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 glyco-engineered 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)

  Discovery/Preclinical Testing

File IND at FDA

Phase 1 Phase 2 Phase 3

File NDA

at FDA

FDA

    

Phase 4

Years 6.5 1.5 2 3.5 1.5

Additional post-marketing testing required by FDA

Test Population Laboratory and animal studies 20 to 100 healthy volunteers

100 to 500 patient volunteers

1,000 to 5,000 patient volunteers

Review process/approval

PurposeAssess safety, biological activity, and formulations

Determine safety and dosage

Evaluate effectiveness, look for side effects

Confirm effectiveness, monitor adverse reactions from long-term use

Success Rate 5,000 compounds evaluated 5 enter trials 1 approved

Investigational New Drug (IND)New Drug Application (NDA) US Food and Drug Administration (FDA)

Clinical Trials:

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 Phagocytosiso Fevero Inflammation ando 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 areo Exquisite antigenic specificity ando The ability to “remember” different types of antigens Adaptive immunity is activated only againsto Invading foreign material and o Never against its own molecules except in autoimmune diseases Therefore, it has the ability to distinguish between self and non-

self 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 Y-shaped 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=isch&sa=X&ei=UhekUpHDIoKShgeB-YG4Cg&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:

(HGPRT- )

(HGPRT+)

(Hybridoma Technology)

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 immuno-

histochemistryo Tissue can be fixed and incubated with the antibodies of interesto These antibodies can then be localized using a 'secondary' antibody

coupled to a gold particle or another enzyme or radioactive labelo That gives a chemical reaction like horse radish peroxidase or beta

galactosidaseo A secondary antibody is frequently made by generating an immune

response to the Fc region of the primary antibody in another specieso Thus, if the primary antibody is a mouse IgGo then the secondary could be a rabbit anti-mouse antibody which has

been linked to β-galactosidaseo 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 side-chains

Secondary antibodies amplify the signal out put

Primary antibody linked with enzyme

Secondary antibody linked with enzyme (conjugated secondary antibodies)

Primary antibody

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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