
monoclonal antibody used on the
cardiovascular system.
•tras- + -tu- + -zu- + -mab:
humanized monoclonal antibody
used against a tumor.
• Pali- + -vi- + -zu- + -mab
humanized mab used against a virus
(RSV)

29. Pharmacokinetics: mAbs
• Routes of administration:
Subcutaneously (Rituximab, Trastuzumab, Adalimumab)
Intramuscularly (Palivizumab)
Intravenously
• IV route: preferred because of 100% bioavailability
• Route for elimination of antibodies
Via uptake & catabolism by reticuloendothelial system &
target tissue.
29

30. P/K: mAbs
•Half-life
Chimeric : 4 –15 days
Humanized: 3 - 24 days
Recombinant human: 11– 24 days
•Human antimouse antibody (HAMA) response
develops 7–10 days following exposure to murine
antibody
30

31. MOA
• 1.Make the cell more visible to the immune system:
• The immune system attacks foreign invaders in your
body, but it doesn't always recognize cancer cells as
enemies. A monoclonal antibody can be directed to attach
to certain parts of a cancer cell. In this way, the antibody
marks the cancer cell and makes it easier for the immune
system to find.
• 2. Block growth signals- cetuximab
• 3.Deliver radiation to cells: By combining a radioactive
particle with a monoclonal antibody, doctors can deliver
radiation directly to the cancer cells.

32. Therapeutic uses
• Immunosuppression
•Autoimmune diseases
•Malignancies
•Antiplatelet therapy
•Infectious diseases
•Asthma
• Osteoporosis
32

33. Monoclonal antibodies in therapeutic use:
• ANTI CD3:
• CD 3 is a co receptor which plays an important role in
T cell receptor signaling.
• MUROMUMAB (OKT3) is a drug which blocks this
receptor. It blocks killing by cytotoxic T cells and many
other T cell functions. It kills cells by blocking vital
functions of CD3, approved for treatment of renal
allograft rejection crisis.
• TEPLIZUMAB is a newer anti CD3 drug. It is
used for
• protecting remaining beta cells in newly diagnosed
type I DM. It is currently in phase III clinical trials.

34. • ANTI CD20:
• RITUXIMAB:
• Its mechanisms of action in killing cancer cells are-
ADCC ,CDC and direct induction of apoptosis with
proven efficacy against wide range of NHL B-cell
malignancies.
• Dose: 375mg/m2 IV infusion weekly for 4 weeks.
• Side effects are fever with rash, dyspnoea and late onset
neutropenia.

35. • OCRELIZUMAB is a newer CD20 drug .
• It targets mature B lymphocytes and thus is an
immunosuppressive drug.
• It is currently in Phase III.
• It is used in RA, SLE, MS and lymphomas.
• OFATUMUMAB
• is also a newer CD20 drug.
• It inhibits early B lymphocyte activation.
• It targets different epitope of that by rituximab.
• It was approved in February 2010 for refractory CLL.

36. Radiolabelled Anti CD20
• Radiolabelling the monoclonal antibodies increases
their efficacy.
• They can also be used for various imaging purposes.
• Yttrium 90-ibritumomab (Y90),
• Indium 111- ibritumomab (In111),
• Iodine 131- tositumomab (I131 sub) are the 3
commonly radiolabelled anti CD20 MAbs.

37. • They show increase in efficacy than their naked
counterparts.
• Effective in relapsed /refractory/advanced cases
of follicular B-cell lymphoma and NHL in the
doses of 0.4 ci/kg IV.
• Its side effects are myelodysplasia and
hematological toxicities

38. ANTI CD 22:
• CD22 is a co receptor important for B cell receptor
signaling.
• EPRATUZUMAB is a drug which blocks CD22
signaling.
• It is currently in phase III clinical trials.
• It is active against malignant B cells and used in SLE.
• It produces cell death by ADCC

39. ANTI CD52:
• CD52 is present on thymocytes, macrophages,
lymphocytes and monocytes.
• ALEMTUZUMAB, is a drug which blocks the CD52
• signaling.
• It kills tumor cells by ADCC, CDC and apoptosis.
• It is Administered IV 30mg/day thrice weekly.
• Premedication with diphenhydramine and
acetaminophen should precede this drug since
hypersensitivity reactions are common.
• It used in B cell and T cell lymphomas and MS.
• Its side effects are T cell depletion and
immunosuppression.

