Transcript Antibodies

 Engineered Monoclonal Antibodies:
 Monoclonal antibodies have demonstrated enormous potential as new classes
of drugs that confer great benefits to patients, and more than 40 therapeutic
antibodies have already been approved for clinical use
 In particular, the past 5 years might be recognized as the period guiding the
new era for “engineered antibodies,” with the successful approval of
numerous antibody–drug conjugates, bispecific antibodies, and glycoengineered antibodies for clinical applications
 These therapeutic antibodies seem to be more promising with improved
properties when compared to their ancestors
 There are several molecules in phase II/III clinical trails and few waiting for
approvals
Table – Phases of trail
 One drug, mogamulizumab (KW-0761 / POTELIGEO/ AMG761) has already
received approval for marketing by Japanese Ministry of Health
• It is defucosylated humanized mab derived from Kyowas Kirin’s
“POTELLIGENT” technology which produces antibodies with enhanced
antibody-dependent cell-mediated cytotoxicity (ADDC) activity
• To be used for patients with relapsed or refractory CC kemokine receptor
4 (CCR4) positive adult T cell leukemia-lymphoma (ATL)
Clinical Trials:
Years
Discovery/Preclinical Testing
Phase 1
Phase 2
Phase 3
FDA
6.5
1.5
2
3.5
1.5
20 to 100
healthy
volunteers
1,000 to 5,000
100 to 500
patient
patient volunteers
volunteers
Test Population Laboratory and animal studies
File IND
at FDA
Purpose
Assess safety, biological
activity, and formulations
Determine
safety and
dosage
Success Rate
5,000 compounds evaluated
5 enter trials
Investigational New Drug (IND)
New Drug Application (NDA)
US Food and Drug Administration (FDA)
Evaluate
effectiveness,
look for side
effects
Confirm
effectiveness,
monitor adverse
reactions from
long-term use
File
NDA Review
process/
at
FDA approval
1 approved
Phase 4
Additional postmarketing
testing required
by FDA
 In addition, a large number of molecules are also in pre-clinical trials and are
expected to progress through various stages of drug development over the
next ten years
 By 2023, the over all market is estimated to be in the range of US $ 4.3 to 6.6
billion
 We will first discuss followings:
i. Immune System
ii. Antibody structure, function, production and uses (polyclonal and
monoclonal)
iii. Glycoslation
i. Immune System:
 The human body provides two levels of protection from infectious
diseases;
• Non-specific resistance or innate immunity and
• Specific resistance or adaptive immunity
 The principal components of innate immunity are;
o
Mechanical and chemical barriers
o
o
o
o
•
Phagocytosis
Fever
Inflammation and
Acute phase proteins
Mechanical barriers are the skin and the mucous membrane while
chemical barriers are
- Acidic gastric secretions
- Low pH of the skin
- Lysozyme
- Gastric and duodenal enzymes
-
Antibodies and inhibitors
= Antibodies (IgA) are also found in tears and saliva
-
= Secretary IgA protects the body surface against invading
pathogenic microbes
Interferons
-
Complement proteins
-
Antimicrobial peptides
 In contrast to innate immunity, adaptive immunity is a more
evolved and specific defense mechanism
 The characteristics of adaptive immunity are
o
Exquisite antigenic specificity and
o
The ability to “remember” different types of antigens
 Adaptive immunity is activated only against
o
Invading foreign material and
o
Never against its own molecules except in autoimmune
diseases
 Therefore, it has the ability to distinguish between self and nonself
 Activation of the Immune Response:
• There are two arms of the immune response:
 Humoral response mediated by B cells and antibodies and
 Cell-mediated response effected by T cells
Fig from Science
Fig 1.1 Roitt 7th Ed
Fig 1.7 Khan 2009
ii. Antibody’s Structure and Function:
 An antibody or immunoglobulin is a glycoprotein have a characteristic Yshaped structure produced by the body's immune system
• When it detects harmful substances called antigens
• Examples of antigens include microorganisms (such as bacteria, fungi,
parasites, and viruses) and chemicals
 Antibodies may be produced when the immune system mistakenly
considers healthy tissue a harmful substance. This is called
an autoimmune disorder
Figs down loaded from web site
https://www.google.com.pk/search?q=antibodies&source=lnms&tbm=isc
h&sa=X&ei=UhekUpHDIoKShgeBYG4Cg&ved=0CAcQ_AUoAQ&biw=1024&bih=629#imgdii=_
 Each type of antibody is unique and defends the body against one specific
type of antigen
Fig 9.2 Glick 2nd Ed / Glick 3rd Ed
(Two Fab and one Fc)
composes ~75 % of
the immunoglobulins
in human serum
 An Fc receptor is a protein found on the surface of certain cells including
• B lymphocytes
• Follicular dendritic cells
• Natural killer cells
• Macrophages
• Neutrophils and
• Mast cells
• that contribute to the protective functions of the immune system
• Its name is derived from its binding specificity for a part of an antibody
known as the Fc (Fragment, crystallizable region)
• Fc receptors bind to antibodies that are attached to infected cells or
invading pathogens
• Their activity stimulates phagocytic or cytotoxic cells
 to destroy microbes or
 infected cells by antibody-mediated phagocytosis or