40. ANTI CD33:
• CD33 is a co receptor found on myeloid cell surface.
• GEMTUZUMAB OZOGAMICIN (MAb linked to a
toxin). Humanized MAb covalently linked to a
semisynthetic derivative of calicheamicin .
• It causes DS DNA breaks and cell death.
• Its dose is 2 doses of 9mg/m2 IV separated by 14 days.
• It is used in AML.
• Its side effects are hematopoietic suppression and vaso
occlusive disorders.

41. • LINTUZUMAB
• is a newer anti CD33 MAb.
• It is currently in phase III for AML.

42. ANTI CD 11a:
• CD 11a is a co receptor found on B cells and important
in cell to cell adhesion and co-stimulation.
• EFALIZUMAB is a drug that blocks CD11a signaling.
• It is approved for the treatment of adult patients with
severe psoriasis.
• It administered by SC injections.

43. ANTI HUMAN EPIDERMAL GROWTH
FACTOR RECEPTOR 2/ NEU:
• Human epidermal growth factor receptor is a factor which
plays an important role in growth of many breast cancers.
• TRASTUZUMAB
• is a drug which blocks this receptor.
• it causes cell death by inhibition of HER2 signaling with
G1 arrest and also by ADCC and apoptosis .
• Its dose is loading dose of 4mg/kg IV followed by 2 mg/kg
weekly.

44. • It is used in HER2-positive metastatic breast
cancers.
• Its side effects are cardiomyapathy and flu like
syndrome.
• PERTUZUMAB is a newer HER2 blocking MAb.
• It is currently in clinical trials

45. ANTI EPIDERMAL GROUTH FACTOR
RECEPTOR (EGFR)
• Epidermal growth factor receptor plays a vital role in
growth of many cancers.
• CETUXIMAB is a drug which blocks EGFR signaling and
causes cell death by ADCC.
• Its dose is loading dose of 400mg/kg infusion followed by
250mg/kg weekly.
• It is used in metastatic colorectal and head and neck
cancers.
• ITS SIDE effects are infusion related toxicity and skin rash.

46. • PANITUMUMAB –
• It is an another EGFR receptor blocking MAb..
• Produces cell death by similar mechanism as
cetuximab.
• It was approved in September 2006.
• It is used in metastatic colorectal and head and neck
cancers.
• Its adverse effects are skin rash and fatigue.
• First MAb approved by FDA developed from transgenic
mice.

47. • MATUZUMAB and NIMOTUZUMAB
• are the other EGFR blocking MAbs
• which are in phases III of clinical trials.

48. ANTI VASCULAR ENDOTHELIAL GROUTH
FACTOR (VEGF)
• Vascular endothelial growth factor plays an important role
in angiogenesis and neo vascularization of various tumors.
• BEVACIZUMAB is a drug which blocks VEGF.
• It inhibits angiogenesis and neovascularization
• in the dose of 5mg/kg IV every 14 days until disease
progression stops.
• It is used in colorectal and head and neck cancers.
• Its side effects are hypertension, pulmonary hemorrhages,
GI perorations and CCF.

49. • RANIBIZUMAB is a newer VEGF blocking drug.
• It is used in neovascular macular degeneration.
• It is injected directly into vitreous cavity.

50. Side effects of monoclonal antibody
• In general, the more common side effects caused by
monoclonal antibody drugs include:
• Allergic reactions, such as hives or itching
• Flu-like symptoms, including chills, fatigue, fever, and
muscle aches and pains
• Nausea
• Diarrhea
• Skin rashes

51. Rare, but more serious side effects of
monoclonal antibody therapy may include:
• Infusion reactions. Severe allergy-like reactions can occur
and in very few cases lead to death.
• Dangerously low blood cell counts. Low levels of red blood
cells, white blood cells and platelets may lead to serious
complications.
• Skin problems
• Bleeding. Some of the monoclonal antibody drugs are
designed to stop cancer from forming new blood vessels.
There have been reports that these medications can cause
bleeding.

52. Agent Target(s) FDA-approved
indication(s)
Ado-trastuzumab emtansine
(Kadcyla)
HER2 (ERBB2/neu) Breast cancer
(HER2+)
HER2 (ERBB2/neu) exon 21
substitution
(L858R) mutations)
Aldesleukin (Proleukin) Renal cell carcinoma
Melanoma
Alemtuzumab (Campath) CD52 B-cell chronic
lymphocytic
leukemia
Brentuximab vedotin (Adcetris) CD30 Hodgkin lymphoma
Anaplastic large cell
lymphoma
Belimumab (Benlysta) BAFF Lupus
erythematosus
Belinostat (Beleodaq) HDAC Peripheral T-cell
lymphoma

53. Bevacizumab (Avastin) VEGF ligand Cervical cancer
Colorectal cancer
Fallopian tube
cancer
Glioblastoma
Non-small cell
lung cancer
Ovarian cancer
Peritoneal cancer
Renal cell
carcinoma
Canakinumab (Ilaris) IL-1β Juvenile idiopathic
arthritis Cryopyrin-
associated periodic
syndromes
Cetuximab (Erbitux) EGFR (HER1/ERBB1) Colorectal cancer
(KRAS wild type)
Squamous cell
cancer of the head
and neck

54. Denosumab (Xgeva) RANKL Giant cell tumor of
the bone
Ibritumomab tiuxetan (Zevalin) CD20 Non-Hodgkin's
lymphoma
Ipilimumab (Yervoy) CTLA-4 Melanoma
Nivolumab (Opdivo) PD-1 Melanoma
Obinutuzumab (Gazyva) CD20 Chronic lymphocytic
leukemia
Ofatumumab (Arzerra, HuMax-
CD20)
CD20 Chronic lymphocytic
leukemia
Panitumumab (Vectibix) EGFR (HER1/ERBB1) Colorectal cancer
(KRAS wild type)
Pembrolizumab (Keytruda) PD-1 Melanoma

55. Pertuzumab (Perjeta) HER2 (ERBB2/neu) Breast cancer (HER2+)
Ramucirumab (Cyramza) VEGFR2 Gastric cancer or
Gastroesophageal
junction (GEJ)
adenocarcinoma Non-
small cell lung cancer
Rituximab (Rituxan, Mabthera) CD20 Non-Hodgkin’s lymphoma
Chronic lymphocytic
leukemia Rheumatoid
arthritis Granulomatosis
with polyangiitis
Siltuximab (Sylvant) IL-6 Multicentric Castleman's
disease
Tocilizumab (Actemra) IL-6R Rheumatoid arthritis
Juvenile idiopathic
arthritis

56. Tositumomab (Bexxar) CD20 Non-Hodgkin's
lymphoma
Trastuzumab (Herceptin) HER2 (ERBB2/neu) Breast cancer
(HER2+) Gastric
cancer (HER2+)

57. TO SUMMARISE :
MAbs are highly specific Abs produced by a clone of
single hybrid cells formed by fusion of B cell with the
tumor cell.
The hybridoma formed yields higher amount of
MAbs.
 MAbs can be produced in vitro and in vivo .
 Animals are utilized to produce MAbs, but these
antibodies are associated with immunogenicity and
ethical problems.

58.  Recombinant DNA technology, genetic
engineering and transgenic animals are used to
produce humanized MAbs or pure human MAbs,
with fewer ADRs
Used for treatment of cancer, autoimmune
disorders, graft rejections, infections, asthma etc.

59. •THANKS