 antibody-dependent cell mediated cytotoxicity
Figs down loaded from website
The main feature of ADCC enhanced antibody technology POTELLIGENT®, originally developed by
Kyowa Hakko Kirin, is its ability to remarkably increase the ADCC activity by reducing the amount of
the fucose in the carbohydrate structure of antibodies, thus causing the extremely effective elimination
of target cells, such as cancer cells
 Antibody Production and Functions:
• Antigens stimulate an immune response via the production of
antibodies
• When a pathogen invades the body, it is engulfed by wandering
macrophages which present the antigenic fragments on its surface
• This macrophage becomes an antigen-presenting cell, and presents
the antigen to helper T cells (TH cells)
• The TH cells bind to the antigen and become activated, and in turn
activate the B cell with the specific antibody for the antigen
• This B cell clones and differentiates into plasma cells and memory
cells
• The plasma cells produce high quantities of specific antibody to the
antigen, whereas memory cells survive in the bloodstream for years
• Upon re-exposure to the antigen, memory cells initiate a faster and
stronger response and thus confer long-term immunity
Fig down loaded from Google/Antibodies/Images
Antibody Production in vivo:
MHC - major histocompatibility complex
Antibodies Functions:
Polyclonal Antibodies Production:
(Hybridoma Technology)
(HGPRT+)
(HGPRT- )
The accumulation of fluid in the peritoneal cavity, causing abdominal swelling
 Antibodies (Polyclonal/Monoclonal) Uses in Basic and Applied
Sciences:
• Antibodies have a wide variety of uses. For example:
 To immuno-localize a particular antigen in a tissue immunohistochemistry
o Tissue can be fixed and incubated with the antibodies of
interest
o These antibodies can then be localized using a 'secondary'
antibody coupled to a gold particle or another enzyme or
radioactive label
o That gives a chemical reaction like horse radish peroxidase or
beta galactosidase
o A secondary antibody is frequently made by generating an
immune response to the Fc region of the primary antibody in
another species
o Thus, if the primary antibody is a mouse IgG
o then the secondary could be a rabbit anti-mouse antibody
which has been linked to β-galactosidase
o Alternatively the antibody can be purified and then conjugated
to another molecule to produce a fluorescent antibody
 to quantitate the presence of an antigen using either a
radioimmunoassay (RIA) or an enzyme linked immuno-absorbent
assay (ELISA)
 to detect a protein after fractionation by SDS-PAGE – a technique
called Western blotting
 to selectively bind to and precipitate an antigen – a technique
called immuno-precipitation
 to purify - a technique called affinity chromatography
 To diagnose and to treat various diseased conditions like cancer
Figs down loaded from Google/Antibodies/Images
iii. Glycosylation:
 It is a process by which sugars/glycans are chemically attached to
proteins to form glycoproteins
 It is a form of co-translational and post translational modification
 Five classes of glycans are produced:
• N-linked glycans attached to a nitrogen of asparagine or arginine sidechains
Secondary antibody linked with
enzyme (conjugated secondary
antibodies)
Primary antibody
linked with enzyme
Primary antibody
Secondary antibodies amplify the signal out put
Western Blotting
Human Epidermal Growth Factor Receptor 2 (HER2/neu)
CDC - Complement-dependent cytotoxicity
ADCC - Antibody-dependent cellular cytotoxicity
Figure 1. Elimination of tumor cells decorated with an ab-derived therapeutic protein via the recruitment
of effector cells. Depending on the type of effector cell and the chosen trigger molecule, the mode of
action can be either antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular
phagocytosis (ADCP), or a cytotoxic T-cell reaction. The depicted cytotoxic agents are a monoclonal ab,
a bispecific chemically coupled F(ab)2, a recombinant bispecific tandem single-chain Fv-fragment
(bsscFv), and a trispecific tandem single chain triplebody (sctb).
Antibodies 2012, 1(1), 88-123; doi:10.3390/antib1010088
Antibody-drug conjugates or ADCs are a new class of highly
potent biopharmaceutical drugs designed as a targeted therapy
for the treatment of people with cancer
Structure of divalent (top) and trivalent (bottom) scFvs, tandem (left) and di/trimerisation format (right).
Divalent (or bivalent) single-chain variable fragments (di-scFvs, bi-scFvs) can be
engineered by linking two scFvs.
Figure 3. Schematic structure of NK-cell recruiting agents. (A) tandem bispecific single
chain Fragment variable (bsscFv); (B) single chain triplebody (sctb); (C) two-chain diabody;
(D) tandem diabody (TandAb); (E) bispecific Tribody (bsTb); (F) bispecific Bibody (bsBb);
(G) dual-affinity re-targeting molecule (DART); (H) mini-ab; (I) immunoligand;
Antibodies 2012, 1(1), 88-123; doi:10.3390/antib1010088
•
O-linked glycans attached to the hydroxy oxygen of serine, threonine,
tyrosine, hydroxylysine or hydroxyproline side-chains
Fig down loaded from Google/glycolsylation/Images
Asparagine / Glutamine
Serine / threonine
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